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Application of stable isotope labeled diphenhydramine to study the pharmacokinetics and metabolism of… Tonn, George Roger 1995

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Application of Stable Isotope Labeled Diphenhydramine to Study thePharmacokinetics and Metabolism of Diphenhydramine in Pregnant, Non-Pregnant, and Fetal Sheep.ByGeorge Roger TonnB.Sc. (Pharm.), University of British Columbia, Vancouver, Canada, 1988.M.Sc. (Pharm.), University of British Columbia, Vancouver, Canada, 1990.A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FORTHE DEGREE OF DOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Faculty of Pharmaceutical Sciences)(Division of Biopharmaceutics and Pharmaceutics)We accept this thesis as conforming to the standard requiredTHE UNIVERSITY OF BRITISH COLUMBIAFebruary, 1995© George Roger Tonn, 1995In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. it is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of Pharmaceutical SciencesThe University of British ColumbiaVancouver, CanadaDate2 7—02-95DE-6 (2188)11II. AbstractDiphenhydramine (DPHM), an antihistamine, has been used in pregnant women;however, information regarding its disposition in human pregnancy is lacking. Recently, detailedpharmacokinetic studies in pregnant sheep have demonstrated that DPHM readily crosses theovine placenta, and is eliminated from the fetus by placental and non-placental pathways. Thepurpose of this study is to investigate the components of the fetal non-placental elimination (i.e.,fetal renal and hepatic), and to compare these to the estimates obtained from adult sheep. Sincestable isotope techniques were to be employed, synthesis of stable isotope labeled DPHM (i.e.,[2H10]DPHM) and its major metabolite diphenylmethoxyacetic acid (i.e.,{2H10]DPMA) wasrequired. Next, gas chromatographic - mass spectrometric methods were developed tosimultaneously measure either DPHM and[2H10]DPHM, or DPMA and[2H10]DPMA. Thecurrent study demonstrates that the measured fetal renal clearance of DPHM contributes only—2% to the observed fetal non-placental clearance. Overall, the total non-placental clearance ofDPHM measured by direct methods (i.e., pulmonary [Yoo, 1989] and renal) can account for10% of the non-placental clearance. Unlike adult sheep, where hepatic extraction of DPHMwas —93%, no significant extraction of DPHM by the fetal liver following umbilical venousadministration was observed. Therefore, fetal hepatic elimination is not likely to account for theremainder of the fetal non-placental clearance. However, fetal hepatic in vitro metabolism ofDPHM (to form N-demethyl DPHM and DPMA) suggests that the fetal liver is capable ofDPHM biotransformation. Thus, the liver and possibly other organs may contribute at least aportion of the fetal non-placental clearance via DPHM biotransformation. It appears that only asmall fraction of the fetal non-placental clearance of DPHM can be accounted for by fetal renal111and pulmonary clearances. While the low renal clearance of intact DPHM is similar both in fetusand mother, large differences in the hepatic uptake andJor metabolism of DPHM were observedbetween mother and fetus. This suggests that the pathways for the non-placental elimination ofDPHM differ in mother and fetus. Despite the advances made in this study, the components ofthe fetal non-placental clearance remains largely unknown, and requires further study.ivIV. Table of ContentsII. Short Abstract jjifi. Table of Contents ivIV. List of Tables ixV. List of Figures xiiVI. List of Abbreviations xviVII. Acknowledgments xxii1. INTRODUCTION 11.1. General Introduction 11.2. Pharmacology of DPHM 21.3. Toxicology of DPHM 31.4. Clinical Applications of DPHM 41.5. Therapeutic Applications of Antihistamines In Pregnancy 41.6. Analysis Methods for DPHM 51.7. Pharmacokinetics of DPHM 61.7.1. Absorption of DPHM 61.7.2. Distribution of DPHM 71.7.3. Metabolism of DPHM 71.7.4. Excretion of DPHM and its Metabolites 91.8. DPHM Disposition in Pregnancy 101.8.1. Disposition and Fetal Effects of DPHM in Pregnant Sheep 111.9. Stable Isotopes 141.10. Rationale and Objectives 161.11. Hypothesis and Specific Aims 172. EXPERIMENTAL 202.1. Materials 202.1.1. Preparation of Stock Solutions and Buffers 222.2. Equipment and Instrumentation 242.2.1. Gas Chromatography with Nitrogen Phosphorus Specific Detection 242.2.2. Gas Chromatography with Mass Spectrometry 252.2.3. Gas Chromatography/High Performance Liquid Chromatography - 25Mass Spectrometry2.2.4. Spectrophotometer 252.2.5. Physiological Monitoring 252.2.6. General Experimental Equipment 262.3. Chemical Synthesis of Standards and Metabolites of DPHM 272.3.1. Synthesis of[2Hiojbenzhydrol 272.3.2. Synthesis of10]DPHM HC1 292.3.2.1. Characterization of[210]DPHM 31V2.3.3. Synthesis of N-demethyl DPHM HC1 and N,N-didemethyl DPHM HC1 312.3.3.1. Characterization of N-demethyl DPHM and N,N-didemethyl DPHM 312.3.4. Synthesis of DPMA and{2H10]DPMA 322.3.4.1. Characterization of DPMA and [2HIO]DPMA 332.3.5. Purity Assessment of Synthesized Standards. 352.4. Analytical Method Development 372.4.1. Development of an Analysis Method for DPHM and[2H10]DPHM 372.4.1.1. Optimization of Mass Spectrometer Parameters 372.4.1.2. Optimization of Gas Chromatographic Parameters 372.4.1.3. Optimization of Extraction Procedure 382.4.1.4. Gas Chromatography - Mass Spectrometer Operating Conditions 382.4.1.5. DPHM and[2H10JDPHM Extraction Procedure 392.4.1.6. Preparation of a Calibration Curve 402.4.1.7. Calculation of Extraction Recoveries of DPHM and[2H10]DPHM 412.4.1.8. Sample Stability Assessment 412.4.1.9. Method Validation 422.4.2. Development of an Analysis Method for DPMA and[2H10]DPMA 432.4.2.1. Optimization of Mass Spectrometer Conditions 432.4.2.2. Optimization of Gas Chromatograph Conditions 432.4.2.3. Optimization of Extraction 442.4.2.4. Optimization of Derivatization 442.4.2.5. Gas Chromatograph - Mass Spectrometer Operating Conditions 452.4.2.6. DPMA and[2H10]DPMA Extraction Procedure 462.4.2.7. Preparation of a Calibration Curve 472.4.2.8. Calculation of Extraction Recovery of DPMA and[2H10]DPMA 472.4.2.9. Sample Stability Assessment 472.4.2.10. Method Validation 482.5. Standard Procedures for Animal Experiments 482.5.1. Animal Handling 482.5.2. Surgical Preparation for Chronic Experimentation 492.5.2.1. Non-pregnant Sheep - Surgical Preparation 492.5.2.2. Pregnant Sheep - Surgical Preparation 512.5.3. Chronic Monitoring of Animals 552.5.3.1. Amniotic, Tracheal, and Vascular Pressures, Heart rate, Blood Flow, 55ECoG and EOG.2.5.3.2. Fetal Urine Measurements 552.5.3.3. Blood Gas Analysis 562.5.3.4. Glucose and Lactate measurements 562.5.4. Dosage Preparation 572.6. Experimental Protocols 572.6.1. Adult Non-Pregnant Studies 572.6.2. Adult Isotope Effect Studies 572.6.3. Fetal Isotope Effect Studies 582.6.3.1. FetalBolus 582.6.3.2. Fetallnfusion 58vi2.6.4. Adult First-Pass Metabolism 592.6.4.1. Adult First-Pass Metabolism During Normoxia 592.6.4.2. Adult First-Pass Metabolism During Mild Hypoxemia 602.6.5. Fetal Hepatic First-Pass Metabolism 602.6.5.1. Fetal Umbilical Venous Bolus 602.6.5.2. Fetal Umbilical Venous Infusion 612.6.6. Paired Fetal/Maternal Infusion 622.6.7. Sample Handling 632.7. In Vitro Experiments 632.7.1. DPHM Red Blood Cell Uptake 632.7.2. Adult and Fetal Hepatic Microsomal Metabolism Experiments 642.7.2.1. Preparation of Adult and Fetal Hepatic Microsomes 642.7.2.2. Protein Concentration and Cytochrome P450 Measurements 662.7.2.3. DPHM and N-demethyl DPHM Quantitation 662.7.2.4. Fetal and Adult Hepatic Microsomal Incubations 672.7.3. Plasma Protein Binding of DPMA 672.8. Data Analysis 692.8.1. Data Reduction 692.8.2. Calculation of In Utero Fetal Weight 692.8.3. Pharmacokinetic Data Analysis 702.8.4. Statistical Analysis 793. RESULTS 813.1. Development of Analytical Methodology 813.1.1. Capillary Gas Chromatographic-Mass Spectrometric Analysis of DPHM 81and[2H10]DPHM3.1.1.1. Optimization of Mass Spectrometer and Gas Chromatograph 813.1.1.2. Optimization of the Extraction Procedure for the DPHM and[2H10IDPHM 85Analysis Method3.1.1.3. Calibration Curve for the DPHM and[2H10]DPHM Assay 903.1.1.4. Extraction Recoveries of DPHM and{10]DPHM 913.1.1.5. Sample Stability Assessment of DPHM[210]DPHM 913.1.1.6. Validation of DPHM and[2H10]DPHM Gas Chromatographic - Mass 93Spectrometric Analysis Method3.1.2. Capillary Gas Chromatographic - Mass Spectrometric Analysis of DPMA 96and[2H10IDPMA3.1.2.1. Optimization of Mass Spectrometer and Gas Chromatography 963.1.2.2. Optimization of Extraction and Derivatization Procedures for DPMA 100and[2H10JDPMA3.1.2.3. Calibration Curve for DPMA and[2H10]DPMA 1023.1.2.4. Extraction Recovery of DPMA and{10]DPMA 1033.1.2.5. Sample Stability Assessment 1033.2.1.6. Method Validation for DPMA and[2H10]DPMA Analysis 1043.2. Animal Experimentation 106vii3.2.1. Disposition of DPHM in Non-Pregnant Sheep 1063.2.2. Isotope Effect Studies 1093.2.2.1. Adult Non-Pregnant Sheep 1093.2.2.2. FetalLambs 1153.2.2.2.1. Bolus Studies 1153.2.2.2.2. Infusion Studies 1193.2.3. Hepatic First-Pass Metabolism Studies 1233.2.3.1. Non-Pregnant Sheep 1233.2.3.1.1. Mesenteric Bolus Administration in Normoxic Conditions 1233.3.3.1.2. Mesenteric Bolus Administration during Mild Hypoxemia 1253.2.3.2. FetalLambs 1283.2.3.2.1. Umbilical Venous Bolus Administration 1293.2.3.2.3. Umbilical Venous Infusions 1323.2.4. Paired Fetal/Maternal Infusions 1353.2.4.1. Estimates of Trans-Placental and Non-Placental Clearances 1353.2.4.2. Metabolism of DPHM to DPMA in Mother and Fetus 1453.2.4.3. Renal Clearance of DPHM,[2H10]DPHM, DPMA and[2H10]DPMA 1493.2.4.4 Fetal Effects following Paired Maternal/Fetal Infusions 1543.3. In Vitro Studies 1573.3.1. Uptake of DPHM in Red Blood Cells 1573.3.2. Plasma Protein Binding of DPMA 1573.3.3. Fetal and Adult Hepatic Microsomal Metabolism of DPHM 1584. DISCUSSION 1634.1. Development of a Gas Chromatographic-Mass Spectrometric Method 163for the Simultaneous Quantitation of DPHM and[2H10]DPHM fromBiological Fluids Obtained from Pregnant Sheep4.2. Development of a Gas Chromatographic-Mass Spectrometric Method for 168the Simultaneous Quantitation of DPMA and[2H10]DPMA in Ovine Plasmaand Urine4.3. Disposition of DPHM in Non-pregnant Sheep 1714.4. Isotope Effect Studies 1734.5. Hepatic First Pass Metabolism of DPHM in Adult and Fetal Sheep 1764.5.1. Hepatic First Pass Metabolism of DPHM in Adult Sheep During Normoxia 1764.5.2. Hepatic First-Pass Metabolism of DPHM in Adult Sheep during Mild 177Hypoxemia4.5.3. Fetal Hepatic First-Pass Metabolism Following Umbilical Venous 180Administration4.6. Paired Maternal-Fetal Infusions of DPHM and[2H10]DPHM 1874.6.1. Fetal Behavioral Effects Following Simultaneous Maternal-Fetal Infusions 187of DPHM and[2H10]DPHM4.6.2. Trans- and Non-Placental Clearances of DPHM in the 189Maternal/Fetal Unit4.6.3. Fetal and Maternal Renal Clearances of[2H10]DPHM and DPHM 197viii4.6.4. Disposition of DPMA and[2H10]DPMA in the Ovine Fetal/Maternal 199Unit following Simultaneous Maternal/Fetal Infusions4.6.5. Fetal and Maternal Renal Clearance of DPMA and[2H10]DPMA 2024.7. Plasma Protein Binding of DPMA 2044.8. In Vitro Metabolism of DPHM in Hepatic Microsomes Prepared from 205Fetal and Adult Sheep5. SUMMARY AND CONCLUSIONS 2085.1. Synthesis of[2H10]DPHM, and Simultaneous Analysis of DPHM and 208[2H10]DPHM in Biological Fluids Obtained From Pregnant Sheep5.2. Synthesis of[2H10]DPMA, and Simultaneous Analysis of DPMA and 208[2H10]DPMA in Biological Fluids Obtained from the Ovine Fetal/Maternal Unit5.3. Lack of Isotope Effects Following Fetal and Maternal[2H10]DPHM 209Administration5.4. Hepatic First-Pass Metabolism of DPHM in Adult and Fetal Sheep 2105.5. Simultaneous Maternal/Fetal Infusions of DPHM and[2H10IDPHM 2125.6. Fetal and Maternal Hepatic Microsomal Metabolism of DPHM 2145.7. Global Summary 2155.8. Conclusions 2166. REFERENCES 220ixIV. List of TablesTable 1: Intra-day variability of DPHM and[2H10]DPHM measurements in plasma, 94amniotic, and fetal tracheal fluid.Table 2: Inter-day variability of DPHM and[2H10JDPHM measurements in plasma, 94amniotic, and fetal tracheal fluid.Table 3: Intra-day variability of DPMA and[2H10]DPMA assay method in 105plasma and urine.Table 4: Inter-day variability of DPMA and[2H10]DPMA assay method in 105plasma and urine.Table 5: Pharmacokinetic Parameters following a 100 mg IV bolus of 107DPHM via the femoral vein in non-pregnant ewes.Table 6: Biliary concentrations of DPHM obtained from periodic 107collections of bile from two non-pregnant ewes following a100 mg femoral venous bolus dose of DPHM.Table 7: Pharmacokinetic parameters of DPHM and{2H10]DPHM 110following simultaneous IV bolus administration of equimolaramounts of DPHM HC1 and[2H10]DPHM HC1 via the femoralvein in two non-pregnant ewes.Table 8: Pharmacokinetic parameters of DPHM and[2H10]DPHM 116following simultaneous administration of equimolar amountsof DPHM HC1 and[2H10]DPHM HC1 via the fetal lateral tarsalvein.Table 9: Pharmacokinetic parameters obtained from non-pregnant sheep 124during a mesenteric first-pass metabolism study following thesimultaneous administration of DPHM or[2H10]DPHM via thefemoral or mesenteric vein.Table 10: Initial and average percentage changes in blood gas status following 126the initiation of mild to moderate hypoxemia induced by nitrogengas infusion into the trachea of non-pregnant sheep.Table 11: Pharmacokinetic parameters obtained from the non-pregnant 128mesenteric first-pass metabolism study following the simultaneousadministration of DPHM or[2H10]DPHM via the femoral ormesenteric vein during mild hypoxemia.xTable 12: Pharmacokinetic parameters calculated using femoral arterial 130plasma drug concentrations during fetal umbilical first passmetabolism experiments following the simultaneousadministration of DPHM and[2H1O]DPHM.Table 13: Pharmacokinetic parameters calculated using carotid arterial 131plasma drug concentrations during fetal umbilical first passmetabolism experiments following the simultaneousadministration of DPHM and[2H10]DPHM.Table 14: Clearances values calculated following simultaneous steady-state umbilical 134and tarsal venous infusions of DPHM or[2H10]DPHMTable 15: Blood gas parameters during simultaneous umbilical venous and 134tarsal venous infusion of DPHM or[2H10]DPHM to steady-state.Table 16: Mean (± SEM) maternal and fetal arterial plasma drug concentrations 137following simultaneous maternal/fetal infusions.Table 17: Pharmacokinetic parameters calculated during paired 138maternallfetal infusionsTable 18: The uncorrected and corrected volumes of distribution of DPHM in 139maternal sheep and[2H10]DPHM in fetal sheep following asimultaneous fetal infusion of[210]DPHM (170 pg/min) andmaternal infusion of DPHM (670 j.tg/min).Table 19: Weight corrected trans- and non-placental clearances 142of DPHM in fetus and mother following a simultaneous infusionof DPHM to mother and[2H10]DPHM to the fetus.Table 20: Non weight corrected and weight corrected trans-placental 143and non-placental clearance parameters of DPHM followingsimultaneous infusions of DPHM and{2H10]DPHM tomother and fetus, respectively. Calculated using the mass balanceapproach.Table 21: A comparison of model dependent estimates of trans-placental 144clearances (2 compartment open model), and model independent(Fick Method) estimates of trans-placental clearances.xiTable 22: Pharmacokinetic parameters calculated from the simultaneous fitting 146of DPHM and DPMA in maternal plasma and[2H10JDPHM and[2H10JDPMA in fetal plasma.Table 23: Fetal and maternal urine flow during paired fetal and maternal 150infusions of DPFIM and[2H10]DPHM, respectively.Table 24: The renal clearances of DPHM and DPMA, and[2H10]DPHM 151and[2H10]DPMA in adult sheep and in fetal lambs.Table 25: Protein and Cytochrome P450 concentrations in fetal and adult 160microsomes.Table 26: Comparison between total, trans-, and non-placental clearances 189from a previous study (Yoo et al., 1993) using time separatedmaternal and fetal infusions, and the current study using simultaneousfetal and maternal infusions of[2H10JDPHM and DPHM.xiiV. List of FiguresFigure 1: Chemical structure of diphenhydramine 1Figure 2: Synthesis of a)[2H10]benzophenone, and b)[2Hio]benzhydrol 29Figure 3: Synthesis of[2H10jDPHM 30Figure 4: Synthesis of DPMA and[2H10]DPMA 34Figure 5: A 2 compartment-open model for the disposition of DPHM in fetal 70or maternal sheep.Figure 6: Schematic representation of the 2 compartment open model for drug 74disposition in the maternal fetal unit.Figure 7: A linked pharmacokinetic model showing 2 compartment kinetics for 78parent drug (DPHM), and 1 compartment kinetics for the metabolite(DPMA) used for the estimation of metabolite pharmacokinetic parametersin mother and fetus.Figure 8: The mass spectra and fragment assignment of a)DPHM and 82b)[2H10]DPHM following electron impact ionization (70 eV) of thepurified standards.Figure 9: The mass spectrum and mass assignments of N-demethyl DPHM 84following GC-MS with electron impact ionization (70 eV).Figure 10: The effect of varying inlet purge times on total area counts of DPHM. 85Figure 11: The effect of varying the initial column temperature on the half-height 86peak width of DPHM.Figure 12: The relative extraction efficiency (peak area counts for DPHM) of 87different solvents for the extraction of DPHM and[2H10]DPHMfrom ovine plasmaFigure 13: Optimized extraction procedure for DPHM and[2H10]DPHM from 88biological fluids obtained from pregnant sheep.Figure 14: Ion chromatograms for DPHM (m/z 165) and[2H10IDPHM (m/z 173) 89in plasma, fetal tracheal fluid, and amniotic fluid. Blanks, spikedstandards (2.0 ng/mL), and biological samples are shown.xliiFigure 15: Calibration curve for DPHM and[2H10]DPHM in plasma. 90Figure 16: The effect of three freeze-thaw cycles on the plasma concentrations 92of DPHM and[2H10JDPHM in spiked plasma samples.Figure 17: The effect of prolonged bench-top storage (22° C) on the plasma 92concentrations of DPHM and[2H10]DPHM in spiked plasma samples.Figure 18: The correlation between concentrations of DPHM and[2H10]DPHM 95measured using the GC-MS method and a previously developedGC-NPD methodFigure 19: The mass spectra of the TBDMS derivatives of DPMA and 97[2H10]DPMA and the mass fragment assignments following GC-MSwith electron impact ionization (70 eV) of the standards.Figure 20: Ion chromatograms of m/z 165 (internal standard), 183 (DPMA), 99and 177([2H10]DPMA) in blank plasma and urine and plasma and urinespiked with 250.0 ng/mL of DPMA, 250.0 ng/mL of[2H10]DPMA, and400.0 ng of the internal standard DPAA.Figure 21: The optimized extraction scheme for DPMA and[2H10]DPMA from 101ovine plasma and urine.Figure 22: Calibration curve for DPMA and[2H10]DPMA extracted from 102ovine plasma.Figure 23: Degradation of DPMA in acidified (0.4 mL of 1.0 M HC1) water, plasma 104and urine matrices (i.e., pH < 1.2).Figure 24: Mean femoral arterial plasma concentrations (± SEM) of DPHM 108following a 100 mg IV bolus via the femoral vein in adult non-pregnantsheep.Figure 25: The mean cumulative amount (± SEM) of DPHM excreted in the 108urine following a 100 mg IV bolus dose of DPHM via the femoralvein in non-pregnant adult sheep.Figure 26: A plot of the representative plasma concentrations of DPHM and 111[2H10IDPHM following an equimolar dose of DPHM HC1 and10]DPHM HC1 administered simultaneously via the femoral veinto ewe 2169 (isotope effect study).xivFigure 27: A representative plot of the cumulative amount of DPHM and 112[2H10JDPHM in urine following an equimolar dose of DPHM HC1and10]DPHM HC1 administered simultaneously via the femoralvein to ewe 2169 (isotope effect study).Figure 28: A representative plot of the plasma concentrations of DPMA and 113[2H10]DPMA following an equimolar dose of DPHM HC1 and10JDPHM HC1 administered simultaneously via the femoral veinto ewe 2169 (isotope effect study).Figure 29: A representative plot of the cumulative amounts of DPMA and 114[2H10]DPMA in urine following an equimolar dose of DPHM HC1and10]DPHM HC1 administered simultaneously via the femoralvein to ewe .2169 (isotope effect study).Figure 30: A representative figure of the arterial plasma concentration of 117DPHM and[2H10]DPHM following a simultaneous IV bolus dose ofDPHM and10JDPHM via the fetal lateral tarsal vein.Figure 31: A representative plot of the amniotic and fetal tracheal fluid 118concentrations of DPHM and[2H10JDPHM following simultaneousadministration of DPHM and10]DPHM via the fetal lateraltarsal vein.Figure 32: Plasma concentrations of DPHM and[2H10]DPHM in fetal and 120maternal plasma in ewes 2181 and 2241 following simultaneousinfusions of 60 ig/min of each DPHM and[2H10]DPHMvia the fetal lateral tarsal vein.Figure 33: Amount of DPHM and[2H10]DPHM in fetal urine in ewes 2181 121and 2241 following simultaneous infusions of 60 jig/mn of eachDPHM and[2H10]DPHM via the fetal lateral tarsal vein.Figure 34: Concentrations of DPHM and[2H10]DPHM in amniotic fluid in 121ewes 2181 and 2241 following simultaneous infusions of 60 jig/mmof each DPHM and[H10jDPHM via the fetal lateral tarsal vein.Figure 35: Concentrations of DPMA and[2H10]DPMA in fetal plasma in ewes 1222181 and 2241 following simultaneous infusions of 60 jig/mn of eachDPHM and{H10]DPHM via the fetal lateral tarsal vein.Figure 36: A representative plot of the plasma concentrations of DPHM and 125[2H10]DPHM in a non-pregnant ewe (E#1 154) following simultaneousmesenteric (DPHM) and femoral venous([2H10]DPHM) administration.xvFigure 37: Representative plots of the plasma concentrations of DPHM and 127[2H10]DPHM in a non-pregnant ewe (E#1 158-above) and (E#102-below)following simultaneous mesenteric (DPHM) and femoral venous([2H10]DPHM) administration during mild hypoxemia.Figure 38: A representative figure showing the fetal femoral and carotid 132arterial plasma concentrations of DPHM and[2H10JDPHMfollowing the simultaneous administration of DPHM via theumbilical vein and[2H10]DPHM via the fetal lateral tarsal vein.Figure 39: Mean (± SEM) femoral arterial plasma concentrations of DPHM and 140[2H10]DPHM in fetus and mother following simultaneous infusionof DPHM (670 tg/min) via the maternal femoral vein and[2H10]DPHM(170 pg/min) via the fetal lateral tarsal vein.Figure 40: A representative plot of the concentrations of DPHM and 141{2H10]DPHM in amniotic fluid and fetal tracheal fluid followingsimultaneous infusion of DPHM (670 tg/min) via the maternalfemoral vein and[2H10]DPHM (170 tg/min) via the fetal lateraltarsal vein.Figure 41: Mean (± SEM) maternal and fetal femoral arterial plasma concentrations 147of DPMA and[2H10]DPMA following simultaneous fetal and maternalinfusions of10]DPHM (170 pg/min) and DPHM (670 .tg/min), respectively.Figure 42: Mean (± SEM) maternal and fetal femoral arterial plasma concentrations 148of DPMA and[2H10]DPMA following simultaneous fetal and maternalinfusions of10JDPHM and DPHM respectively.Figure 43: Mean (± SEM) cumulative amounts of DPHM in maternal urine 152and[2H10]DPHM in fetal urine following simultaneous fetal infusion of{10]DPHM (170 ig/min) and maternal infusions of DPHM (670 ig/min).Figure 44: Mean (± SEM) cumulative amounts of DPMA in maternal urine and 153[2H10]DPMA in fetal urine following simultaneous fetal and maternalinfusions of[2H10]DPHM (170 .tg/min) and DPHM (670 tg/min)respectively.Figure 45: Mean (± SEM) high, intermediate, and low electrocortical activity in 155fetal sheep following simultaneous fetal and maternal infusions of[2H10]DPHM and DPHM, respectively.xviFigure 46: Mean (± SEM) fetal heart rate and fetal breathing movements 156following simultaneous fetal and maternal infusions of[2H10JDPHM and DPHM, respectively.Figure 47: Time course for DPHM to distribute into red blood cells 158Figure 48: Time course of DPHM disappearance and N-demethyl DPHM 161production in adult microsomes.Figure 49: Time course of DPHM disappearance in fetal microsomes. 161Figure 50: Production of N-demethyl DPHM in fetal and maternal microsomes 162following a 90 minute incubation.Figure 51: Production of DPMA in fetal and maternal microsomes following 162a 90 minute incubation.Figure 52: An anatomical sketch of the fetal liver in sheep 181xviiVI List of Abbreviationsp. MicronAlpha, an exponential rate constant (apparent rate of distribution)Beta, an exponential rate constant (apparent rate of elimination)o Coefficient of variability estimated by maximum likelihood non-linearcurve fitting using Adapt IICoefficient of variability estimated by maximum likelihood non-linearcurve fitting using Adapt II°C Degree Celsius.tg MicrogramMicroliter[2H10]DPHM Stable isotope (deuterium) labeled diphenhydramine[2H10]DPMA Stable isotope (deuterium) labeled diphenylmethoxyacetic acidDeuterium2H0 Deuterium oxideACS American Chemical Societyad. lib. ad libitum - at willAMN AmnioticANOVA Analysis of varianceAUC Area under the plasma concentration vs. time curveAUMC Area under the first moment curveca ApproximateCA Fetal carotid arteryCp Steady-state plasma concentrationCLff Total drug clearance from the fetal compartmentCLfm Trans-placental clearance of drug from the fetal to maternalcompartmentCLfo Non-placental clearance of drug from the fetal compartmentxviiiCLmf Trans-placental clearance of drug from the maternal to fetalcompartmentCLmm Total drug clearance from the maternal compartmentCLmo Non-placental clearance of drug from the maternal compartmentcm CentimeterCp Plasma concentrationCLT Total body clearanceDOS Disc Operating SystemDPAA Diphenylacetic acidDPHM DiphenhydramineDPMA Diphenylmethoxyacetic acidDSC Differential scanning calorimetryE Extraction ratioE# Ewe numberECoG Electrocortical activityEDTA Ethylenediaminetetraacetic acidEl Electronic impact ionization modeEoG Electroocular activityeV Electron voltsF BioavailabilityFA Fetal femoral arteryFm Fraction of drug converted to metabolite divided by the apparent volumeof distribution of the metabolite (DPMA)fm Fraction of drug converted to metabolite (DPMA)fr. French (designation of catheter sizes)g GramGC Gas chromatographyGFR Glomerular filtration rateH’-NMR Proton nuclear magnetic resonanceH2 Hydrogen gasxixHC1 Hydrochloric acidHP Hewlett PackardHPLC High performance liquid chromatographi.d. Internal diameteri.e., id est; that isJV Intra VenousK10 Apparent first-order rate constant describing the elimination of drug fromthe central compartmentK12 Apparent first-order rate constant describing the transfer of drug from thecentral compartment to the peripheral compartment in a twocompartment modelK21 Apparent first-order rate constant describing the transfer of drug from theperipheral compartment to the central compartment in a twocompartment modelKC1 Potassium chlorideKf Apparent first-order rate constant describing the formation of metabolitefrom parent drugKg KilogramKm apparent first-order rate constant describing the elimination of metaboliteKo Drug infusion rate to the motherKo’ Drug infusion rate to the fetuskPa KilopascalsLC Liquid chromatographLD50 Median lethal doseLOQ Limit of quantitationM Molar (moles/litre)m Meterm/z Mass to charge ratioMA Maternal arterialmg MilligramxxMHz Megahertzmm Minutenim Millimetermlvi MillimolarMDRT Mean dispositional residence timeMS Mass spectrometryMSD Mass selective detectormsec MillisecondMTBSTFA N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamideMTRT Mean total residence timeMV Maternal femoral veinNADH -nicotinamide-adenine dinucleotide disodium saltNADPH f3-nicotinamide-adenine dinucleotide tetrasodium saltNaOH Sodium hydroxideNCI Negative chemical ionizationng NanogramNPD Nitrogen-phosphorous detectoro.d. Outer diameterpcg PicogramPo2 Partial pressure of oxygen in the bloodPCI Positive chemical ionizationPco2 Partial pressure of carbon dioxide in bloodPFTBA PerfluorotributylaminepH Negative logarithm of hydrogen ion concentrationPFBBr. Pentafluorobenzyl bromideP.S.I. Pounds per square inchPTFE Polytetrafluoroethylenep-TSA para-toluene sulfonic acidQH Liver blood flowQum Umbilical blood flowxxiRBC Red blood cellsSD Standard deviationSEM Standard error of meanSIM Selected ion monitoringT Time (duration of infusion)t TimeT112 Half-lifeTEA TriethylamineTLC Thin layer chromatographyTR Tracheal fluidTRIS Tris(hydroxymethyl) aminomethaneU International units (insulin dosages)USP United States PharmacopeiaUV Umbilical VeinV VoltsVc Apparent volume of distribution of central compartmentVd13 Apparent volume of distributionVd Apparent steady-state volume of distributionVm Apparent volume of distribution of metaboliteX g Times gravity (centrifugal force)EXu Cumulative amount of drug in urineEMu Cumulative amount of metabolite in urinexxiiVII AcknowledgmentsThere are a number of people without whom this project would not have been possible,and I would like to take this opportunity to thank them for their contributions to the workconducted in this thesis, and their emotional support throughout these years.Firstly, I would like to acknowledge the support and guidance of my supervisors Drs. J.E.Axelson and D.W. Rurak, and my supervisory committee: Drs. F.S. Abbott, J. Diamond, M.J.Douglas, and R.L. Thies.There are also a number of people from our lab who made coming to work each day apleasure. I would like to thank Drs K.W. Riggs, S.D. Yoo, M.R. Wright, K.Y. Yeleswaram, S.Panesar, and A. Borel for their friendship, guidance, and a shoulder to lean on particularly duringmy early training. Thank you to all the well qualified technicians, Mr. E. Kwan, Mrs. C. Hall,Dr. A. Szeitz, Mr. R. Burton and Mrs. B. McErlane who showed a great deal of patience inteaching me many new skills, all the time with a smile on their faces. Thanks to my colleaguesA. Douroudian, J.D. Gordon, J. Kim, K. Stobbs, W.P. Tan, S. Kumar, W. Tang, and Y. Palaty.My family has played a key role in my ability to complete this degree; my mom and stepfather, my brothers Gerry, Harry, and Eddie and their families. I am eternally indebted to you foryour love and support. In addition, my in-laws, Dick and the late Dinie Christiaanse, have alsobeen a great support for me.Mieke, I would not have been able to complete this degree without your daily love andsupport. I am truly and eternally grateful for you support, understanding, trust, and love.This thesis is dedicated to the memory of my loving father Edmund Tonn. Dad, I wishthat you could see me now; I hope that you are proud.11. Introduction1.1. General IntroductionDiphenhydramine [2-(diphenylmethoxy)-N,N-dimethylethylamine, DPHM (Molecularweight 255)] is a potent reversible H1 receptor antagonist of the ethanolamine class (Douglas,1985). It is a weak base with a pKa of 9.0, thus, at a physiological pH (i.e., pH 7.4) the drug isalmost completely ionized. DPHM is also highly lipophilic with a octanol/water partitioncoefficient of 1862. DPHM (Figure 1) was one of the first H1 receptor antagonists to bemarketed in the late 1 940s, and is still marketed today in Canada as either a single entity product(Benadryl® or Nytol®) or in combination with other drugs in cough and cold preparations.HC—O—CH2HN(CH3)Figure 1: Chemical structure of diphenhydramine21.2. Pharmacology of DPHMDPHM binds reversibly to both peripheral and central H1 receptors with little or nobinding to H2 receptors (Douglas, 1985). As a result, it antagonizes many of thepharmacological actions of histamine at these receptors. This includes histamine-stimulatedsmooth muscle contraction (e.g., gastrointestinal and respiratory smooth muscle),vasoconstriction, and, more importantly, vasodilation (Douglas, 1985). The drug also reducesvascular permeability, and thus limits edema, wheal, and flare formation following localizedexposure of the dermis to histamine (Simons et al., 1990, Douglas 1985, and Bilzer et al., 1974).As a result of binding to H1 receptors in the central nervous system (CNS), DPHM can elicitCNS depression (somnolence, slowed reaction times, and diminished alertness) at normaltherapeutic doses (Douglas, 1985, Gengo et al., 1989, and Gengo, et al., 1990). In fact, attherapeutic doses somnolence has been reported in over half of the human subjects studied(Preston et al., 1992, Roehrs et al., 1993). However, at high or toxic doses, DPHM can provokeCNS stimulation (Douglas, 1985). In addition to the H1 receptor antagonist activities, DPHMalso possesses atropine-like anticholinergic, local anesthetic, antitussive, and antinauseaactivities (Douglas, 1985, Packman et al., 1991). Moreover, it binds to monoamine oxidases inrat and mouse brain and lung. This binding has been shown to alter the normal concentrations ofvarious biogenic amines in these tissues (Chiavegatto and Bernardi, 1991, Shishido et al., 1991,Yoshida et al., 1989, Yoshida et al., 1990).31.3. Toxicology of DPHMThe IV median lethal dose (LD50)of DPHM is 42-46 mg/Kg in rats, 98 mg/Kg in mice,10-11 mg/Kg in rabbits, and 42-46 mg/Kg in dogs, while the oral LD50 is 500-545 mg/Kg in rats,and 164-167 mg/Kg in mice (Rieveschl and Gruhzit, 1945, and Gruhzit and Fisken, 1947).Following subcutaneous administration, the LD50 in rats ranges from 3.8 mg/cm2 in 4 day oldrats to 20.7 mg/cm2 in 40 day old rats, demonstrating that the LD50 of DPHM increases with agein rats (Lee, 1966). The reason for this may be that more drug is absorbed followingsubcutaneous administration in younger compared to older rats (Lee, 1966). The general toxicsymptoms include excitement, tremors, convulsions, and respiratory and cardiac failure(Rieveschl and Gruhzit, 1945, and Gruhzit and Fisken, 1947). In humans, DPHM has asubstantial margin of safety (i.e., toxicity has been reported to occur at 6-40 times the normaltherapeutic dose); however, toxicities in both adults and children are common (Douglas, 1985).Toxicity has been reported in children with chicken pox following topical DPHM administration(Chan and Wallander, 1991, Huston et at., 1990, and Bernhard and Madison, 1991). Accordingto these reports, children were agitated, confused, and hallucinating. In more severe poisoning inchildren, the previously noted symptoms are often followed by convulsions, cardiovascular andpulmonary collapse, and death (Douglas, 1985). In adults, DPHM poisoning is relativelycommon and symptoms include sedative and anticholinergic effects, and coma. In more severecases of poisoning, cardiovascular and pulmonary collapse and death have been reported (Clarkand Vance, 1992).DPHM, like chiorpheniramine and mepyramine, is embryo toxic, but it does not appear tobe teratogenic in rats (Naranjo and de Naranjo, 1968, and Schardein et al., 1971). These data are4supported by data from retrospective human studies (Saxen, 1974). However, in combinationwith morphine, DPHM appears to potentiate fetal malformations (lulliucci and Gautieri, 1971).1.4. Clinical Applications of DPHMDPHM is used in the treatment of various allergy-mediated diseases. It is effective in thesymptomatic treatment of urticaria, seasonal rhinitis and conjunctivitis, and allergic dermatoses(Moscati and Moore, 1990, and Douglas, 1985). The drug is also marketed as an “over thecounter” hypnotic. However, by virtue of DPHM’ s hypnotic effects, its use for the treatment ofallergy-mediated conditions is decreasing with the advent of newer non-sedating antihistamines.DPHM has also been used in various cough and cold preparations either as a single entityproduct or in combination with other pharmacological agents. In addition, it has been used totreat nausea and vomiting of various etiologies, including motion sickness and cancerchemotherapy (Grunberg et at., 1988, and Roila et al., 1989). Recently, DPHM has also beenused as a local anesthetic agent for the repair of minor lacerations and wounds (Ernst et at.,1993).1.5. Therapeutic Applications of Antihistamines In PregnancyAlthough the use of drugs during pregnancy is generally discouraged, certaincircumstances arise where drug treatment becomes necessary. Antihistamines have been usedduring pregnancy for the treatment of specific pregnancy related conditions, such as pregnancyrelated urticaria and severe nausea and vomiting (Forfar and Nelson, 1973). However, some of5the uses of antihistamines during pregnancy are essentially extensions of their therapeuticapplications in the non-pregnant population, such as the treatment of insomnia, symptomatictreatment of cough and colds, and alleviation of the symptoms of allergic rhinitis (Piper et al,1987). A recent survey demonstrated that drugs are used in approximately 15% of pregnantwomen for the treatment of cold and flu, 7% for nausea and vomiting, 2% for insomnia, and 6%for skin related conditions (de Jong-van den Berg et al., 1993). Moreover, according to drug usesurveys between 1963 and 1987, antihistamines were used by -2O% of pregnant women(Peckham and King, 1963, Forfar and Nelson, 1973, Hill, 1973, Doering et at., 1978,Brockelbank et al., 1978, Rayburn et al., 1982, and Piper et al., 1987), and among antihistaminicagents, DPHM was used in —23% of the documented cases (Piper et at., 1987). These findingssuggest that a significant number of human fetuses may be exposed to this drug at some timeduring their gestation.1.6. Analysis Methods for DPHMMany methods have been reported for the analysis of DPHM in biological fluids. Theseinclude UV (Wallace et al, 1966) and fluorescence (Glazko, 1974) spectrophotometry, highperformance liquid chromatography (HPLC) with either UV detection (Selinger et at., 1990) orfluorescence detection (Webb and Eldon, 1991), gas chromatography (GC) analysis using flameionization detection (Albert et at., 1974, Barni Comparini et at., 1983, and Chiarotti et at., 1983),nitrogen phosphorus specific detection (NPD) (Bilzer and Gundert-Remy, 1973, Baugh andCalvert, 1976, Abernethy and Greenblatt, 1983, Lutz et at., 1983, Meatherall and Guay, 1984,and Yoo et at., 1986), and mass spectrometry (MS) (Chang et at., 1974, Carruthers et at., 1978,6Rohdewald and Milsmann, 1986, Maurer and Pfleger, 1988, and Walters-Thompson andManson, 1992). Although some of these methods possess the required sensitivity for the analysisof DPHM in biological fluids obtained from pregnant sheep, only methods employing MS couldprovide the necessary differentiation between DPHM and stable isotope labeled (SIL) DPHM.Since stable isotope techniques were to be applied, this differentiation between SIL DPHM andunlabeled DPHM was necessary. However, the GC-MS methods published to date focus on arelatively non-specific small mass fragment ion (i.e., mlz 58) (Chang et al., 1974, Carruthers etal., 1978, Rohdewald and Milsmann, 1986, Maurer and Pfleger, 1988, and Walters-Thompsonand Manson, 1992). This fragment would not be capable of differentiation between DPHM andSIL DPHIvI, particularly if the labels were on the aromatic rings of the molecule. Therefore,development of a method capable of simultaneously measuring DPHM and STh DPHM wasrequired.1.7. Pharmacokinetics of DPIIM1.7.1. Absorption of DPHMDPHM is rapidly absorbed in humans following oral administration, with maximalplasma concentrations attained between 1-4 hours following administration (Carruthers et al.,1978, Gielsdorf et al., 1986, Blyden et al., 1986, Luna et al., 1986, and Simons et al., 1990).Peak plasma concentrations following a 50 mg oral dose were between 40 and 80 ng/mL(Carruthers et al., 1978, Gielsdorf et al., 1986, Luna et al., 1986). The drug undergoes asubstantial first-pass effect following oral administration in humans, with bioavailability between70.43 to 0.78 (Albert et at., 1975, Carruthers et al., 1978, Spector et at., 1980, and Blyden et al.,1986).1.7.2. Distribution of DPHMThe plasma protein binding of DPHM has been reported to be as high as 98% in humans(Albert et at., 1975). However, more recent studies have suggested that the plasma proteinbinding of the drug is somewhat less (i.e., —78-85%) (Spector et at., 1980, Meredith et at., 1984).In addition, Spector et at. (1980) demonstrated that there is a difference in the plasma proteinbinding between Orientals and Caucasians (i.e., binding was 76% in Caucasians and 85% inOrientals). It has also been demonstrated that the binding of DPHM decreases from 78% to 67%in chronic liver disease (Meredith et al., 1984). Since binding of DPHM to human serumalbumin is only —30% (Drach et al., 1970), it is likely that other plasma proteins, such as o-1-acid glycoprotein (known to bind basic drugs) may also play a role in the binding of this agent(Kremer et al., 1988). However, the binding of DPHM has not been extensively studied in eitherhumans or animals. The tissue distribution pattern following either oral, intraperitoneal, andintravenous administration to either rats or guinea pigs showed that the highest tissue drugconcentrations were in the lung, followed by the spleen, brain, liver, muscle, and heart (Glazkoand Dill, 1949a). The high tissue distribution of DPHM in the lung may be due to the highbinding of this drug to monoamine oxidases, as demonstrated in perfused rat lung and in isolatedrat lung mitochondria (Yoshida et at., 1989 and 1990).81.7.3. Metabolism of DPHMIn rat, guinea pig, and rabbit, DPHM is extensively degraded in liver homogenates, and toa lesser extent in lung and kidney homogenates (Glazko and Dill, 1949b). The degradation in ratliver appears to be rapid, with approximately 75% of the added amount of DPHM degradedwithin 120 minutes. Similar findings were reported by Lee (1966), who demonstrated thatDPHM degradation occurs rapidly, and that the rates did not differ between hepatic homogenatesprepared from older (40 days) and younger (15 days) rats. The large first-pass effect followingoral administration and the reduced clearance of DPHM in patients with chronic liver diseasesuggests that hepatic elimination of DPHM may also be an important route of elimination inhumans (Meredith et al., 1984, Albert et al., 1975, Carruthers et al., 1978, and Blyden et al.,1986). The hepatic elimination of this drug may partially occur via N-demethylation, sinceDPHM was found to be rapidly N-demethylated in rat liver microsomes (Roozemond et al.,1965). DPHM and some closely related analogs (i.e., orphenadrine) form metabolic-intermediatecomplexes with the rat liver cytochrome P450 2B1 and 2C6 isoforms (Bast et al., 1990, Rekka etal., 1989, and Reidy et al., 1989). The formation of these complexes may inhibit themetabolism, and thus, the elimination of other drugs. Recently Hussain et al. (1994)demonstrated that the co-infusion of DPHM with diltiazem in perfused rat livers resulted in aninitial sharp increase in the diltiazem perfusate concentration, followed by 45% higher steadystate concentrations of diltiazem compared to control values. This was thought to be due to bothdisplacement of diltiazem from hepatic tissue binding sites and the inhibition of diltiazemmetabolism by DPHM (Hussain et al., 1994). DPHM has also been shown to undergo Ndeamination in hepatic microsomes prepared from rats, guinea pigs, and rabbits (Yamada et al.,91993). These studies demonstrated that DPHM was deaminated to form methylamine,suggesting that the drug must be demethylated prior to being deaminated (Yamada et al., 1993).In humans, rhesus monkeys, and dogs, the metabolites identified in vivo suggest thatDPHM undergoes successive N-demethylations to give N-demethyl DPHM and N,N-didemethylDPHM, followed by deamination to yield diphenylmethoxyacetic acid (Drach and Howell, 1968,Chang et al., 1974, Glazko et al., 1974, and Drach et al., 1970). Diphenylmethoxyacetic acid(DPMA, Molecular weight 242) has been demonstrated to form glycine conjugates in dogs andglutamate conjugates in rhesus monkeys (Drach and Howell, 1968, Drach et al., 1970). DPMAand its conjugates are the most prominent urinary metabolites identified in dogs (-42%), andrhesus monkeys (—60%) (Drach et al., 1970). In addition, significant quantities (i.e., 10-20%) ofthe N-oxide metabolite of DPHM were found in the urine in all species examined to date (Drachand Howell, 1968, Drach et al., 1970, Chang et al., 1974). The in vivo metabolism of DPHM inthe rat has not yet been completely established, and many of the metabolites in this speciesremain unidentified (Drach and Howell, 1968). Recently, DPHM was shown to form aquaternary ammonium glucuronide conjugate in humans (Luo et al., 1991 and 1992). In humans,Glazko et al. (1974) and Blyden et al.(1986) demonstrated that plasma concentrations of the Ndemethylated metabolites declined in a fashion similar to intact DPHM. However, DPMA wasfound to accumulate in plasma for up to 24 hours following the oral administration of DPHM,and exceed plasma concentrations of DPHM and N-demethyl DPHM by 10 fold (Glazko et al.,1974).101.7.4. Excretion of DPHM and its MetabolitesOnly a small fraction of the DPHM dose administered is excreted as intact drug in theurine of rats (— 4-6%), rabbits (<3%), dogs (—4%), and monkeys (—3%) (Glazko et al., 1949,Drach et al., 1970, and Parry and Calvet, 1982). Similarly, only a small portion (2-4%) of intactDPHM is excreted in humans (Albert et al., 1975, Meredith et al., 1983). However, a largeportion of DPHM’ s metabolites are excreted in urine. The percentage of urinary metabolitesexcreted is —35% of the dose in rats, and —49% in humans (Glazko et al., 1949, and Drach et al.,1970). The role of biliary excretion of DPHM andlor its metabolites has not yet been reported ineither laboratory animals or humans.1.8. DPHM Disposition in PregnancyWith the exception of one case, which documented neonatal DPHM withdrawalsymptoms, information regarding DPHM effects and disposition in human pregnancy is absent(Parkin, 1974). Due to ethical and technical constraints, detailed studies of DPHMpharmacokinetics and pharmacodynamics in pregnancy cannot be conducted in humans.Therefore, several approaches have been developed to assess the extent of fetal exposure andplacental transfer of drugs. Single point estimations of drug concentrations in cord blood fromhumans following birth provide clinically relevant information; however, these estimates arehighly dependent on the time of sampling relative to that of drug administration (Anderson et al.,1980, and Levy and Hayton, 1973). The perfused human placenta has also been used to detailthe placental transfer of drugs, however, the development of leaks in the placenta duringperfusion can provide misleading findings (Faber and Thornburg, 1983). Several small animal11species, such as rats, guinea pigs, and rabbits have been utilized to examine drug disposition inthe fetal/maternal unit. However, a limitation of this approach is that serial blood samplescannot be obtained due to the small fetal blood volume in these species (Rurak et al., 1991).Chronic catheterized preparations, either sheep or primates, have been utilized for detailedmaternal and fetal pharmacokinetic and pharmacodynamic studies (Rurak et at., 1991). Sheepare the most commonly employed species; however, they possess an epithelialchorial placentawhich is less permeable to hydrophilic endogenous compounds and drugs, unlike thehemochorial placenta found in humans, primates, and several small animal models (Rurak et at.,1991, and Faber and Thornburg, 1983). Therefore, fetal/maternal pharmacokinetic experimentsconducted in sheep with polar hydrophilic compounds may not provide quantitative data that isrelevant to the situation in humans (Rurak et at., 1991). However, the placental transfer ofhydrophobic compounds, such as DPHM, does not appear to be limited in the sheep placenta(Yoo et at., 1986, Rurak et at., 1991).1.8.1. Disposition and Fetal Effects of DPHM in Pregnant SheepYoo et at. (1986) demonstrated that DPHM undergoes rapid placental transfer to the fetallamb, with maximum fetal levels occurring within 5 minutes following maternal bolusadministration. There was also extensive fetal exposure to the drug following maternal bolusadministration (i.e., AUC fetal/AUC maternal = 0.85). The lipophilic nature of the drug and therapid and extensive exposure of the fetus following maternal administration suggests that transplacental transfer of DPHM occurs by simple diffusion. Further, it was shown that DPHM doesnot persist in the fetal circulation, since the apparent terminal elimination half-life was similar in12both mother and fetus (i.e., 52 vs. 46 minutes, respectively). Similar to other amine drugs,DPHM was found to accumulate in fetal tracheal and amniotic fluids, with levels in the trachealfluid four fold greater than fetal plasma concentrations (Yoo, 1989, Riggs, et at., 1987, andRurak et at., 1991). The administration of DPHM via the amniotic cavity resulted in preferentialfetal uptake (Rurak et at., 1994). The routes of the fetal drug uptake identified are fetalswallowing, and uptake via the fetal membranes. These data suggest that DPHM present in theamniotic fluid could be recirculated in the fetal lamb via these mechanisms (Yoo et at., 1989).Time-separated fetal and maternal infusions to steady-state, utilizing a two compartment-open model (Szeto et at., 1982), demonstrated that both mother and fetus can eliminate DPHMvia placental and non-placental pathways (Yoo et at., 1993). In the fetal lamb, the rate of non-placental clearance was approximately three fold greater than that observed in the mother on aweight corrected basis (Yoo et at., 1993). This suggests that the fetal lamb, per Kg, is moreefficient in eliminating the drug than the mother. The elimination of DPHM via the fetal lung,although resulting in high tracheal fluid levels, accounts for only a small portion (-.8%) of thefetal elimination of the drug (Rurak et at., 1991). To date, this is the only specific route of fetalnon-placental elimination that has been explained for DPHM. Thus, for this drug, the bulk of thenon-placental clearance remains to be elucidated. DPHM is not unique in this regard. Otherdrugs, including ritodrine, labetalol, acetaminophen, metoclopramide, meperidine, and morphineall undergo substantial fetal non-placental clearance (Wright et at., 1991, Yeleswaram et at.,1993, Wang et at., 1986, Riggs et at., 1990 and Szeto et at., 1982), yet the components of theirnon-placental clearance remain largely unknown. Wang et at. (1986) demonstrated that -.97% ofthe maternal non-placental clearance could be accounted for by metabolic and renal pathways;however, in the fetal lamb only —33% of the non-placental clearance of acetaminophen could be13accounted for by these pathways. Olsen et al. (1988) also demonstrated that only -63% ofmorphine infused to the fetus resulted in the formation of morphine-3-glucuronide. Similarresults were also noted for ritodrine and labetalol (Wright et al., 1991, and Yeleswaram et al.,1993). Thus, an understanding of the fetal non-placental components responsible for DPHMelimination may also provide some insight into the non-placental clearance of other drugs.Yoo et al. (1993) also demonstrated that a difference exists for DPHM trans-placentalclearances from fetus to mother (CLfm) and from mother to fetus (CLmf). The CLfm was 2-3fold greater than the CLmf. Despite correcting these clearance estimates (i.e., CLmf and CLfm)for differences between the fetal and maternal plasma protein binding (i.e., —86% is bound in theewe and only 72% in the fetal lamb), the differences, although somewhat lower, still remained.The reason for this phenomenon is not currently known, however, the magnitude of the greaterdifference in CLfm compared to CLmf correlates with greater drug lipophilicity, and thus,placental permeability (CLfm) (Yoo et al, 1993).Ideally, with the 2-compartment-open model employed by Yoo et al. (1993), the fetal andmaternal infusions should be conducted simultaneously; however, without labeled drug, time-separated infusions are required (Szeto, 1982, Szeto et al., 1982, Yoo et al., 1993). Due to therapid elimination of DPHM from both mother and fetus, only a 48 hour washout period wasrequired in these studies. The effects of this washout period on the disposition of DPHM in thedynamic fetal/maternal unit are not clear; however, the short washout period would have likelyminimized these effects.During both fetal and maternal infusions, DPHM elicits substantial fetal behavioraleffects in sheep. These fetal effects appeared to vary in relation to the fetal plasmaconcentrations achieved. Rurak et al. (1988) demonstrated that at lower fetal drug14concentrations (—36 ng/mL) achieved with maternal drug administration, the fetal effects wereconsistent with CNS depression (i.e., decreases in low voltage ECoG pattern, low voltage ECoGpatterns associated with rapid eye movements, and the overall incidence of fetal breathingmovements). At higher plasma concentrations achieved with fetal drug infusion (—448 nglmL),transient declines in Po2 and pH, associated with tachycardia and vigorous fetal breathingmovements were observed during the initial portion of the infusion. In addition, there was a fallin low voltage ECoG activity and a marked increase in intermediate voltage ECoG pattern(Rurak et at., 1988).1.9. Stable IsotopesThe past two decades have seen a steady rise in the use of stable isotope labeledcompounds to investigate drug pharmacokinetics and metabolism in both laboratory animals andman (Browne, 1990). Stable isotopes are, as the name implies, stable forms (non-radioactive) ofan atom which differ only in atomic mass due to differing numbers of neutrons in the nucleus.Stable isotope labeling refers to the substitution of an atom in a molecule of interest with itscorresponding stable isotope (e.g., 13C, 170, 180 15N, and 2H). In most cases, the stable isotopelabeled (Sit) analog of the original molecule will have identical physical and chemical propertiesto the unlabeled molecule, but will differ in molecular mass. The most widely used analyticalmethodology to differentiate between Sit molecules and their unlabeled counterparts employsmass spectrometry coupled with either GC or HPLC (Baillie, 1981). To enhance the selectivity,and thus discern between labeled and unlabeled drug, the mass spectrometer is run in theselective ion monitoring mode (51M). This simply means that the mass spectrometer is15programmed to focus on individual fragment ions for the Sit drug and the unlabeled drug. Thus,SJM can provide both the necessary differentiation between a Sit and an unlabeled molecule(selectivity) and the required sensitivity (sub-nanogram range) for maternal and fetalpharmacokinetic studies.The use of Sit compounds in pharmacokinetic experiments provides several advantagesover traditional experimental designs (Browne, 1990, and Baillie, 1981). The simultaneous coadministration of Sit and an unlabeled counterpart in pharmacokinetic studies essentially allowstwo experiments to be conducted on one occasion (i.e., the control and the test). Thissignificantly reduces the inter-day variability and the influence of time dependent changes onpharmacokinetic parameters (Baillie, 1981, Browne, 1990, and Eichelbaum, 1982). In addition,this technique can also reduce the number of exposures to the drug, samples to be analyzed, andnumber of experimental days (Browne, 1990). These advantages essentially translate into areduction in the number of subjects/animals required for the equivalent degree of statisticalpower, and a reduction in cost and time.The key advantage of using stable isotope techniques in the study of drug disposition inpregnancy is the elimination of possible time-dependent effects on pharmacokinetic parametersdue to the dynamic nature of the fetal/maternal unit (Battaglia and Meschia, 1986). An increasein the statistical power resulting in a possible reduction in the number of study subjects is also ofparamount importance since the acquisition, preparation, and maintenance of chronicallyinstrumented pregnant sheep and other similar preparations (e.g., primates) is very costly. Inaddition, this approach allows for better utilization of animal resources. Since two experimentscan be conducted on one occasion, more experiments can be conducted on one animal during thenarrow time window available for experimentation (i.e. 7-21 days) before the ewe delivers. By16the same virtue, conducting both the control and test experiments on one occasion increases theprobability of conducting successful experiments (i.e., there is less loss of experimental data dueto catheter failures or fetal death occurring between test and control administrations than whenusing only unlabeled drug).There are also limitations to the use of stable isotope techniques. The largest impedimentis the lack of accessibility to Sit technology (i.e., instrumentation, SIL drug, and analyticalmethodology) (Browne, 1990). Moreover, the key assumption made following the simultaneousco-administration of the SIL and the unlabeled compound is that the SIL compound displays“equivalent” disposition characteristics to the unlabeled compound (i.e., absorption, distribution,metabolism, and excretion) (Baillie, 1981, Van Langenhove, 1986, Browne, 1990, andChasseaud and Hawkins, 1990). If this is not the case, the resulting “isotope effect” (i.e., thedifference between the labeled and the unlabeled drug) would severely limit the utility of the Sitdrug for pharmacokinetic studies. Therefore, prior to conducting an experiment utilizing thesimultaneous administration of Sit and unlabeled drug, the absence of an isotope effect must beverified (Wolen, 1986, Baillie, 1981, and Van Langenhove, 1986). However, where possible, itis also important to investigate the metabolic profile following administration of the SIL andunlabeled drug to ensure that the observed pharmacokinetic equivalence of the intact drug alsocorresponds to the metabolites generated (i.e., to rule out possible metabolic shifting)(Eichelbaum et al., 1990).1.10. Rationale and ObjectivesThe investigation of DPHM disposition in pregnant sheep has demonstrated that the drugrapidly and readily crosses the ovine placenta. In addition, these studies have shown that it is17eliminated by both placental and non-placental means, and that the weight corrected fetal non-placental clearance of DPHM exceeds that of the adult by 3 fold. The components of this largefetal non-placental clearance of DPHM is not clear, since only —8% of the fetal non-placentalclearance can be accounted for (fetal pulmonary uptake). This situation is not unique to DPHM;that is, for all drugs that have been studied in sheep, the routes of fetal non-placental clearancehave not been fully elucidated. Moreover, for some compounds it is clear that the routes ofelimination in the mother cannot account fully for fetal clearance of the drug. Thus, accountingfor the remainder of the fetal non-placental clearance of DPHM may provide a more generalinsight into drug disposition in the fetal lamb. In addition, the elimination of DPHM by fetalnon-placental means could involve fetal andlor placental drug metabolism, and result in theformation of possible active andlor toxic metabolites which may distribute, accumulate, andpossibly even persist in the fetus. This further necessitates investigations into the fetal non-placental clearance of DPHM. However, since the metabolism of DPHM has not yet beendocumented in sheep, studies examining the elimination of the drug in adult sheep must beconducted to provide comparative data. Therefore, the objectives of the current study were toexamine and contrast the fetal and maternal hepatic and renal contribution towards theelimination of DPHM in chronically instrumented pregnant sheep. Since stable isotopetechniques have distinct advantages for the study of pharmacokinetics in the maternal/fetal unit,they were used in the current study. However, prior to the application of stable isotopetechniques, a labeled analog of DPHM must be synthesized, and an analytical method developedwhich could simultaneously measure DPHM and STh DPHM. In addition, the absence of anisotope effect for Sit DPHM must be verified in adult and fetal sheep. Once these tasks have18been completed, the components of the fetal and maternal non-placental clearance can beinvestigated.1.11. Hypothesis and Specific AimsThe working hypothesis of this thesis was:Hepatic elimination of DPHM and renal excretion of the drug and its metabolites contributesignificantly to the overall non-placental clearance of DPHM in the fetal lamb.To test this hypothesis, the specific aims of this project were to:1. Synthesize a stable isotope analog of DPHM, namely,[2H10]DPHM, and to develop asensitive and specific GC-MS method for the simultaneous quantitation of DPHM and[2H10jDPHM.2. Synthesize a stable isotope analog of a prominent DPHM metabolite (i.e., DPMA),namely[2H10]DPMA, and to develop a sensitive and specific GC-MS method for thesimultaneous quantitation of DPMA and[2H10IDPMA.3. Test for the presence of isotope effects in the disposition of[2H10]DPHM in maternalsheep following bolus administration, and fetal sheep following both bolus administrationand fetal infusion.194. Apply stable isotope methodology to determine the hepatic first-pass metabolism ofDPHM in non-pregnant adult sheep following mesenteric (portal venous) bolusadministration.5. Apply stable isotope methodology to determine the hepatic first-pass metabolism ofDPHM in fetal lambs following umbilical venous bolus administration and infusion.6. Utilize simultaneous maternal and fetal infusions of DPHM and[2H10]DPHM to measuretrans-placental and non-placental clearances in the ovine fetal/maternal unit, respectively.7. Use stable isotope techniques to characterize the disposition of DPMA in both maternaland fetal sheep following simultaneous maternal/fetal infusions of DPHM and[2H10]DPHM, respectively.8. Calculate the contribution of DPHM and[2H10JDPHM, DPMA, and[2H10]DPMA renalelimination towards the measured non-placental maternal and fetal clearances.9. Assess the fetal behavioral effects following simultaneous infusions of DPHM and[2H10JDPHM to mother and fetus, respectively.10. Compare the metabolism of DPHM in hepatic microsomes prepared from fetal and adultsheep.202. Experimental2.1. MaterialsReference standards, chemicals, reagents and other materials used during this thesis projectare listed below, along with information on purity (where applicable), and the source. Unlessotherwise specified, the materials were used without prior purification or modification. Thematerials utilized were: diphenhydramine hydrochloride [2-(diphenylmethoxy)-N,N-dimethylethylamine] (>99% purity), orphenadrine hydrochloride [N,N-dimethyl-2-[(2-methylphenyl)phenyl-]ethylamine] (>99% purity), diphenylacetic acid (> 98% purity)(SigmaChemical Co., St. Louis, MO, U.S.A.); thiopental sodium injectable 1 g/vial; sodium chloride forinjection USP (Abbott Laboratories, Montreal, Que.); injectable ampicillin (250 mg/vial)(Novopharm, Toronto, Ont.); injectable gentamicin sulfate (40 mg/vial) (Schering Canada, Ltd,Pointe Claire, Que.); injectable atropine sulfate (0.6 mg/mL) (Glaxo Laboratories, Montreal,Que.); heparin 1000 units/mL (Organon Canada Ltd., West Hill, Ont.); halothane (AyerstLaboratories, Montreal, Que.); lidocaine 2% (Astra Pharma Inc., Mississauga, Ont.). Allinjectable drug formulations were obtained from the Pharmacy Department, Grace Hospital,Vancouver, B.C.Other materials used during the course of this project were: deuterated benzene([2H6]benzene, 99.5% purity) (MSD Isotopes, Montreal, Que.); anhydrous aluminum chloride,anhydrous sodium sulfate, anhydrous magnesium sulfate, bromoacetic acid, carbon tetrachioride,diethyl ether, disodium hydrogen orthophosphate (dibasic, ACS reagent grade), ethyl alcohol,hydrochloric acid, isopropyl alcohol, magnesium chloride, HPLC grade methanol, petroleum21ether, potassium dihydrogen orthophosphate (monobasic, ACS reagent grade), potassiumchloride, sodium metal, sodium hydroxide pellets (ACS reagent grade), and para-toluenesulfonic acid (BDH, Toronto, Ont.); deuterium oxide (99.9% purity) (Aldrich Chemical Co.,Milwaukee, WI, U.S.A.); triethylamine (TEA), N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) and pentafluorobenzyl bromide (PFBBr) (sequanal grade)(Pierce Chemical Co., Rockville, IL, U.S.A.); ethylacetate, n-hexane, methylene chloride, andtoluene all distilled in glass (Caledon Labs., Georgetown, Ont.); ethylenediaminetetraacetic acid(EDTA), sucrose, tris[hydroxymethyjaminomethane (Trizma® Base) (Sigma Chemical Co., St.Louis, MO, U.S.A.); 13-nicotinamide-adenine dinucleotide (reduced) disodium salt Ca. 98%(NADH), and f3-nicotinamide-adenine dinucleotide phosphate (reduced) tetrasodium salt ca.98% (NADPH) (Boehringer Mannheim Canada, Laval, Que.).Deionized, high purity water was produced on-site by reverse osmosis and subsequentfiltration using a Milli-Q® water system (Millipore, Bedford, MA, U.S.A.). This water will bereferred to as distilled water in the remaining sections of this thesis.Ultra-high purity helium, hydrogen, and zero air (Matheson Gas, Edmonton, Alta.), andnitrogen USP (Union Carbide Canada Ltd., Toronto Ont.) were utilized.Also used were: needles and plastic disposable LuerLok® Syringes for drug administrationand sample collection (Becton-Dickinson Canada, Mississauga, Ont.); disposable plastic pipettetips (National Scientific Supply Company, Inc., San Rafael, CA, U.S.A.); nylon syringe filters(O.22i1) (MSI, Westboro, MA, U.S.A.); borosilicate glass pasteur pipettes (Johns Scientific,Toronto, Ont.); heparinized blood gas syringes (Marquest Medical Products Inc., Englewood,CO, U.S.A.); heparinized Vacutainer® tubes (Vacutainer Systems, Rutherford, NJ, U.S.A.); 15mL Pyrex® disposable culture tubes (Corning Glass Works, Corning, NY, U.S.A.);polytetrafluoroethylene (PTFE) lined screw caps (Canlab, Vancouver, B.C.); Silicone rubbertubing for catheter preparation (Dow Corning, Midland, MI, U.S.A.); PTFE-coated stainless steelwire for electrode preparation (Cooper Corp., Chatsworth, CA, U.S.A.); cellophane dialysismembrane “sacks” (molecular weight cutoff = 12,000 daltons) (Sigma Chemical Co., St. Louis.MO, U.S.A.); and cryovials and closures (Nalge Company, Rochester, NY, U.S.A.).2.1.1. Preparation of Stock Solutions and BuffersStandard stock solutions of diphenhydramine (DPHM) and[2H10]diphenhydramine([2H10]DPHM) were prepared with accurately weighed portions of DPHM hydrochloride (HCI)and[2H10]DPHM HCI. These weighed portions were dissolved in distilled water and diluted in aserial fashion to yield final concentrations of 200.0 ng/mL and 208.0 ng/mL (mass corrected forthe mass of the stable isotope label) of DPHM and[2H10]DPHM free base, respectively. Thestock solution of orphenadrine, the internal standard for the DPHM/[210]DPHM assay, wasprepared in a similar fashion to yield a final concentration of 1.0 ig/mL. The aqueous standardsolutions of diphenylmethoxyacetic acid (DPMA) and[2H10]diphenylmethoxyacetic acid([2H10]DPMA) were prepared with distilled deionized water to yield final concentrations of500.0 ng/mL and 520.0 ng/mL (mass corrected for the mass of the stable isotope label),respectively. Diphenylacetic acid (DPAA), the internal standard for the DPMAI[2H10]DPMAassay, was initially dissolved in methanol. An aliquot of the methanolic solution was dilutedwith distilled deionized water to give a final concentration of 2.0 jig/mL. All stock solutionswere stored at 4° C, and were used for no longer than six months. There was no evidence ofdegradation of these standard stock solutions during this time period. Standard solutions of all23the above analytes were also prepared in methanol for extraction recovery studies. Thesesolutions were prepared and used on the same day to prevent evaporation of the methanol.A solution of TEA in toluene (0.0125M) was prepared by diluting TEA with toluenedistilled in glass. Four to five pellets of sodium hydroxide (NaOH) were added to the solution toensure dryness. This solution was stored at 4°C.A 1.0 M NaOH solution was prepared by dissolving sodium hydroxide pellets withdistilled water, and hydrochloric acid (1.0 M) was prepared by diluting ACS reagent gradeconcentrated HCI acid with distilled water.Isotonic phosphate buffer (0.1 M, pH 7.4) was prepared from accurately weighed portionsof potassium phosphate (mono-basic), disodium phosphate, and sodium chloride. If necessary,the pH was adjusted to pH 7.4 with small aliquots of 1.0 M NaOH or HC1.The 0.05 M Tris-HC1: 1.15% potassium chloride (KC1) [pH 7.4 at 4°C] buffer wasprepared by dissolving Trizma® base and KCI with cooled distilled water (4°C). The pH wasadjusted using 1.0 M HCI. The 10mM EDTA: 1.15% KC1 (pH 7.4) buffer solution wasprepared by dissolving the appropriate quantities of EDTA and KC1 with distilled water (4°C).The pH was adjusted with 1.0 M HC1. A 0.25 M sucrose solution was prepared by dissolving aweighed portion of sucrose with distilled water. These solutions were stored at 4 °C and usedprior to 3 months in the case of the Tris-KC1 and EDTA-KC1 buffers. The sucrose buffer wasprepared on a monthly basis.242.2. Equipment and Instrumentation2.2.1. Gas Chromatography with Nitrogen Phosphorus Specific DetectionA Hewlett Packard (HP) model 5890 (Series II) gas chromatograph was equipped with asplit-splitless capillary inlet system, a HP Model 7673 autoinjector, a nitrogen-phosphorusspecific detector, a Vectra® 386 SX computer equipped with HP 3365 MS DOS® workstationsoftware (Version A.02.0l), a cross-linked fused silica capillary column (25 m X 0.31 mm i.d.,film thickness, 0.25.t, 5% phenylmethylsilicone) (Ultra-2), a 4 X 78 mm borosilicate glass inletliner (Hewlett Packard Ltd., Avondale, PA, U.S.A.); and a silicone rubber septa (ThermogreenLB-2®, Supelco, Bellafonte, CA, U.S.A.).2.2.2. Gas Chromatography with Mass SpectrometryA HP model 5890 (Series II) gas chromatograph was equipped with a split-splitlesscapillary inlet system, a HP Model 7673 autoinjector, a HP Model 597 IA quadrapole massselective detector, and a Vectra 486 25T Computer equipped with MS DOS® HP Model G1O3OAworkstation software, a cross-linked fused silica capillary colunm (25 m X 0.31 mm i.d., filmthickness, 0.25ii, 5% phenylmethylsilicone; HP Ultra-2; DPMAJ[H10]DPMA assay method, orDB-1701 30 m X 0.25 mm i.d., 0.25 i, 5% phenylmethylsilicone and 7% cyanopropylsilicone;J&W Sci’ntific, Folsom, CA, U.S.A; DPHMI[H10]DPHM assay method), a 4 X 78 mmborosilicate glass inlet liner, and a Thermogreen LB2® silicone rubber septa.252.2.3. Gas Chromatography/High Performance Liquid Chromatography - MassSpectrometryA HP Model 5989 MS Engine consisting of a HP 5890 Series II gas chromatograph with aHP 5989 quadrapole mass spectrometer capable of negative chemical (Nd), positive chemical(PCI) and electron impact (El) ionization modes, and a HP 1090 microbore high performanceliquid chromatograph was employed. For LC/MS, thermospray sample introduction was used.LC/MS analysis was conducted using direct flow injections via the thermospray interface. (i.e.,no LC column was used). The carrier phase was composed of 50% ammonium acetate buffer 10mI’l pH 7.0: 50% acetonitrile. The thermospray capillary temperature was maintained at 120C, and the fragmenter was turned off. The ion source was run in the positive ion scanning mode(Mass Range m/z 120 to 400).2.2.4. SpectrophotometerA HP 8452A diode array spectrophotometer equipped with a Vectra® computer interfacewas used for all spectrophotometric measurements.2.2.5. Physiological MonitoringA Beckman R-7 11 Dynograph Recorder (Beckman Instruments, Inc., Palo Alto, CA,U.S.A.) was equipped with disposable DTX transducers (Spectramed, Oxnard, CA, U.S.A.),cardiotachometers (Model 9857, Sensormedics, Anaheim, CA, U.S.A.), and transit-time bloodflow transducers (Transonic Systems Inc., Ithaca, NY, U.S.A.). An Apple lie computer and26computer data acquisition system consisting of an interactive systems analog to digital converter(Daisy Electronics, Newton Square, PA, U.S.A.), and a clock card (Mountain Software, Scott’sValley, CA, U.S.A.) were connected in series with the recorder. Blood chemistry measurementswere made with an IL 1306 pH/Blood gas analyzer (Allied Instrumentation Laboratory, Milan,Italy), a Hemoximeter (Radiometer, Copenhagen, Denmark), and a 2300 STAT plusglucose/lactate analyzer (Y.S.I. Inc. Yellow Springs, OH, U.S.A.).2.2.6. General Experimental EquipmentOther equipment utilized included: a vortex-type mixer (Vortex-Genie), and an incubationoven (Isotemp model 350) (Fisher Scientific Industries, Springfield, MA, U.S.A.); an JEC model2K centrifuge (Damon/EEC division, Needham Hts., MA, U.S.A.); a rotating-type mixer(Labquake model 4 15-110, Lab Industries, Berkeley, CA, U.S.A.); an infusion pump (Harvardmodel 944, Harvard Apparatus, Millis, MA, U.S.A.); a DIAS Roller pump (DIAS, Ex154, DIASInc. Kalamazoo, MI, U.S.A.); a1H-NMR (Bruker AC-200 [200MHz], U.B.C. ChemistryDepartment); a differential scanning calorimeter (Series 99 Thermal Analyzer, Dupont ClinicalInstruments, Wilmington, DE, U.S.A.); a high speed centrifuge model 12-21, an ultra-centrifugemodel L8-60M or L5-50, IA- 17 fixed angle rotor, Ti 50.2 fixed angle rotor (BeckmanInstruments, Inc., Palo Alto, CA, U.S.A.).272.3. Chemical Synthesis of Standards and Metabolites of DPHM2.3.1. Synthesis of[2H10jbenzhydrolStable isotope labeled benzhydrol was synthesized in two steps (Figure 2). The initial stepinvolved the synthesis of[2H1o]benzophenone. This method was similar to a method for thesynthesis of unlabeled benzophenone (Marvel and Sperry, 1941). Into a three neck 250 mLPyrex® round-bottom flask equipped with a Teflon® coated magnetic stirring rod, 7.6 ganhydrous aluminum chloride and 16.7 mL of dry carbon tetrachioride were added. Athermometer, a separatory funnel, and a reflux condenser equipped with a water and HCI trapwere added onto the round bottom flask. The reaction was started with the addition of 0.8 mL of{2H6]benzene. The reaction mixture was cooled using an ice bath to maintain the temperature inthe reaction flask between 10-15° C. A mixture of[2H6]benzene (9.2 mL) and carbontetrachloride (9.2 mL) was added in a drop-wise fashion to the reaction mixture from theseparatory funnel (2-3 hours). Following the addition of the[2H6]benzene/carbon tetrachloridemixture, the reaction mixture was stirred for an additional three hours with the temperaturemaintained between 10-15° C. The reaction mixture was allowed to stand for 12 hours duringwhich it reached room temperature. The excess carbon tetrachioride was removed from thereaction mixture with a Pasteur pipette. The reaction mixture was then cooled and 25 ml. ofdeuterium oxide was slowly added. Following the addition of the deuterium oxide, the reactionmixture was extracted using diethyl-ether. The organic layer was transferred to a clean 250 rnLround bottom flask and dried using anhydrous sodium sulfate. The organic layer wassubsequently filtered and the solvents were removed under vacuum. The resulting product waspurified using column flash chromatography (in a glass column of dimensions 6 cm X 75 cmpacked with Silica gel 60; mesh 240-400; mobile phase is 97% n-hexane: 3% diethyl ether).28Fractions eluting from the column were collected, and those fractions shown to include theproduct (i.e., via thin layer chromatography) were pooled, dried with anhydrous sodium sulfate,and filtered. The solvent was removed under vacuum. The product was re-crystallized overnightfrom benzene (4° C). The yield of the vacuum dried[2Hiojbenzophenone was 60% based on themass of[2H6]benzene. The product,[2H10]benzophenone, was converted to[2H10]benzhydrol, asdescribed earlier for unlabeled benzhydrol (Wisegogle and Sonneborn, 1941) (Figure 2). Thepurified{2H10]benzophenone (—6 g) was added to a clean 250 mL round bottom flask along with6 g of NaOH, 6 g Zinc dust, and 60 mL ethyl alcohol. A thermometer, a Teflon® coatedmagnetic stirrer, and a reflux condenser were attached to the round bottom flask. The mixturewas gradually heated to 70° C, with stirring, and the temperature was held for 2 hours. Thereaction mixture was filtered with suction, and the residue washed with two 3 mL aliquots ofheated ethyl alcohol. The resulting filtrate was poured into ice cold 1.0 M HCI. The resultingcrystals were filtered with suction, washed with ice cold distilled water, and dried under vacuum.This reaction was almost complete with a yield of 95%. The purified product,[2Hiojbenzhydrol, was used for the subsequent synthesis of stable isotope labeled compounds(i.e.,[2H10]DPHM and[2H10]DPMA).2)Figure 2:2.3.2.2 2H0+2HCI1000Synthesis of A.)[2Hio]benzophenone, and B.)[2H10]benzhydrolSynthesis of[2H10]DPHM HC1The synthesis of[2H10jDPHM involved a one step reaction, as reported earlier for tritiurnlabeled DPHM (Figure 3) (Blackburn and Ober, 1967). Into a clean three neck 250 ml. roundbottom flask 2.5 g of purified[2H10]benzhydrol, 1.5 niL dimethylarninoethanol, 2.7 g paPatoluene sulfonic acid, 27 niL tetrachioroethane, and 41 mL toluene were added. A Tef1oncoated magnetic stirrer, a reflux condenser, a thermometer, and a water trap were added onto thereaction flask. The reaction was conducted under a nitrogen atmosphere. The reaction va2 2A.2[H6]+ 0014B.2020NaO H/ZnEthyl Alcohol2H5HO—OH2030initiated by gradually heating the reaction mixture to 125° C with stirring. The reaction mixturewas refluxed for 48 hours, cooled to 60° C, and diluted with 90 mL petroleum ether and 30 mLdistilled water. The reaction mixture was transferred to a 500 mL separatory funnel. The bottomtwo aqueous layers were removed and transferred to a clean separatory funnel. The top organicphase was washed with two 20 mL aliquots of distilled water, and the washings were combinedwith the aqueous fraction. A 7.0 mL aliquot of 50% NaOH was added to the aqueous phase.Next, the aqueous phase was extracted with one 60 mL and two 30 mL portions of diethyl ether.The combined ether phase was washed with two 20 mL portions of water and then dried twicewith 4 g portions of anhydrous magnesium sulfate. This mixture was filtered and the etherremoved under vacuum. A 2 mL aliquot of isopropyl alcohol was added to the resulting oil,followed by an addition of 2.6 mL of isopropyl alcohol saturated with HC1 (i.e., HC1 gas wasbubbled through the isopropyl alcohol). This was followed by the addition of 70 mL anhydrousdiethyl ether. The mixture was allowed to stand at 4° C overnight to crystallize. The followingmorning the mixture was filtered and the resulting crystals dried under vacuum. The productwas re-crystallized twice: once, using 2 mL of isopropyl alcohol and 60 mL anhydrous diethylether; the second time, using acetone and heat. The yield of this reaction was 50%.p-TSA+ HOCH2CHN(3)125°C2Figure 3: Synthesis of[2H10]DPHM312.3.2.1. Characterization of[2H10JDPHMIdentification of the{2H10]DPHM HCI was confirmed using H-NMR (Bruker AC-200[200MHz], Dept. of Chemistry, University of British Columbia)[ ‘H-NMR: (D20) 6 5.38 (s, IH,CHO), 3.58 (t, 2H, OCH2), 2.62 (t, 2H, CH2), 2.28 (br s, 6H, N(CH32)J(Appendix 2). Inaddition, GC/LC/MS was also used in the identification of[2H[o]DPHM. Characteristicfragments of[2H10]DPHM following GC-MS/EI were [m/z (% abundance)]: m/z 58(100),73(59), 159(23), 173(48), 177(46), and 193(1). Direct flow injection HPLC-MS, which wasconducted using a thermospray interface with the ion source in the positive ion mode, identifiedthe ion m/z 266 which corresponded to the [M+HJ fragment of[2H10}DPHM. In addition, asharp melting point at 167° C (literature MP for DPHM is 166-170° C) was determined for[2H10]DPHM HCI using differential scanning calorimetry (DSC).2.3.3. Synthesis of N-demethyl DPHM and N,N-didemethyl DPHMN-demethyl diphenhydramine and N,N-didemethyldiphenhydramine were synthesized andpurified as outlined above in section 2.3.2; however, rather than dimethylaminoethanol,methylaminoethanol and aminoethanol were used for the synthesis of N-demethyl DPHM andN,N-didemethyl DPHM, respectively.322.3.3.1. Characterization of N-demethyl DPHM and N,N-didemethyl DPHMThe structures of N-demethyl DPHM and N,N-didemethyl DPHM were characterizedusing GC-MS (Scan). Characteristic ion fragments of N-demethyl DPHM were [m/z (%abundance)]: m/z 59(100), 152(3 1), 165(79), 167(66), and 183(28), and for N,N-didemethylDPHM were [mlz (% abundance)]: m/z 152(33), 165(73), 167(100), and 183(11). In addition,H’-NMR was also used to confirm the structures of N-demethyl DPHM HC1 and N,Ndidemethyl DPHM HC1 (Bruker AC-200 [200MHz], Dept. of Chemistry, University of BritishColumbia; N-demethyl DPHM HC1 [‘H-NMR: (D20) 7.40 (m,1OH, Aromatic), 5.53 (s, IH,CHO), 3.70 (t, 2H, OCH2), 3.25 (t, 2H, CH2), 3.23 (s,3H, N(CH3), 2.18 (s, 1H, NH)]; N,Ndidemethyl DPHM HC1 [‘H-NMR: (D20) 7.40 (m,1OH, Aromatic), 5.58 (s, IH, CHO), 3.70(t, 2H, OCH2), 3.25 (t, 2H, CH2)]). The melting points of N-demethyl DPHM HC1 and N,Ndidemethyl DPHM HC1 were 159 and 178° C, respectively, as determined by DSC.2.3.4. Synthesis of DPMA and[2H10]DPMAThe method for the synthesis of DPMA and[2H10]DPMA was adapted from a methoddescribed earlier to synthesize only DPMA shown in figure 4 (Djerassi and Scholz, 1948). Into aclean 250 mL three neck round bottom flask with a Vigreux distillation column, droppingseparatory funnel, a glass inlet tube, and a thermometer were added 0.7 g freshly cut sodiummetal and 12 mL methanol. A gentle stream of dried nitrogen gas was passed through the glassinlet tube. This mixture was stirred, and gradually a mixture of 5.5 g of either[2H10]benzhydrolor benzhydrol in 24 mL of dry toluene was slowly added. The temperature of the reaction vesselwas slowly increased to 140° C until a moderate rate of distillation was achieved. Aliquots of33dry toluene were periodically added to keep the mixture liquid. Once the temperature in theVigreux column had reached 900 C, the column was replaced with a reflux condenser. A 2.1 gportion of bromoacetic acid was added, followed by 20 mL of dry toluene. The reaction mixturewas then refluxed for 2 hours. The reaction mixture was cooled, and 30 mL of distilled waterwas added. The reaction mixture was added to a clean 500 mL separatory funnel. The aqueouslayer was alkalinized (i.e., above pH 13.0) by the drop wise addition of 10 M NaOH. Thetoluene and aqueous layers were separated. The aqueous phase was washed with diethyl ether (2aliquots of 25 mL), acidified by the drop-wise addition of 10 M HC1 (i.e., pH 1), and extracted (3aliquots of 20 mL) with diethyl ether. The ether layer was washed with two aliquots of 20 mL ofdistilled water or until the washings were neutral (i.e., pH —7.0). The ether layer was then driedwith anhydrous sodium sulfate and filtered. The ether was removed under vacuum. Theresulting oil of either DPMA or[2H10]DPMA was purified using colunm flash chromatography(in a glass column of dimensions 6 cm X 75 cm packed with silica gel 60; mesh 240-400; mobilephase 50% diethyl ether: 48% hexane: 2% isopropyl alcohol) followed by re-crystallization withhexane and acetone. The yields of vacuum dried DPMA and[2H10]DPMA were 28% and 30%respectively.2.3.4.1. Characterization of DPMA and[2H10]DPMAThe identification of DPMA and[2H10]DPMA was confirmed using1H-NMR (Bruker AC200 [200MHz], Department of Chemistry, University of British Columbia): [DPMA - ‘H-NMR:(CDCL3); 4.20 (S, 2H, OCH2); 5.50 (S, IH, CH); 7.30 (M, 1OH, ArH)j and[2H10]DPMA - ‘HNMR: (CDCL3);6 4.20 (S, 2H, OCH2), 5.50 (S, IH, CH)]. In addition, gas chromatographymass spectrometry (GC-MS), using both electron impact and negative chemical ionization, was34used in the identification of DPMA and[2H10JDPMA. Pure DPMA and[2H10]DPMA weredissolved in toluene, derivatized with MTBSTFA, and injected into the GC-MS (El), which wasused in the scanning mode. Characteristic fragments following GC-MS (El) for DPMA were[m/z (% abundance)]: m/z 152 (20), 165(38), 167(100), 183(93), and 299(4), and for[2H10]DPMA were: 159(15), 173(19), 177(100), 193(95), and 309(4), respectively. Aliquots ofDPMA and[2H10]DPMA dissolved in toluene were derivatized with PFBBr. These PFBBrderivatives of DPMA and[2H10]DPMA were subjected to GC-MS in the negative chemicalionization (NCI) mode with methane as the reagent gas. The total ion scan showed only onefragment for DPMA ( m/z 241) and[2H10]DPMA (m/z 251), corresponding to the loss of thepentafluorobenzyl group from the molecular ion (i.e., [M- 181 ]). Sharp melting points at 77 and78° C were measured for DPMA and[2H10}DPMA, respectively, using DSC.I NaNC—OH + BrCH2COO H2COOHaNa2H+ BrCH2COO HC—O—CH2000H2H5Figure 4: Synthesis of DPMA and[2H10]DPMA352.3.5. Purity Assessment of Synthesized Standards.The purity of the DPHM HC1 and the synthesized[2H10}DPHM HC1 standards wereassessed in the following fashion. Firstly, aqueous solutions of DPHM HCI or[2H10JDPHM HCJ(100 ig/mL) were extracted with 2% isopropyl alcohol: 98% hexane with 0.05 M triethylamine(TEA). The organic phase was dried and reconstituted with 0.05 M TEA in toluene. An aliquotof the reconstituted samples (i.e., DPHM and[2H10jDPHM) was subjected to gaschromatography with nitrogen/phosphorus specific (NPD) detection, while the other aliquot ofthe reconstituted samples was assessed using GC-MS (scan mode). Only one chromatographicpeak, other than those present in the blank, could be detected using GC-NPD and GC-MS/EI(total ion chromatogram) following injection of the prepared DPHM HC1 and[2H10]DPHM HC1standards. Standard aqueous solutions of DPHM HC1 and[2H10]DPHM HC1 (100 ig/mL) weresubjected to LC-MS via direct sample introduction into the LC-MS source through athermospray interface. DPHM and[2H10]DPHM did not fragment extensively under theseconditions, and therefore, only one ion was observed in each sample, which corresponded to the[M+1j ions of DPHM, (m/z 256) and[2H10]DPHM (m/z 266). Thermal analysis was conductedusing DSC. Data obtained showed only one sharp peak corresponding to a melting point of 167°C for both DPHM and[2H10]DPHM. The absence of other peaks during the thermal analysisalso suggests the lack of any polymorphic forms, and/or solvates of the DPHM and[2H10]DPHMHC1 standards.The purity of N-demethyl DPHM and N,N-didemethyl DPHM was determined using GCMS (Scan) and GC-NPD, as described above. The N-demethyl DPHM HCI and N,N-didemethylwere dissolved in 0.05 M TEA in toluene and injected directly into the GC. N-demethyl DPHMwas deemed pure, since only one peak was detected in the chromatogram using both GC-MS36(Scan) and GC-NPD; however, the N,N-didemethyl DPHM HCI demonstrated a peak whicheluted just following the metabolite peak. This peak was reduced by further re-crystallization,but was never eliminated (peak area counts with GC-NPD and GC-MS were <2% of themetabolite). Thermal analysis using DSC showed only one sharp peak for N-demethyl DPHMand N,N-didemethyl DPHM. The absence of other peaks during the thermal analysis alsosuggests the lack of any polymorphic forms, and/or solvates of the N-demethyl DPHM HC1 andN,N-didemethyl DPHM HCI standards.The purity of DPMA and[2H10]DPMA was assessed in the following fashion. DPMA and[2H10]DPMA were dissolved in toluene, an aliquot was removed and derivatized withMTBSTFA, and this aliquot was injected directly into the GC-MSIEI. The total ionchromatogram showed only the peaks corresponding to the tert-butyldimethylsilyl (TBDMS)derivatives of DPMA and[2H10]DPMA. No chromatographic peaks other than those present inthe blank were detected. In addition, both DPMA and[2H10}DPMA were dissolved in distilledwater. These samples were extracted with toluene from an acidified aqueous matrix. Thetoluene was dried under a gentle stream of nitrogen gas. The reconstituted residue wasderivatized with either MTBSTFA (GC-MS[EI) or PFBBr (GC-MS/NCI). The total ionchromatograms showed only the chrornatographic peaks corresponding to the compounds inquestion. Thermal analysis of DPMA and[2H10]DPMA conducted using DSC showed only onesharp peak corresponding to their respective melting points. The lack of other peaks duringthermal analysis also suggests the lack of any polymorphic forms, and/or solvates and hydrates ofthe DPMA and[2H10JDPMA standards.372.4. Analytical Method Development2.4.1. Development of an Analysis Method for DPHM and[2H10JDPHM2.4.1.1. Optimization of Mass Spectrometer ParametersThe fragment ions for selected ion monitoring (SIM) of both DPHM and[2H10]DPHMwere chosen based on the retention of the stable isotope label and good abundance. The massspectrometer was tuned using perfluorotributylamine (PFTBA), using a variety of tuningalgorithms to optimize the sensitivity of the assay (i.e., programmed auto tune [m/z 69, 219, and502], mid-mass tune [m/z 69, 219, and 265) and a manual tune [m/z 100, 131, and 219]). Themass spectrometer dwell time was set to allow 15 scans/chromatographic peak.2.4.1.2. Optimization of Gas Chromatographic ParametersTwo capillary columns were tested for the quantitation of DPHM and[2H10]DPHM,namely, a HP Ultra-2, and a J&W DB-1701. The influence of injector purge times on analytepeak area counts was examined by varying purge times (i.e., 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5minutes) and repeated injections of a standard sample. The effect of the rate of column heliumflow on column efficiency was also tested using a variety of column head pressures (7.5, 10,12.5, 15, 17.5, and 20.0 P.S.I.). In addition, the effect of the injector temperature on the analytepeak area counts was measured (200, 225, 250, and 275 °C). Finally, the influence of the initialcolumn temperature on half-height peak width was examined; a variety of initial columntemperatures were assessed, namely, 70, 80, 90, 100, 110, 120, 130, 140, and 150° C.382.4.1.3. Optimization of Extraction ProcedureA variety of solvents were tested for the extraction efficiency and suitability (i.e., ease ofextraction and selectivity of the solvent) for the DPHM and[2H10]DPHM analysis method.Spiked plasma samples were basified with 1.0 M NaOH and extracted with either toluene,methylene chloride, hexane, or 98% hexane: 2% isopropyl alcohol. The peak area response andthe chromatographic base lines (i.e., lack of interfering chromatographic peaks) were used tochoose the most appropriate solvent for extraction. In addition, a variety of hexane:isopropylalcohol solvent compositions (i.e., 0, 2, 5, 10, and 20% isopropyl alcohol) were examined in asimilar fashion as described above. The influence of silanization of glassware, and the additionof triethylamine (TEA) at a variety of concentrations (i.e., 0.025, 0.05, 0.10, and 0.20 M) on peakarea counts were also examined. Once the appropriate extraction solvent system wasdetermined, the effect of different extraction times (i.e., 5, 10, 15, 20, 25, and 30 minutes) on therelative extraction recovery of DPHM and{2H,0}DPHM was investigated.2.4.1.4. Gas Chromatograph - Mass Spectrometer Operating ConditionsA 2.0 !.LL aliquot of prepared sample was injected through a Thermogreen® LD-2 siliconerubber septa into the gas chromatograph splitlsplitless inlet equipped with a Pyrex® glass inletliner (78 mm X 4 mm i.d.) in the splitless mode (purge time 1.5 minutes). Chromatographicseparation of DPHM,[2H10]DPHM, and orphenadrine from endogenous materials and thedemethylated metabolites of DPHM and[2H10]DPHM was achieved using a 30 m J&W DB391701 0.25 mm i.d. (0.25 t film thickness) capillary column. Column head pressure was set at12.5 P.S.I. The gas chromatographic operating conditions were as follows: The injection porttemperature was held at 1800 C. The initial oven temperature was maintained at 140° C for Iminute, then the oven temperature was ramped at 30° C/minute to 200° C. The oven temperaturewas again ramped at 17.5° C/minute from 200° C to 265° C, where it was held for 5.0 minutes.The temperature program resulted in a total run time of 12.7 minutes. The transfer linetemperature was held at 280° C. The mass spectrometer (MS) was manually tuned with thetuning reagent perfluorotributylamine (PFTBA) to ions m/z 100, 131, and 219. The GC-MSoperating in the electron impact ionization mode (voltage 70 eV) with selective ion monitoring(El-SIM) was used to quantitate DPHM and[210]DPHM by monitoring ions m/z 165 and 173,respectively. The dwell time was set at 50 msec. for each ion being monitored, to ensureadequate sampling of the chromatographic peak of interest (i.e., 15 scans/peak). The electronmultiplier voltage was programmed to + 300 V relative to the tune value during the elution of thecompounds of interest. The voltage was programmed to reset to -1000 V relative to the tunevalue at all other times to maximize the life span of the electron multiplier.2.4.1.5. DPHM and[2H10]DPHM Extraction ProcedureSamples were prepared for analysis by a single step liquid/liquid extraction procedure.Aliquots of biological samples (0.1 - 1.0 mL) including maternal and feial plasma, amnioticfluid, tracheal fluid and urine were individually pipetted into clean borosilicate test tubes. Thesamples were made up to volume (1.0 mL) with distilled water, internal standard (orphenadrine200 ng), and 0.5 mL of 1 M NaOH were then added to the test tube along with 7.0 mL of solvent40(0.05 M TEA in 2% isopropyl alcohol: 98% hexane). The samples were capped and mixed for20 minutes, cooled for 10 minutes at 50 C in a freezer in order to break any emulsion formedduring mixing, and centrifuged for 10 minutes at 3000 x g. The organic phase was transferred toa clean test tube and dried in a water bath at 300 C under a gentle stream of nitrogen gas. Thedried samples were reconstituted with 150 pL of 0.05 M TEA in toluene. The reconstitutedsamples were then transferred to clean borosilicate microvial inserts which were placed instandard borosilicate autosampler vials, from which a 2.0 jiL aliquot was used for injection.2.4.1.6. Preparation of a Calibration CurveAn eight point calibration curve was constructed from the aqueous standard solutions ofDPHM and[2H10]DPHM to yield concentrations of 2.0, 5.0, 10.0, 20.0, 50.0, 100.0, 150.0 and200.0 ng/mL using a prepared stock solution of 200.0 ng/mL of DPHM and[2H10]DPHM. Theblank plasma, fetal tracheal fluid, or amniotic fluid was added to the calibration curve samples.The samples were extracted and quantitated as described above. Weighted linear regression(weighting function 1/y2) was performed between the mean drug response (DPHM or[2H10JDPHM peak arealinternal standard [orphenadrine] peak area) and the spiked drugconcentrations of DPHM or[2H10jDPHM.2.4.1.7. Calculation of Extraction Recoveries of DPHM and[2H10IDPHMExtraction recoveries of both DPHM and[2H10]DPHM were determined at low, moderate,and high concentrations (2.0, 50.0, and 200.0 nglmL, respectively) from a variety of biological41matrices (maternal plasma, amniotic fluid, and tracheal fluid). Two groups of samples were usedto assess extraction recovery, namely, a test group and a control group. Both groups of samplescontained blank biological matrix (plasma, amniotic fluid, and fetal tracheal fluid) and internalstandard. However, the samples from the test group were spiked with DPHM and[2H10]DPHMto yield final concentrations of 2.0, 50.0, and 200.0 ng/mL, whereas the samples in the controlgroup were not spiked with DPHM and[2H10JDPHM at this point of the experiment. Followingliquid-liquid extraction of both the test and control group samples, aliquots of DPHM and[2H10]DPHM standards, made up in methanol, were added to the control group samples to yielddrug concentrations of 2.0, 50.0, and 200.0 ng/mL. Control and test samples were then dried,reconstituted, and chromatographed as described above. The concentrations of the test andcontrol samples were determined from standard curves extracted from the correspondingbiological matrices. The extraction recovery was calculated as the ratio of the measuredconcentration of the test samples over the measured concentration of the control samples at thelow, medium, and high concentrations.2.4.1.8. Sample Stability AssessmentNumerous studies were carried out in order to determine the stability of the samples duringstorage and analysis. The freezer stability of these samples was assessed by spiking blankmaternal plasma with DPHM and[2H10]DPHM at known concentrations of 5.0, 25.0, and 100.0ng/mL, and storing these samples at -20° C for up to 12 months. Samples were periodicallyremoved (i.e., 0.5, 1, 2, 4, 6, 12 months) and the concentration of DPHM and[2H10]DPHMmeasured. These measured concentrations where then compared to the known concentrations.42The freeze-thaw stability of DPHM and[2H10]DPHM in plasma was assessed in the followingmanner. Blank plasma samples were spiked with 58 ng/mL of DPHM and{2H10JDPHM. Thesesamples were frozen at -20° C and thawed at 22° C on the bench-top. This cycle was continuedfor a total of three cycles. On the final cycle, the samples were frozen at -20° C and stored untilanalysis. The bench-top stability of DPHM and[2H10]DPHM in a plasma matrix was determinedin the following fashion. Blank plasma was spiked with DPHM and[2HIOJDPHM to yield aconcentration of 58 nglmL. The samples were left on the bench-top at 22° C for various periodsof time (i.e., 0, 1, 2, 4, 6, 12, and 24 hours). The samples were immediately frozen at -20° C andstored frozen until the time of analysis. A stability study of the extracted and derivatizedsamples stored on the autosampler tray of the GC-MS was conducted. Prepared samples atconcentrations of 2.0 and 200.0 ng/mL of DPHM and[2H10]DPHM were extracted and analyzed.These samples were repeatedly injected at 24 hour intervals for a total of 72 hours (i.e., 0, 24, 48,and 72 hours).2.4.1.9. Method ValidationIntra-day variability was determined by quantitating six replicates at concentrations of 2.0,20.0, 100.0, and 200.0 ng/mL on one experimental day. Inter-day variability was determined byquantitating one sample in duplicate at concentrations of 2.0, 20.0, 100.0, and 200.0 ng/mL onsix different experimental days.The GC-MS method for the quantitation of DPHM and[2HIO]DPHM was independentlycross-validated by quantitating samples of DPHM and[2HIO]DPHM individually by the GC-MSmethod developed, and by a published capillary gas chromatographic analysis for the43quantitation of DPHM using a GC-NPD (Yoo et at., 1986). In order to be able to quantitate bothDPHM and[2H10]DPHM using GC-NPD, calibration curves and samples were preparedindividually, that is, DPHM and[2H10JDPHM were not present together in the same sample (onlyfor cross validation samples). This was done because the GC-NPD method could notdifferentiate between DPHM and[2H10]DPHM, if they were present in the same sample.2.4.2. Development of an Analysis Method for DPMA and[2H10]DPMA2.4.2.1. Optimization of Mass Spectrometer ConditionsThe mass spectrometer conditions were optimized as described above (2.4.1. 1.)2.4.2.2. Optimization of Gas Chromatograph ConditionsTwo capillary columns were tested for use in the quantittion of DPMA and[2H10]DPMA(i.e., HP Ultra-2 and J&W DB-1701). The effect of the rate of column helium flow on columnefficiency was also tested using a variety of column head pressures (7.5, 10, 12.5, and 15 P.S.I.).In addition, the effect of the injector temperature on the analyte peak area counts was measured(250, 260, 270, 280, 290, 3000 C). Finally, the influence of the initial column temperature onpeak width was examined (150, 160, 170, 180, 190, 200° C).442.4.2.3. Optimization of ExtractionTwo solvents were investigated for the extraction of DPMA and[2Ht0JDPMA from ovineplasma and urine matrices. The peak area counts and the quality of the chromatography wereassessed following drug extraction with ethyl acetate and toluene from acidified urine andplasma matrices.2.4.2.4. Optimization of DerivatizationTwo derivatization techniques were examined during the development of the DPMA and[2H10]DPMA analysis method. Pentafluorobenzyl or tert-butyldimethylsilyl derivatives wereformed using the reagents pentafluorobenzylbromide (PFBBr) and N-methyl-N-(tertbutyldimethylsilyl)trifluoroacetamide (MTBSTFA). Samples of DPMA and[2H10]DPMA wereextracted from acidified (pH —-1.5) biological matrices (plasma and urine). The toluene layer,removed following the extraction, was dried and the residues derivatized. In the case of the PFBderivatives, 200 pL of a solution containing 1 part PFBBr and 100 parts acetone, and a —0.5 galiquot of anhydrous sodium sulfate were added to the dried residue. These samples were cappedand incubated at 600 C for 1 hour. Residual PFBBr was neutralized with the addition of 0.5 mLof distilled water and vortex mixed for — 30 seconds. The organic layer was removed and driedunder a gontle stream of nitrogen gas at 40° C and reconstituted with 200 tL toluene. To formthe TBDMS derivatives the residue resulting from the evaporation of the organic extractionsolvent was reconstituted with 200 tL toluene and 50 pL of MTBSTFA. This mixture wasincubated for 60 minutes at 60° C. The effect of various volumes of MTBSTFA (i.e., 5, 10, 25,50 and 100 tL) on DPMA,[2H10]DPMA, and DPAA peak areas was also assessed. In addition,45incubation times for the MTBSTFA derivatization procedure were also optimized (i.e., timestested 30, 60, 90, 120 minutes).2.4.2.5. Gas Chromatograph-Mass Spectrometer Operating ConditionsA 1.0 jiL aliquot of prepared sample was injected through a Thermogreen® LD-2 siliconerubber septum into the split/splitless capillary inlet equipped with a Pyrex® glass inlet liner (78mm X 4 mm i.d.) operated in the splitless injection mode with a purge time of 1.5 minutes.Chromatographic separation of the analytes([2H10]DPMA, DPMA, and DPAA) fromendogenous materials was achieved using a 25 meter HP Ultra-2 0.2 mm i.d. (0.33 t filmthickness) capillary column. Column head pressure was optimized at 15 P.S.I. (corresponding to0.6 mL/minute at the initial temperature). The gas chromatographic system operating conditionswere as follows: The injection port temperature was held at 280° C. The initial oventemperature was maintained at 125° C for 1 minute, the oven temperature was ramped at 12.5°C/minute to 280° C, where it was held for 4.0 minutes. The temperature program resulted in atotal run time of 17.4 minutes. The transfer line temperature was held at 285° C. The MS wasmanually tuned using the tuning reagent perfluorotributylamine (PFTBA) to ions m/z 100, 131,and 219. The GC-MS operating in the electron impact ionization mode (voltage 70 eV) withselective ion monitoring (El-SIM) was used to quantitate DPAA, DPMA, and[2H10]DPMA bymonitoring ions m/z 165, 183 and 177, respectively. The dwell time was set at 125 msec for eachion being monitored to ensure adequate sampling of the chromatographic peak of interest (i.e., 15Scans/chromatographic peak). The electron multiplier voltage was programmed to + 200 Vrelative to the tune value during the elution of the compounds of interest. The voltage was46programmed to reset to -1000 V relative to the tune value at all other times to maximize the lifespan of the electron multiplier.2.4.2.6. DPMA and[2H10}DPMA Extraction ProcedureSamples were prepared for analysis using a single-step liquid-liquid extraction. Aliquotsof biological samples (0.10-1.00 mL plasma and 0.05-1.00 mL urine) were pipetted into cleanborosilicate test tubes. The samples were made up to a volume of 1.0 mL with distilled water. A200 .tL aliquot of internal standard (diphenylacetic acid: DPAA 2.0 j.tg/mL), 400 tL of 1.0 MHC1, and 5.0 mL of toluene were added to the biological sample. The test tubes were cappedwith PTFE-lined lids and mixed for 20 minutes, cooled for 10 minutes at -20° C in a freezer (tobreak any emulsion formed during mixing), and centrifuged at 3000 x g. The organic layer wastransferred to clean test tubes and evaporated to dryness in a water bath at 40° C under a gentlestream of nitrogen gas. The dried samples were reconstituted with 200 !IL dry toluene (toluenestored on anhydrous sodium sulfate), and 25 iL of the derivatizing reagent MTBSTFA wasadded. The tubes were capped, mixed for one minute on a vortex mixer, and incubated at 60° Cfor one hour. After the samples cooled, the derivatized mixture was transferred to cleanborosilicate microvial inserts (placed in standard borosilicate autosampler vials) from which a1.0 iL aliquot was used for injection.2.4.2.7. Preparation of a Calibration CurveA seven-point calibration curve was constructed from aqueous standard solutions ofDPMA and[2H10]DPMA to provide concentrations of 2.5, 5.0, 10.0, 25.0, 50.0, 125.0, and 250.047ng/mL with 400 ng of DPAA added as the internal standard. Aliquots of blank plasma or urinewere added to the calibration curve samples. The samples were extracted and quantitated asdescribed above. Weighted linear regression (weighting function = l/y2) was performed betweenthe drug response (DPMA or[2H10JDPMA peak arealinternal standard (DPAA) peak area) andthe spiked drug concentrations of DPMA or[2H10]DPMA.2.4.2.8. Calculation of Extraction Recovery of DPMA and[2H10JDPMAExtraction recoveries of DPMA and[2H10JDPMA from plasma and urine were bothdetermined at low, moderate, and high concentrations (5.0, 50.0, and 500.0 ng/mL). Extractionrecoveries of DPMA and[2H10]DPMA were determined as outlined in section 2.4.1.6. forDPHM and[2H10]DPHM.2.4.2.9. Sample Stability AssessmentNumerous studies were carried out in order to determine the stability of the samples duringstorage and analysis. The freezer stability of these samples was determined by spiking blankplasma with DPMA and[2H10]DPMA at a known concentration of 100.0 ng/mL. These sampleswere stored at -20° C, removed at specific intervals (1, 2, 4, and 6 months), and analyzed. Thefreeze-thaw stability, bench-top stability, and injector stability of DPMA and{2HIO}DPMA inplasma samples were assessed as described in section 2.4. 1.8. for DPHM and[2H10}DPHM.Because DPMA and[2H10]DPMA appeared to be sensitive to acid, the stability of the samples inan acidified matrix (i.e., acidified to the same degree as during the extraction procedure) was48examined. Briefly, spiked samples in a distilled water, plasma, and urine matrix were acidifiedwith 400 tL of 1.0 M HC1. These samples were vortex mixed for one minute and left on thebench-top for the following periods of time: 0, 0.5, 1.0, 2.0, 4.0, 6.0, and 21.0 hours. Followingthe desired incubation time, the internal standard was added and the samples extracted asdescribed above. The degradation half-lives of DPMA and[2H10}DPMA were determined in thebiological matrices examined.2.4.2.10. Method ValidationIntra-day variability was determined by quantitating four replicates at concentrations of 2.5.25.0, 125.0, and 250.0 ng/rnL using the GC-MS method reported above for the quantitation ofDPMA and[2H10JDPMA on one experimental day. Inter-day variability was determined byquantitating one sample in duplicate at concentrations of 2.5, 25.0, 125.0, and 250.0 ng/inL onfour different experimental days.2.5. Standard Procedures for Animal Experiments2.5.1. Animal HandlingBoth pregnant and non-pregnant ewes of Suffolk, Finn, and Dorset mixed breed were usedin these studies. The animals were brought into the animal unit at the Children’s VarietyResearch Center at least 1 week prior to surgery, and kept in groups of two or more in large pensin full view of one another. The animals received a standard diet and free access to water.49Ethical approval for the studies was obtained from the Animal Care Committee of the Universityof British Columbia, and the procedures used were in accordance to the guidelines of theCanadian Council of Animal Care.2.5.2. Surgical Preparation for Chronic Experimentation2.5.2.1. Non-pregnant Sheep - Surgical PreparationNon-pregnant Dorset, Suffolk, and Finn mixed breed ewes were used in these studies. Theewes were allowed free access to water; however, food was withheld for approximately 18 hoursprior to surgery. Aseptic techniques were employed throughout the surgical procedure.Approximately 20 minutes following intravenous atropine (6.0 mg) administration to controlsalivation, anesthesia was induced with intravenous sodium pentothal (1.0-1.5 g). The animalswere intubated with an endotracheal tube and anesthesia was maintained through the ventilationof the animal (12 cycles/minute) with a mixture of halothane (1-2%), nitrous oxide (70%) andbalance oxygen. An intravenous bolus injection of 500 mg ampicillin via the jugular vein wasfollowed by the intravenous drip administration of a solution of 5% dextrose (500 mL)containing 80 mg gentamicin, at a rate of 5.0 - 10.0 mL/minute. The ewe’s abdomen, flank, andgroin were shaved, and the surgical areas sterilized with 10% povidone-iodine topical solution,while other areas were covered with sterile sheets and drapes. Subcutaneous injections of 2%lidocaine were made along the site of the incision. Prior to implantation, all catheters were filledwith heparinzed saline (12 U/mL). Silicone rubber catheters (1.02mm i.d. 2.15mm o.d.) wereimplanted in the femoral artery and vein. An abdominal incision to the right of the umbilicus50was made to gain access to the gall bladder, where a catheter was placed to allow for bilecollection. In addition, a (0.64mm i.d. 1.19 mm o.d.) catheter was implanted in a small branchof the mesenteric vein, with the catheter tip lying in the main mesenteric vein in the direction ofthe hepatic portal vein of the ewe. The catheters were exteriorized, tunneled subcutaneouslythrough an incision on the flank, and secured in a denim pouch. The catheters were filled withfresh heparinized sterile saline and capped when not in use. Tracheal catheters for N2 gasinfusion were implanted in order to induce hypoxemia. The neck of the ewe was shaved, andsterilized with 10% povidone-iodine topical solution. A small incision was made near themidline of the neck 4-5 cm below the larynx. The trachea was exposed. The endotracheal tubewas deflated and a small hole was cut through the trachea between the cartilage rings of thetrachea. A polyvinyl tube (5 mm i.d.) was rapidly inserted and glued into place with TissueGlue® (3M, Minneapolis, MN, U.S.A.). The endotracheal tube was then re-inflated. The tubewas anchored to the surrounding tissue with 2-0 silk and the incision closed. Ampicillin (500mg) and gentamicin (40 trig) were administered prophylactically lM on the day of surgery and thefollowing four days. The ewes were allowed to recover for at least five days before they wereused in experiments. Just prior to each experiment, a size 16 fr. Foley® catheter was insertedinto the bladder via the urethra for total urine collection.2.5.2.2. Pregnant Sheep - Surgical PreparationTime dated pregnant Dorset, Suffolk, and Finn mixed breed ewes were operated onbetween 115 and 125 days of gestation (term = 145 days). Ewes were allowed free access towater, but food was withheld for approximately 18 hours prior to surgery. Aseptic techniques51were employed throughout the surgical procedure. Following intravenous atropine (6.0 mg)administration to control salivation, anesthesia was induced with intravenous sodium pentothal(1.0-1.5 g). The animals were intubated with an endotracheal tube and anesthesia wasmaintained through the ventilation (12 cycles/minute) of the animals with a mixture of halothane(1-2%), nitrous oxide (70%), and balance oxygen. An intravenous bolus injection of 500 mgampicillin was given via the jugular vein, followed by an intravenous drip of 5% dextrosesolution (500 mL) containing 80mg of gentamicin, at a rate of 5.0- 10.0 mL/minute. The ewe’sabdomen, flank, and groin were shaved, and the surgical areas sterilized with 10% povidoneiodine topical solution, while other areas were covered with sterile sheets and drapes.Subcutaneous injections of 2% lidocaine were given along the site of the incision. A midlineabdominal incision was made in the ewe and the uterus identified. Access to the head of thefetus was gained through an incision of the uterine wall in an area devoid of placental cotyledonsand major blood vessels. With a small incision (1 cm) the fetal trachea was exposed. Again, allcatheters were filled with heparinized (12 U/mL) normal saline prior to implantation. Thesilicone rubber catheter (1.02mm i.d. 2.16mm o.d.) was inserted into the trachea through asmall incision through the fetal skin 1-2 cm below the larynx (See Appendix 3). The catheterwas inserted through a small hole in between two rings of cartilage, and was then advanced 4-5cm into the trachea towards the fetal lung. The tracheal catheter did not obstruct lung fluidefflux from the airway into the pharynx. The catheter was anchored onto the skin overlying theincision with a piece of 3-0 silk attached to the catheter. A drop of Tissue Glue® was applied onthe catheter’s point of entry to the trachea. The fetal carotid artery was then dissected free fromother tissue and three pieces of 3-0 silk were passed underneath the vessel. After the vessel wastied off with the distal silk, the proximal portion of the vessel was temporarily constricted. Then,52a partial cut was made on the vessel between the middle and the distal sutures. Approximately 3-4cm of the silicone rubber catheter (0.64mm i.d. 1.19mm o.d.) was threaded through the cut onthe vessel towards the ascending aorta (See Appendix 3). The catheter was secured to the vesselwith all three silk sutures, and a drop of Tissue Glue® was applied to the site of entry. Thecatheter was anchored to the adjacent muscle of either side of the incision with the sutures. Thetracheal incision was then closed with 2-0 silk. To implant the electro-cortical and electrooccular electrodes, an incision was made laterally across the entire width of the fetal skull. Theskin was peeled back and the fetal skull was exposed. Small holes were drilled (0.25 mm)biparietally through the skull. Electrodes consisting of multistranded stainless steel wire,insulated with Teflon® except at the tip and threaded through a — 5 mm small plastic disc, wereinserted through these holes onto the dura. To seal the hole, the plastic disc was glued to theskull using Tissue Glue®. A third electrode wire (ground wire) was then sutured to thesurrounding skin tissue. The incision was sutured back together using 2-0 silk. To insert theelectro-ocular electrodes, small incisions were made (—1 cm) above the orbital ridge on eitherside of the fetal head. The electrode wires were driven through the orbital ridge of the zygomaticbone, with the uninsulated portion of the wire sutured through the bone and glued with TissueGlue®. Next, an amniotic catheter (1.02 mm i.d. 2.16mm o.d.) was placed in the amniotic fluidand anchored to the neck of the fetus. The fetus was then placed back into the uterus, after whichthe uterus was closed with a continuous 2-0 gut chromic suture, and then oversewn. Next, asecond uterine incision was made to expose the fetal hindquarters. An incision (2 cm) was madeabove the femoral arterial pulse in the groin. The vessel was prepared for catheterization asdiscussed above for the carotid artery. Approximately 5-6 cm of the catheter was threadedthrough the cut on the femoral artery towards the descending aorta. Both right and left fetal53femoral arteries were catheterized in a similar fashion. Following catheterization, the catheterswere secured and anchored as described above for the carotid artery. In a similar manner,silicone rubber catheters were placed into the right and left fetal lateral tarsal veins.Approximately 11-12 cm of the catheters were threaded into the vessel in order to reach theinferior vena cava. A Transonic® flow probe was placed around the common umbilical artery tomeasure fetal umbilical flow. To implant the flow probe, a flank incision (3-4 cm) was made 2-3cm lateral to the spine, from just central to the kidney to the iliac crest. The common umbilicalartery was approached retroperitoneally and dissected clear of other tissues. A transit-time bloodflow transducer, size 4SB was then placed around the artery, and the flow transducer cable wasanchored to surrounding tissue. In addition, the ipsilateral internal iliac artery was ligated toreduce the non-placental components of the common umbilical arterial flow. The incision wasclosed in layers, and finally the skin was sutured with 2-0 silk. To catheterized the fetal bladder,a suprapubic incision was made. The fetal bladder was exposed and a purse string suture of adiameter of 2 cm was made using 3-0 silk. A small cut was made into the bladder inside thepurse string and the catheter was inserted. The purse string suture was tied. An additional 2-0suture was wrapped around the catheter and the fetal bladder tissue. The catheter was anchoredto both the fetal abdominal muscle wall and the abdominal skin. The incision was closed inlayers and finally the skin was sutured using 3-0 silk. To catheterize the common umbilical vein.a small incision was made in the umbilicus overlying one of the two umbilical veins. The tissuesurrounding the umbilical vein was carefully dissected away from the surface of the vessel. Twosutures (5-0 silk) were placed, in parallel, through the vessel wall at right angles to the long axisand separated by 1 mm. Using an 18-gauge needle, a hole was made in the vessel wall betweenthe sutures, and a silicone catheter (0.51 mm i.d. 0.94 mm o.d.) was inserted —2 cm, so that the54tip of the catheter lay in the intra-abdominal common umbilical vein. The sutures were tied in a“figure 8” fashion around the catheter and a drop of Tissue Glue® was applied. The catheter wasanchored to the skin overlying the umbilicus and to skin of the fetal abdomen. The incision wasthen closed with 3-0 silk. Two additional amniotic catheters were anchored to the fetalabdominal skin. The fetus was then gently returned to the uterus. Amniotic fluid lost duringsurgery was replaced with irrigation saline (Tranvenol Canada Inc., Mississauga, Ont.), afterwhich the uterine incision was closed with a continuous 2.0 gut chromic suture, and thenoversewn. The catheters were flushed with heparinized normal saline (12 U/mL), tunneledsubcutaneously, and exited through a small incision on the maternal right flank. The ewe’smidline abdominal incision was closed in layers and the flank incision was sewn up as well.Finally, the maternal femoral artery and vein were catheterized (1.02 mm i.d. 2.15 n-im o.d.). Allcatheters were capped and stored in a denim pouch which was secured in place with twoadhesive bandages on the right flank. The ewe’s abdomen was then wrapped with elastic crepebandages. All vascular catheters were flushed daily with 2 mL of heparinized normal saline. Inthe case of the umbilical venous catheter, an additional 0.5 mL of heparin (1000 U/mL) was usedto prevent clot formation. Ampicillin (500 mg) and gentamicin (80 mg) were administeredintramuscularly to the ewe on the day of surgery and for the first four days following surgery,while ampicillin 500 mg and gentamicin (20 mg) were administered IV to the fetus at the time ofsurgery. Ampicillin (500 mg) and gentamicin (20 mg) were also administered into the amnioticcavity on a daily basis until delivery. The ewes were moved into holding pens in the company ofother sheep and were allowed to recover for at least 5 days following surgery. On the day of theexperiment, a 16 fr. Foley® catheter was inserted into the maternal bladder via the urethra andattached to a polyvinyl bag for cumulative urine collections.552.5.3. Chronic Monitoring of Animals2.5.3.1. Amniotic, Tracheal, and Vascular Pressures, Heart rate, Blood Flow, ECoG andEOG.Prior and during experiments, amniotic, tracheal pressure and fetal and maternal arterialpressures were continuously monitored using disposable DTX transducers. Fetal and maternalheart rates were measured from the arterial pulse or from arterial blood flow by means ofcardiotachometers (Model 9857). Electrocortical and electro-ocular signals were recorded usinga type 9806A AC/DC coupler. Umbilical flow was measured with a transit time flowmeter(Model T201, Transonic Systems, Ithica, NY, U.S.A.). All variables were recorded using aBeckman R-7 11 polygraph recorder in conjunction with a computerized data acquisition system(Kwan, 1989). The sampling rate was 2.5 Hz. At the end of each minute, the measurementswere averaged, fetal arterial pressure corrected for amniotic pressure, and the values displayed onthe computer monitor. Every 30 minutes, the minute average measurements were automaticallytransferred to floppy diskettes for subsequent analysis.2.5.3.2. Fetal Urine MeasurementsFetal urine collections were made using a computer controlled roller pump assemblydeveloped in our laboratory. This setup incorporated a disposable DTX transducer connected toa gravity fed urine reservoir (a sterile 10 mL syringe barrel) to which the fetal bladder catheterwas connected. As the pressure in the reservoir increased above a preset trigger pressure (usually563 mm Hg), the computer activated the roller pump (DIAS, Ex154, DIAS Inc. Kalamazoo, MI,U.S.A.) which would pump a calibrated volume of urine from the reservoir back to the amnioticcavity (via the amniotic catheter) during control periods, or into sterile sample collection syringesduring experiments. The cumulative volume pumped per minute, which equals fetal urineproduction per minute, was recorded and stored on diskette.2.5.3.3. Blood Gas AnalysisBlood pH, Po2, and Pco2 were measured using an IL 1306 pH/Blood gas analyzer (AlliedInstrumentation Laboratory, Milan, Italy), with temperature correction to 39°C for maternalblood and 39.5°C for fetal samples. Blood oxygen saturation and hemoglobin content weremeasured using a Hemoximeter (Radiometer, Copenhagen, Denmark).2.5.3.4. Glucose and Lactate measurementsGlucose and lactate measurements were made using a 2300 STAT plus glucose/lactateanalyzer.2.5.4. Dosage PreparationDPHM HCI (Sigma Chemical Co. St. Louis, MO), and[2H10jDPHM HC1 were weighed toobtain the correct dose for administration. The weighed doses were dissolved in sterile 0.9%sodium chloride for injection and then filtered through a 0.22 i nylon syringe filter into a capped57empty clean sterile injection vial. All doses were made on the morning of the experiment, andwere not stored for more than 6 hours at 40 C.2.6. Experimental Protocols2.6.1. Adult Non-Pregnant StudiesA 100 mg IV bolus of DPHM (over one minute) was administered via the femoral vein tofive surgically prepared non-pregnant sheep. Serial arterial blood samples were drawn at -5, 5,10, 15, 20, 30, 45, 60, 90, 120, 150, 180, 240, 300, 360, 480, 600 and 720 minutes. Urine wascollected at -5, 15, 30, 45, 60, 90, 120, 150, 180, 240, 360, 480, 600, and 720 minutes (in oneanimal, collection was extended to 24 hours). The urine pH and volume was measured andrecorded. In two of the animals, bile was collected over 10 minute intervals at 5, 15, 30, 45, 60,90, 120, 150, 180, 240, 360, 480, 600, and 720 minutes. The concentration of DPHM in thesebiological samples was measured using a previously published GC-NPD method (Yoo et al.,1986).2.6.2. Adult Isotope Effect StudiesIn the adult isotope effect control experiments equimolar amounts of DPHM and2[H10]DPHM equivalent to approximately 50 mg each of DPHM and [H10]DPHM free basewere simultaneously administered as an intravenous bolus via the maternal femoral vein over oneminute. Serial maternal femoral arterial blood samples (5 mL) were collected at -5, 5, 10, 15, 20,30, 40, 50, 60, 75, 90, 105, 120, 150, 180, 240, 310, 480, 600, and 720 minutes. Urine wascollected at -5, 15, 30, 45, 60, 90, 120, 150, 180, 240, 360, 480, 600, 720, and 1440 minutes.58The urine pH and volume were measured and recorded. The concentrations of DPHM and[2H10]DPHM in the biological samples collected were measured using the developed GC-MSmethod described above (2.4.1.).2.6.3. Fetal Isotope Effect Studies2.6.3.1. Fetal BolusThe fetal control experiments involved the simultaneous administration of DPHM and[2H10]DPHM equivalent to approximately 5.0 mg each of DPHM and[2H10]DPHM free base viathe fetal lateral tarsal vein. Fetal femoral arterial blood, amniotic fluid, and fetal tracheal fluidsamples were collected at -5, 5, 10, 15, 20, 30, 40, 50, 60, 75, 90, 120, 150, 180, 210, 240, 300,360, 480, 600, and 720 minutes. The concentrations of DPHM and[2H10]DPHM in thesebiological samples were measured using the GC-MS method described above (2.4.1).2.6.3.2. Fetal InfusionTwo control experiments were conducted to verify the absence of an isotope effect betweenthe disposition of[2H10]DPHM and the metabolite[2H10]DPMA, compared to DPHM andDPMA. Equimolar doses of DPHM and[2H10JDPHM were simultaneously administered via thefetal lateral tarsal vein as a 2.0 mg loading dose followed immediately by a 90 minute infusionwith an infusion rate of 60 p.g/minute each of DPHM and[2H10]DPHM. Serial samples werecollected from the fetal femoral and carotid arteries and maternal femoral arteries at -5, 5, 15, 30,5945, 60, 75, and 90 minutes. Amniotic fluid and fetal urine samples were also collected at -5, 30,60, and 90 minutes. The concentrations of DPHM and[2H10JDPHM in the collected biologicalsamples were measured using the GC-MS method described above (2.4.1.). In addition, theconcentrations of DPMA and[2H10]DPMA were measured in these samples using the GC-MSmethod described above (2.4.2.).2.6.4. Adult First-Pass Metabolism2.6.4.1. Adult First-Pass Metabolism During NormoxiaIn the adult first-pass metabolism study equimolar amounts of DPHM and[2H10]DPHM(approximately equivalent to 50 mg each of DPHM and[2H10jDPHM) were administeredsimultaneously via different routes (i.e., femoral vein - control and mesenteric vein - test), overone minute. For example, DPHM would be administered via the mesenteric vein simultaneouslywith{2H10]DPHM via the femoral vein. It should be noted that in each animal the route ofadministration of DPHM and[2H10jDPHM was alternated so that[2H10]DPHM and DPHM werenot administered via the same routes of administration in every animal. This procedure wasincorporated into the experimental protocol to avoid any subtle isotope effects not detected bythe isotope effect studies in non-pregnant and fetal sheep. Serial maternal femoral arterial bloodsamples (5 mL) were collected at -5, 5, 10, 15, 20, 30, 40, 50, 60, 75, 9G, 105, 120, 150, 180,240, 360, 480, 600, and 720 minutes. The concentrations of DPHM and[2HIOJDPHM weremeasured in these samples using the GC-MS method described above (2.4.1.).602.6.4.2. Adult First-Pass Metabolism During Mild HypoxemiaThe experimental protocol for the hypoxemia first-pass experiments was similar to that forthe normoxic experiments (2.6.4.1). However, 15 minutes prior to the start of drugadministration and for the first 6 hours following drug administration, nitrogen gas was infusedat 7 L/minute via the tracheal catheter to reduce the inspired 02 concentration. The maternalarterial blood gas status was periodically monitored. When the Po2 rose above 70 mm Hg, thenitrogen flow was increased up to 12 L/minute. Six hours following the bolus dosing, thenitrogen gas flow was turned off, and the oxygen status rapidly returned to base line. Theconcentrations of DPHM and[2H10]DPHM were measured in these samples using the GC-MSmethod described above (2.4.1.).2.6.5. Fetal Hepatic First-Pass Metabolism2.6.5.1. Fetal Umbilical Venous BolusThe fetal umbilical first-pass experiments involved the simultaneous administration ofDPHM and[2H10]DPHM (equivalent approximately to 5.0 mg (E#499, E#l 143, E#543, andE#975) and 2.5 mg (E#208 and E#989) each of DPHM and[2H10]DPHM free base via differentroutes (e.g., DPHM administered via the umbilical vein and[2H10]DPHM administered via thefetal lateral tarsal vein). An additional precaution of alternating the route of administration ofDPHM (i.e., tarsal or umbilical venous) and[2H10]DPHM (i.e., umbilical or tarsal venous) wasincorporated into the experimental protocol to avoid any subtle isotope effects not detected bythe isotope effect studies in non-pregnant and fetal sheep. In all cases, the injection of drug was61given over one minute. Serial samples of fetal femoral (2 mL) and carotid (2 mL) arterial blood,fetal tracheal fluid (2 mL) and amniotic fluid (5 mL) were collected at -5, 5, 10, 15, 20, 30, 40,50, 60, 75, 90, 105, 120, 150, 180, 210, 240, 300, 360, 480, 600, and 720 minutes. Theconcentrations of DPHM and[2H10]DPHM were measured in these samples using the GC-MSmethod described above (2.4.1.).2.6.5.2. Fetal Umbilical Venous InfusionThe simultaneous fetal umbilical and tarsal venous infusion experiments involved theadministration of an IV bolus loading dose of 2.0 mg of DPHM and[2H10]DPHM immediatelyfollowed by a 90 minute infusion of approximately 60 rig/minute of DPHM and[2H10]DPHM viathe umbilical and tarsal veins, respectively. Again, the routes of administration of DPHM and[2H10]DPHM were alternated in subsequent animals. Serial blood samples were drawn from thefetal femoral and carotid arterial, and the fetal tracheal and amniotic catheters at -5, 5, 15, 30, 45,60, 75, and 90 minutes. In these studies, paired femoral and carotid arterial blood gas sampleswere taken and measured. The concentrations of DPHM and[2H10]DPHM were measured inthese samples using the GC-MS method described above (2.4.1.).2.6.6. Paired Fetal/Maternal InfusionPrior to drug administration, 10 sterile (20 mL) heparinized syringes were used to draw up15 mL of drug free maternal arterial blood. These samples were stored refrigerated (4° C) untilrequired for fetal blood replacement (syringes were removed from the refrigerator to allow blood62to warm up to room temperature just prior to infusion). Fetal blood withdrawn by sampling wasslowly replaced following sample collection with drug free maternal blood at 1.0 hour intervalsduring the infusion and at 2.0, 6.0, 12.0, and 24 hours post infusion via a 10 minute infusion tothe fetal femoral artery. DPHM was administered as a 20 mg IV loading dose over a 1.0 minuteinterval immediately followed by an infusion of 670 igIminute via the maternal femoral vein.Simultaneously a fetal 5.0 mg IV loading dose of[2H10]DPHM was given over a 1.0 minuteinterval immediately followed by an infusion of[2H10]DPHM at 170 jig/minute, given via thefetal lateral tarsal vein. Simultaneous fetal and maternal blood samples were collected from thefetal femoral and carotid arteries, umbilical vein, and maternal femoral artery. Blood samples(fetal [2.5 mU and maternal [5.0 mL] arterial samples) were collected at -5, 30, 60, 90, 120, 150,180, 210, 240, 270, 300, 330, and 360 minutes during the infusion and at 30, 60, 120, 180, 240,360, 480, 720, 1080, 1440, 1800, and 2400 minutes post infusion. In animals in which carotidand umbilical venous catheters were functional, samples were collected at -5, 30, 60, 90, 120,150, 180, 210, 240, 270, 300, 330, and 360 minutes, but were not collected post-infusion.Amniotic and tracheal fluids were sampled at -5, 60, 120, 180, 240, 300, and 360 minutes duringthe infusion and at 60, 120, 180, 240, 360,480, 720, 1080, 1440, 1800, and 2400 minutes postinfusion. Cumulative collections of maternal urine were made at -5, 60, 120, 180, 240, 300, and360 minutes during the infusion and at 60, 120, 180, 240, 360, 480, 720, 1080, 1440, 1800, and2400 minutes post infusion; urine volume and pH were measured throughout the experimentalperiod. Fetal urine was collected at -5, 60, 120, 180, 240, 300, and 360 minutes during theinfusion and at 60, 120, 180, 240, 360, 480, and 720 minutes post infusion in a sterile 60 mLsyringe from which the volume was measured. The fetal urine, except a 5 mU aliquot, wasreturned back to the amniotic cavity via the urine collection apparatus. At the end of each63sample, the fetal urine collection syringe was removed and replaced with a new sterile syringe.The concentrations of DPHM and[2H10]DPHM were measured in these samples using the GCMS method described above (2.4.1.). The concentrations of DPMA and[2H10JDPMA weremeasured using the developed method described in section 2.4.2.2.6.7. Sample HandlingMaternal and fetal blood samples were placed into heparinized glass tubes and gentlymixed, with care taken to avoid contact of the blood with the rubber stopper of the Vacutainer®.These samples were then centrifuged at 3000 X g for 10 minutes. The plasma supernatant wasremoved and placed into clean borosilicate test tubes which were capped with PTFE-lined caps.Amniotic and tracheal fluid samples, and fetal and maternal urine samples were placed directlyinto clean borosilicate test tubes which were capped with PTFE-lined caps. All samples werestored frozen at 2O0 C until the time of analysis ( 3 months from sample collection).2.7. In Vitro Experiments2.7.1. DPHM Blood Cell UptakeTwo experiments were conducted; one measured the time course necessary to attainequilibrium for blood cell (BC) uptake of DPHM, and the second experiment measured the effectof temperature on the uptake of DPHM into the BCs. The experimental protocol for the uptakestudies involved spiking heparinized whole blood from non-pregnant ewes to obtain a totalDPHM concentration of 2.0 igIrnL, incubating the samples at 39° C for designated periods oftime, followed by immediate separation of plasma from the BCs by centrifugation. All samples64were stored frozen at 200 C prior to analysis. The concentration of DPHM was measured inboth the BC and plasma fraction using a previously published GC-NPD method (Yoo et al.,1986). The ratio of DPHM concentration in plasma to BC was plotted as a function of time todetermine the extent and rate of DPHM uptake into BC.The experiment investigating the effect of temperature on DPHM uptake into BCs was toensure that sample handling techniques (i.e., allowing the collected samples to cool to roomtemperature prior to separating the BC and plasma fractions) did not change the plasma DPHMconcentration due to BC uptake. Whole blood was spiked to yield a total drug concentration of2.0 .tg/mL. One group of samples was incubated at 22° C while the second group of sampleswas incubated at 39° C for 30 minutes. Following incubation, the samples were immediatelycentrifuged at 3000 X g for 2 minutes, and the plasma and BC fractions were separated. Prior toanalysis, these samples were stored frozen at 200 C. The concentration of DPHM in both theplasma and BC fraction in these samples was determined, using a previously published GC-NPDmethod (Yoo et al., 1986).2.7.2. Adult and Fetal Hepatic Microsomal Metabolism Experiments2.7.2.1. Preparation of Adult and Fetal Hepatic MicrosomesAdult hepatic microsomes were prepared from yearling male lambs (roughly 1 year old) atthe time of slaughter at Pitt Meadows Meats, Pitt Meadows, B.C., Canada. The animals werestunned and then killed by exsanguination. The animals were disemboweled shortly after theywere killed (2-5 minutes). The livers were removed and a lateral slice was made across the liver.The liver slice was immediately rinsed and placed into ice cold Tris KC1 buffer pH 7.4 fortransportation. Fetal hepatic microsomes were prepared from fetal lambs at the time of cesarean65section. The ewes were anesthetized as described in section 2.4.3.2. The sex, gestational age,and fetal weight are shown in Appendix 1. A midline incision was made, and the uterusexposed. An incision was made into the uterus and the fetus was exposed. A midline incisionwas made on the fetus and the fetal liver exposed. The umbilical vein was clamped and 2.5 mLof Euthanol® was immediately injected into the fetal heart. Following fetal death, the liver wasimmediately removed, cut into slices, rinsed free of blood, and immersed in ice cold Tris KC1 pH7.4 buffer for transport. Following the cesarean section, the uterine incision was closed and theabdomen of the ewe closed as described in section 2.4.3.2. The livers were again rinsed with icecold Tris KC1 pH 7.4 buffer to remove any remaining traces of blood, and then minced. Allprocedures were conducted on ice. The minced liver tissue (fetal or adult) was thenhomogenized in Tris KCI pH 7.4 buffer with a Potter-Elvehjem tissue grinder. A total of tenhomogenization passes were made; five with the loose-fitting pestle for the initialhomogenization, and five passes with the tight fitting pestle for the final homogenization. Thehomogenate was transferred to low speed centrifuge tubes and centrifuged at 9000 X g for 20minutes at 40 C. The supernatant was filtered through four layers of “cheese” cloth. Thesupernatant was then transferred to high speed centrifuge tubes and the pellet from the low speedspin was discarded. The supernatant was centrifuged at 105,000 X g for 60 minutes at 4° C. Theresulting supernatant was discarded and the lipid was wiped from the tube. The pellet wascarefully resuspended to avoid the inclusion of the glycogen portion of the pellet and placed in aclean homogenization tube. The microsomal pellet was resuspended in EDTAIKC1 pH 7.4buffer with five passes of the loose fitting pestle. The resulting homogenate was transferred to ahigh speed centrifuge tube and centrifuged at 105,000 X g for 60 minutes at 4° C. Thesupernatant was discarded and the lipid wiped from the inside of the centrifuge tube. The pellet66was resuspended in (four equivalent volumes to pellet) 0.25 M sucrose solution andhomogenized with four passes with the loose fitting pestle. The homogenate was transferred intocryo-test tubes and stored at -70° C until use.2.7.2.2. Protein Concentration and Cytochrome P450 MeasurementsThe protein concentration of the adult and fetal microsomal preparations was determinedusing a modified Lowry method (Markwell et al., 1978). Cytochrome P450 was determinedusing a spectrophotometric assay based on the absorption of Cytochrome P450 bound to carbonmonoxide, as described by Omaru et al., 1968.2.7.2.3. DPHM and N-demethyl DPHM QuantitationThe quantitation of DPHM and N-demethyl DPHM was carried out using methodologydescribed earlier (Abernethy and Greenblatt, 1983, and Blyden et al., 1986), with somemodification. GC-MS analytical instrumentation was used rather than GC-NPD. Reoptimization of this methodology resulted in similar extraction and instrumentation parametersas those used in the original GC-MS assay for[2H10]DPHM and DPHM. Fragment ions m/z 165and 167 were used to quantitate DPHM and N-demethyl DPHM. The standard curve for Ndemethyl DPHM used concentrations of 2.5 -50.0 ng/mL.672.7.2.4. Fetal and Adult Hepatic Microsomal IncubationsInitial experiments to optimize conditions for microsomal incubations were carried out inthe adult hepatic microsomes. These experiments included optimization of the amount ofmicrosomal protein to be added, and optimization of the amount of substrate to be added to theincubation mixture. The microsomal incubations were conducted in a clean test-tube using 0.5mL of 0.2 M phosphate pH 7.4 buffer, 10 p.L of 300 mM MgC12, 1 mg of microsomal protein,and 10 jiL of both 10 mM NADPH and NADH. The reaction mixture was then made up tovolume (so that the final volume would be 1.0 mL) with distilled water. The test-tube containingthe microsomal suspension was gently mixed, to avoid frothing, on a vortex mixer, and placed ina water bath at 39° C for 15 minutes as a pre-incubation. The reaction was started with theaddition of 0.5 llmoles of substrate. The reaction was terminated at 0, 2.5, 5.0, 7.5, 10.0, 15.0,20.0, 30.0, 45.0, and 60.0 minutes by the addition of 0.5 mL 1% ice cold HC1. The tubes werethen immediately capped and frozen until the time of analysis. These tubes were assayed forDPHM and N-demethyl DPHM. Additional incubations were conducted using normal andboiled (control) adult and fetal microsomes. These incubations were conducted as outlinedabove, but were allowed to continue for 90 minutes. They were then assayed for both Ndemethyl DPHM and DPMA.2.7.3. Plasma Protein Binding of DPMAThe plasma protein binding of DPMA was measured in fetal and maternal plasma usingequilibrium dialysis [Plexi-glass® dialysis cells (1.0 mL capacity) with cellophane dialysismembranes (molecular cut-off 12,000 daltons, Sigma Chemical Co., St. Louis, MO, U.S.A.)].68Maternal and fetal plasma for these experiments were obtained from two additional sheep set upfor other experiments. The fetal and maternal drug-free plasma was obtained on non-experimentdays and pooled. Aliquots of fetal and maternal plasma were spiked with DPFIM and DPMA toyield total drug and metabolite concentrations of 1.0 J.tg/mL. Spiked fetal and maternal plasmawas dialyzed against 0.1 M phosphate pH 7.4 buffer. Dialysis was carried out in a temperaturecontrolled water bath at 39° C. Time-to-equilibrium studies were conducted with sampling timesof 2, 4, 6, 8, 12, 24, and 36 hours. In each sample, the pH and the post dialysis volume weremeasured. Dialyzed buffer and plasma were transferred to clean borosilicate test-tubes, cappedwith PTFE-lined lids, and frozen at -20° C. Non-specific binding was determined by dialyzingblank buffer against spiked buffer (1.0 j.ig/mL). Concentrations of DPMA were measured inboth buffer samples; the sum of these samples was then compared to the spiked metaboliteconcentration. The difference was taken as the extent of non-specific binding. The plasmaprotein binding of DPMA in pooled fetal and maternal plasma was determined in five replicatesby dialyzing spiked fetal and maternal plasma against blank buffer for 8 hours. Then both bufferand plasma were removed, volumes and pH measured, and the samples stored as describedabove. DPMA concentrations were determined in both plasma and buffer samples. The freefraction was taken as the ratio of free metabolite concentration (buffer) over total metaboliteconcentration (plasma).692.8. Data Analysis2.8.1. Data ReductionData obtained from fetal monitoring was reduced in the following fashion. The data wasobtained as minute averages on disk. This data was transferred to a spread-sheet (MicrosoftExcel®), and 10 minute averages were calculated; the results are presented as the mean ± SEM,unless otherwise stated.2.8.2. Calculation of in Utero Fetal WeightFetal weights in utero at the time of experimentation were estimated from the weight atbirth and the time interval between the experiment and the birth using equation 1 (Gresham etal., 1972):Eq 1: Log(fetal weight in utero) = Log(birth weight) - 0.153*(number of days betweenexperiment and birth).2.8.3. Pharmacokinetic Data AnalysisThe model used to fit the data obtained from the 1.0 minute infusion wasa 2 compartment open model with infusion input and elimination occurring from the centralcompartment shown below (Figure 5) (Gibaldi and Perrier, 1982).70KoFigure 5: A 2 compartment open model for the disposition of DPHM in fetal ormaternal sheep.The equation for the plasma concentration of DPHM of[2H10JDPHM is shown in equation 2:Eq2: Cp = Ko(K21)(1eaT)et +Vcappx(ct-) Vcapp3(c-f)where T is the duration of the infusion, and is equal to t during the duration of the infusion, andfollowing the infusion T is a constant equal to the total duration of the infusion (1.0 minute).This equation gives Cp (the plasma concentration at a particular time (t)), where o and 3 aredisposition constants, VCapp is the apparent volume of distribution of the central compartment,and K12 and K21 are the rate constants describing the transfer of drug from the central toperipheral and peripheral to the central compartments, respectively. Ko is the infusion rate. Theconcentration vs. time data was fit using the ADAPT II computer fitting program. A maximumlikelihood fitting algorithm with a power variance model was utilized, as shown in equation 3,Ki 071where ö and y are constants describing the variance of the measured concentration (Cp)(D’Argenio and Schumitzky, 1988).Eq.3: Var=52CpCp max was extrapolated from the fitted equation to the end of the infusion (i.e., 1.0minute.). The area under the plasma concentration vs. time curve (AUCo,) was calculatedusing a hybrid trapezoidal method. The linear trapezoidal method was used to calculate the AUCfrom time zero to the maximum plasma concentration one minute following the infusion(AUCoc max), followed by the log-linear trapezoidal method to calculate the AUC frommaximum plasma concentration to the last measured plasma concentration (AUCc max—Cp last).Finally, the AUC under the terminal portion of the disposition curve (AUCCp(t) last—*’o ) wascalculated as Cp last/f3. The total AUC0was calculated as shown by equation 4 (Purves,1992):Eq 4: AUC0= AUCoc max + AUCc max>Cp last + AUC Cplast-+.oThe area under the first moment curve (AUMC) was calculated in a similar fashion, that is, ahybrid method of linear and log-linear trapezoidal functions was used. The total body clearance(CLT) was calculated as shown in equation 5 (Gibaldi and Perrier, 1982):Eq. 5: CLT=Dose/AUCo72The steady state volume of distribution (Vd) was calculated as shown in equation 6 (Perrierand Mayersohn, 1982):Eq. 6: Vd = (Dose/AUCo)*MDRTThe volume of distribution area (Vdj) was calculated as shown in equation 7 (Gibaldi andPerrier, 1982):Eq. 7: Vd = Dose43*AUCoThe total mean residence time (MTRT) was calculated as shown in equation 8 (Weiss, 1992):Eq. 8: MTRT = AUMCOJAUCOOThe mean disposition residence time (MDRT) is calculated as shown in equation 9 (Weiss,1992), where T/2 is the correction factor for drug administration by infusion:Eq. 9: MDRT = MTRT-T/2The systemic availability (F) of DPHM or[2H10]DPHM administered either via the mesentericvein in adult sheep or via the umbilical vein in the fetal lambs (i.e., the test routes) wascalculated as shown in equation 10 (Gibaldi and Perrier, 1982):73Eq. 10: F = AUC0(test route)/AUCo (control route)The fetal total body clearance (CLT) following simultaneous infusion to steady-state wascalculated as shown in equation 1 1(Gibaldi and Perrier, 1982):Eq. 11: CLT = Ko/CpssWhere Cpss is the steady-state plasma concentration.The fetal or maternal plasma concentration vs. time for the simultaneous maternal and fetalinfusion experiments was fit using a 2 compartment open model with elimination occurring fromthe central compartment (Gibaldi and Perrier, 1982) (Figure 5). The concentration vs. time datawas fit using the ADAPT II computer fitting program using a maximum likelihood fittingalgorithm (variance model for Cp; Var = ö*(Cp) + y where and y are estimated variancecoefficients) (D’Argenio and Schumitzky, 1988). Cp0 was extrapolated from the fitted equation.The area under the plasma concentration vs. time curve (AUC0)was calculated using thelinear trapezoidal method (Gibaldi and Perrier, 1982).A 2 compartment-open model was used to describe the disposition of DPHM and[2H10]DPHM in the maternal and fetal sheep, respectively (Figure 6). This model assumessteady-state plasma concentrations, and that drug elimination occurs from both the maternal andfetal compartments. Trans- and non-placental clearances of DPHM were calculated as described(Szeto, 1982, and Szeto et al., 1982) in equations 13-18 below:74Figure 6: Schematic representation of the 2 compartment open model for drug dispositionEq. 13:Eq. 14:Eq. 15:Eq 16:Eq 17:in the maternal fetal unit.CLmm = Ko/[Cmss - Cfss*(Cmss’/Cfss’)]CLff= Ko’/[Cfss’ - Cmss’*(Cfss/Cmss)]CLmf CLff*(CfsslCmss)CLfm = CLmm*(Cmss’ICfss’)CLmo = CLmm - CLmfko ko’CLmfmaternalCLmo CLfoEq 18: CLfo = CLff - CLfm75where CLmm and CLff are the total body clearances from the mother and fetus, respectively,CLmf is the trans-placental clearance from the maternal to fetal compartment, CLfm is the transplacental clearance from the fetal to the maternal compartment, and CLmo and CLfo are thenon-placental clearances from the mother and fetus, respectively. By using the values for theinfusion rates to the mother (Ko) and fetus (Ko’) and the steady-state drug concentrations in themother and fetus following maternal infusion (Cmss and Cfss) and fetal infusion (Cmss’ andCfss’), all the clearances were calculated. Trans-placental clearances were also calculated usingthe Fick method in two animals in which umbilical flow and umbilical venous concentrations ofDPHM and[2H10]DPHM were measured. The calculations for the trans-placental clearances areshown in equations 19 and 20 below:Eq. 19: CLmf = Qum*(C(DPHM)uv - C(DPHM)fa)/C(DPHM)faEq. 20: CLfm = Qum*(C([2H10]DPHM)fa -C([210]DPHM)uv)/C([PHM)faIn addition, this 2 compartment model was also used with the integrated form, using themass balance approach (Edling and Jusko, 1986). The various trans- and non-placentalclearances were calculated as shown in equations 21-24 below:Eq. 21: CLmo = Dose(M)*AUCff Dose(F)*AUCmfAUCmrn*AUCff - AUCmf*AUCfm76Eq. 22: CLfo = Dose(F)*AUCmm - Dose(M)*AUCfmAUCmm*AUCff- AUCmf*AUCfmEq. 23: CLmf= Dose(F)*AUCmfAUCmm*AUCff - AUCmf*AUCfmEq. 24 CLfm = Dose(M)*AUCfmAUCmm*AUCff- AUCmf*AUCfmwhere CLmo, and CLfo are the elimination clearances of DPHM from the mother and fetus,respectively. CLmf and CLfm are the transfer clearances of DPHM from mother to fetus andfetus to mother, respectively. AUCff and AUCmm are the areas under the plasma concentrationvs. time curve of fetal DPHM following fetal administration and of maternal DPHM followingmaternal administration, while AUCmf and AUCfm are the areas of fetal DPHM followingmaternal administration and maternal DPHM following fetal administration, respectively. Forthe infusion data the total mean residence time (MTRT) was calculated as shown above;however, because this dosage form was administered both as a bolus and a simultaneousinfusion, the mean disposition residence time (MDRT) is calculated as shown in equation 25:Eq. 25: MDRT = MTRT-(fo X dt)/Z(dose)Where the correction factor ((1o” X dt)/E(dose)) for a loading dose bolus and simultaneousinfusion is equal to (Ko*T2/2*(Ko*T + Xo). The steady state volume of distribution (Vd8)was77calculated as shown in equation 7 (Perrier and Mayersohn, 1982). An additional correction wasmade to the fetal steady-state volume of distribution. This was to account for the portion of thefetal dose which is transferred from the fetus to the mother across the placenta. The fetal doselost due to placental transfer (Dose’) is calculated as shown in equation 26:Eq. 26: Dose’ = CLmm*AUCfmThe fraction of the fetal dose (f’) which remains in the fetus is calculated as shown in equation27:Eq. 27: f’ = (Total Fetal Dose - Dose’)ITotal Fetal DoseThus, the corrected fetal apparent steady-state volume of distribution (Vd’) is calculated asshown in equation 28:Eq. 28: Vdss’ = (f’ *Total Fetal Dose/AUCff)*MDRT78Fetal and maternal plasma metabolite data was fit using the model outlined below (Figure7). A linked pharmacokinetic model (i.e., where parent drug and metabolite data weresimultaneously fit) was used to reduce the bias in the pharmacokinetic parameter estimates dueto individually modeling parent drug and metabolite. A 1 compartment model was assumedfrom the metabolite, since the data obtained could not support a higher order model for themetabolite.IKoor KoK12Kout_______Central PeripheralK21KfMetaboliteKmFigure 7: A linked pharmacokinetic model showing 2 compartment kinetics for parent drug(DPHM), and 1 compartment kinetics for the metaholite (DPMA) used for theestimation of metabolite pharmacokinetic parameters in mother and fetus.The plasma concentration data for DPHM and the corresponding metabolite was fit usingthe maximum likelihood algorithm with the ADAPT II computer fitting program to thedifferential equations 29-31 shown below (D’Argenio and Schumitzky, 1988).Eq. 29: dX(l)/dt= Ko+ K21*X(2) - (K12 +KlO)*X(l)79Eq. 30: dX(2)/dt K12*X(1) - K21*X(2)Eq. 31: dX(3)/dt = Fm*K10*X(1) Km*X(3)Where Ko is the infusion rate of DPHM to mother and fetus, K2 1, K 12, and K 10 are the rateconstants for the transfer of drug from the tissues to the central compartment, from the centralcompartment to the tissues, and from the central compartment out, respectively. Km is the rateconstant for the elimination of the metabolite DPMA. Fm = fmlVm, fm = KfI(K10), where Kl0= Kout + Kf), and where Vm is the volume of distribution of the metabolite, and Kf is theformation rate constant for the metabolite. The output equations for the concentration of DPHMand DPMA are Cp(DPHM) = X(l)/VCapp, and Cp(DPMA) = X(3). The variance model forCp(DPHM) and Cp(DPMA) used in the fitting of these equations were linear variance models(i.e., Var(DPHM) = Cp(DPHM)*ö(1) + y(1), and Var(DPMA) = Cp(DPMA)*(2) + ‘y(2)).The renal clearance of DPHM and DPMA in both mother and fetus were calculated asshown below in equations 32 and 33 (Gibaldi and Perrier, 1982):Eq. 29: CL(DPHM)Een = Xu(DPHM)/AUCo(DPHM)Eq. 30: CL(DPMA)ren MU(DPMA)/AUC0o( M )where ZXu and 2Mu are the total cumulative amount of DPHM or DPMA excreted in the urine,respectively.802.8.4. Statistical AnalysisValues are expressed as mean values ± standard error of the mean (SEM). A paired sampleT-test was conducted to test for differences in the ratio of the AUC0of DPHM and[2H10}DPHM, pharmacokinetic parameters for isotope effect control studies, and thepharmacokinetic parameters following the simultaneous maternal/fetal infusions (Zar, 1984).Where the requirements of a two sample T-test could not be assumed, the non-parametricequivalent (i.e., Mann Whitney U-test) was used. The time required to reach steady-state wasdetermined by using three groups of mean concentration values (i.e., 150 and 180, 240 and 270,and 330 and 360 minutes) with a repeated measures ANOVA (Zar, 1984). Statistical differencesbetween the fetal effects from the control periods and the test period were determined byrepeated measures ANOVA (Zar, 1984).813. Results3.1. Development of Analytical Methodology3.1.1. Capillary Gas Chromatographic-Mass Spectrometric Analysis of DPHM and[2H10]DPHM.3.1.1.1. Optimization of Mass Spectrometer and Gas ChromatographDPHM and[2H10]DPHM both undergo extensive fragmentation following GC-MS/EI(Figure 8). These molecules appear to fragment at the ether linkage; hence, the molecular ions ofeither DPHM or[2H10]DPHM were absent. The fragmentation of DPHM and[2H10]DPHMresults in three predominant mass to charge ratio (m/z) fragment ions; 58, 165, and 167.forDPHM, and 58, 173, and 177 for[2H10IDPHM. Selected ion monitoring (SIM) was used tooptimize both the selectivity and sensitivity of this analytical method. Two fragment ion pairs(i.e., m/z 167-DPHM and 177-[2H0]DPHM, and m/z 165-DPHM and 173-[2H0jDPHM) allowedfor differentiation between the labeled and unlabeled drugs, and showed the abundance necessaryto achieve the required sensitivity. Ions in/z 165 and 173 were ultimately monitored for thequantitation of DPHM and[2H10]DPHM. To further optimize the mass spectrometer, a manualtuning algorithm was employed. This tuning algorithm for the mass spectrometer uses thefragment ions mlz 100, 131, and 219, which are derived from the tuning reagent PFTBA. Thisprocedure further enhanced the sensitivity of the assay by 20 fold over the auto tune algorithm.The dwell time was set at 50 msec to provide at least 15 scans/chromatographic peak.82Figure 8: The mass spectra arid fragment assignments of a)DPHM andb)[2H10JDPHM followingelectron impact ionization (70 eV) of the purified standards.aABUNDANCE4500004000001500002000002500002000001500001000005000058CH0165152l15I 1T58CH,1<:::227201 2569 20922 2•8 406200 250 300 350 400m/z16000013 15000014000003000011000010000010000090000ABU 70000ND60000A 50000NC 40000E300002000010000173159110LL” &1 1417330 SOC iOo237193- 267 20650 100 150 200 350 200 230m/z83The fragmentation of N-demethyl DPHM resulted in a mass spectrum which was similarto that of DPHM, that is, predominant ions with a m/z of 165 and 167 were observed (Figure 9).In addition, this metabolite was found to co-elute with DPHM on a cross linked 5%phenylmethyl silicone coated capillary column, resulting in the inability to reliably quantitate theparent drug in the presence of the metabolite. To remedy this problem, a capillary column with aliquid phase coating of 7% cyanopropyl:5% phenylmethylsilicone (DB-1701) was chosen. Onthis column, the N-demethyl metabolites eluted between the internal standard andDPHMJ[2H10]DPHM. Optimal column performance for the chromatography of DPHM and[2H10]DPHM was found to occur at a helium carrier gas flow corresponding to a column headpressure of 15 P.S.I. To enhance sensitivity, the splitless mode of sample introduction waschosen. Purge time (i.e., the time during which the volatilized sample is introduced onto thecapillary column) of less than 1.25 minutes resulted in a decrease in sensitivity, while a purgetime greater that 1.5 minutes did not offer a substantial increase in sensitivity (Figure 10). Anoptimal purge time of 1.5 minutes was chosen. Inlet temperatures between 200 and 275° C didnot appear to affect the chromatography, or the apparent sensitivity of the assay method;therefore, an inlet temperature of 225° C was used. In contrast to inlet temperature, initialcolumn temperature did have a substantial effect on the chromatography of DPHM and{2H10]DPHM (Figure 11). Temperatures below 140° C were found to result in wide peaks,complex (i.e., dirty) chromatograms, and decreased resolution between DPHM,[2H10IDPHM,the N-demethylated metabolites, and orphenadrine (the internal standard). An optimized initialcolumn temperature of 140° C was used for the remainder of the assay development.550000165 167167 0500000 165CH2NHCH3450000H_Cr400000 L350000300000V0- 2500002 00000 1521831500001000007750000105139207 3540 11L [ j - 253 2i 328 405 446I’ I’ ‘I50 100 150 200 250 300 350 400m/zFigure 9: The mass spectrum and mass assignments of N-demethyl DPHM followingGC-MS with electron impact ionization (70 eV).851.000.80U,4-C 0.600=0.40I0.200.000.00 0.50 1.00 1.50Purge Activation Time (minutes)Figure 10: The effect of varying inlet purge times on total area counts of DPHM.The optimized chromatographic parameters and temperature program used for the assayresulted in retention times for DPHM and[2H10]DPHM of 7.67 and 7.64 minutes, respectively.The internal standard (orphenadrine) eluted at 8.05 minutes. This temperature program resultedin a total analysis time of 12.7 minutes.3.1.1.2. Optimization of the Extraction Procedure for the DPHM and[2H10]DPHMAnalysis Method.The choice of the optimum solvent system for the extraction for the DPHM and[2H10]DPHM from biological samples was made on the basis of relative extraction efficiency,selectivity (lack of interfering chromatographic peaks), and ease of extraction. Both methylenechloride and 98% hexane: 2% isopropyl alcohol showed greater relative extraction efficienciesthan either toluene or hexane (Figure 12). The 2% isopropyl alcohol: 98% hexane mixtureappeared to be more selective than methylene chloride, since the chromatographic baselines of86blank biological matrices extracted with the former solvent were free from interfering peaks. Inaddition, the extraction conducted with the isopropyl alcohol:hexane mixture was easier, sincethe organic layer requiring transfer was on top of the aqueous layer, unlike with methylenechloride. The addition of 0.05 M TEA to the extraction solvent resulted in a large increase in therelative extraction efficiency (i.e., a 4 fold increase) (Figure 12). Further increases in eitherisopropyl alcohol or TEA concentration in the extraction solvent did not result in an additionalimprovement in efficiency, nor did the use of silanized glassware.—.-—DPHM —o- [2HO]DPHM1.00 -0.900.800.70 -E 0.60 -0.500.400.30 -G) 0.20 -00.10 -0.00 I I50 100 150 200Initial Oven Temperature (°C)Figure 11: The effect of varying the initial column temperature on the half-height peakwidth of DPHM.0087— No TEA 0.05M TEAMeCI2 Toluene Hexane Hexane + 2%IPAFigure 12: The relative extraction efficiency (peak area counts for DPHM) of differentsolvents for the extraction of DPHM and[2H10]DPHM from ovine plasma2502000)—15010The effect of mixing time on the relative extraction efficiency of DPI{M with 0.05 MTEA in 2% isopropyl alcohol: 98% hexane was examined. The peak area counts of DPHMincreased from 5 minutes to 15 minutes, but did not increase further following 15 minutes ofextraction. An optimal mixing time of 20 minutes was chosen for the extraction of DPHM and[2H10]DPHM.The optimized GC-MS parameters, in addition to the optimized extraction procedure(Figure 13), resulted in an analytical method which was free from chromatographic interferenceresulting from co-extracted endogenous materials (Figure 14). In addition, this method providedthe necessary sensitivity and selectivity to quantitate both DPHM and[2H10]DPHM in biologicalsamples obtained from pregnant sheep (i.e., amniotic fluid, fetal tracheal fluid, and plasma).88BIOLOGICAL SAMPLE-Fetal or maternal plasma-Amniotic fluid-Fetal Tracheal fluid1.Make up to 1.0 mL with distilled water+Internal Standard (orphenadrine 200 ng)+0.5 mL iN NaOH+7.0 mL organic solvent(0.05 M triethylamine in 2% isopropyl alcohol:98% hexane)Mix 20 minutes‘IFreeze 10 minutes at -20° C1Centrifuge 10 minutes at 3000 x gOrganicAqueous(Waste)Evaporate to dryness under N2at 30° C.Reconstitute with 0.15 mL0,05M Triethylamine in toluene12.0 L for injection.Figure 13: Optimized extraction procedure for DPHM and[2H10IDPHM from biologicalfluids obtained from the pregnant sheep.PLASMAm/z165AMNIOTICFLUIDm/z165TRACHEALFLUIDm/z165A BII.S.HDPHM__jNSAMPLE___J.CARDL__E APLASMAmlz173AMNIOTICFLUIDmlz173TRACHEALFLUIDmlz173B U N D A2H1ODPHMN CSAMPLEESTANDARD__________________________......__.._._........__.._..._BLANKRETENTIONTIME(MINUTES)Figure14:IonchromatogramSforDPHM(nVz165)and[2HIO]DPHM(nt/z173)inplasma,fetaltrachealfluid, andamnioticfluid.Blanks,spikedstandards(2.0ng/ml ),andbiologicalsamplesareshown.903.1.1.3. Calibration Curve for the DPHM and[2H10]DPHM Assay.The calibration curves for DPHM and{2H10]DPHM showed good linearity over the rangefrom 2.0 ng/mL to 200.0 ng/mL in all of the biological matrices examined. A sample calibrationcurve from plasma is shown in figure 15. The coefficients of variation did not exceed 10%(C.V.) for each point of the calibration curve in plasma, fetal tracheal fluid, and amniotic fluid.The regression coefficients in plasma, fetal tracheal fluid, and amniotic fluid were, in mostinstances, greater than 0.999. The weighting function 1/Y2 was used. The regression equationfor DPHM was Y = 0.0049X + (-0.00 16), and for[2H10JDPHM was Y = 0.0033X + (-0.00 13).The slope of the DPHM calibration curve was greater than the slope for the[2H10]DPHMcalibration curve, reflecting the difference in the relative abundance of the fragment ions m/z 165and 173, respectively (Figure 8).DPHM o[2HIOJDPHM1.000.80- T.00.60- Tcc-r00.40 T0.20-00.00 r I I I0 50 100 150 200Amount Added (ng)Figure 15: Calibration curve for DPHM and[2H10]DPHM in plasma (Mean ± S.D.)913.1.1.4. Extraction Recoveries of DPHM and[2H10JDPHM.The extraction recovery of DPHM and[2H10]DPHM following liquid-liquid extractionfrom ovine plasma, fetal tracheal fluid, and amniotic fluid using 0.05 M TEA in 98% hexane: 2%isopropyl alcohol was nearly complete. No apparent concentration dependent changes in theextraction recoveries were noted, since the extraction recovery at the three differentconcentrations tested (i.e. 2.0, 50.0, and 200.0 ng/mL) were the same. The mean (± S.D.)recovery of DPHM and[2H10]DPHM from plasma was 98 ± 2 and 105 ± 3%, from amniotic fluidwas 100 ± 5 and 110 ± 6%, and from fetal tracheal fluid was 97 ± 5 and 104 ± 5%, respectively.3.1.1.5. Sample Stability Assessment of DPHM and[2H10]DPHM.The samples containing DPHM and[2H10]DPHM stored in the freezer at -20° C appearedstable for up to 12 months, since the concentrations of drug measured in these samples did notdeviate from the known concentration. Following three freeze-thaw cycles, no differences in theconcentrations of DPHM and[2H10]DPHM were noted between the control and freeze-thawsamples (Mann-Whitney U test, P>0.05) [See Figure 16]. The slope of the linear regression of themeasured concentration of plasma DPHM and[2H10]DPHM vs. time was not significantly differentfrom zero (Two Sample T-test; P> 0.05) following bench-top storage for up to and including 24hours (See Figure 17). The final sample stability test was conducted with the extracted samples onthe auto-sampler tray of the GC-MS.-JE0)CC0CuI.1CU)0C0C)DPHM [2H10]DPHM92Figure 16: The effect of three freeze-thaw cycles on the plasma concentrations ofDPHM and[2H10]DPHM in spiked plasma samples (Mean ± S.D., n=4).-JE0)CC0.1Cu1.1CU)0C0()Figure 17: The effect of prolonged bench-top storage (22° C) on the plasmaconcentrations of DPHM and{2HIOJDPHM in spiked plasma samplesCONTROL FREEZE-THAW. DPHM —0--- [2H10]DPHM7060504030201000 10 20Time (hours)30(n=2).93The extracted samples were periodically injected into the GC-MS during large sequence runs for aperiod of up to 72 hours. No differences in the ratios of DPHM and[2H10]DPHM were detected.3.1.1.6. Validation of DPHM and[2H10jDPHM Gas Chromatographic- MassSpectrometric Analysis Method.The validation of this method involved estimation of both intra- and inter-day variability.In addition, the assay was cross validated with a previously published method for the quantitationof DPHM (Yoo et al., 1986). The estimates of intra-day variability for DPHM and[2H10]DPHMwere below 17% at 2.0 ng/mL, and below 8% at all other concentrations examined in all threebiological matrices tested (Table 1). The measured inter-day variability for DPHM and[2H10]DPHM was below 15% at 2.0 ng/mL and below 10% for all other points (Table 2). Apublished GC-NPD method was used in the cross validation studies (Yoo et at., 1986). Theconcentrations of DPHM and[2H10]DPHM were determined independently, since the GC-NPDcould not differentiate between DPHM and[2H10]DPHM. When the concentrations of DPHMand[2H10]DPHM measured by the GC-NPD method were plotted against the concentrations ofDPHM and[2H10]DPHM independently measured by the GC-MS method, the correlation wasexcellent (r=1.000 DPHM and r=0.999[2H10]DPHM) (Figure 18).CID CDLI)0 0 CD C) CD Cl) 0 0HCD —— CD Cl) -t CD CD Cl) Cl) 0 C)—L’J’L’JL’JbbbbL\)-‘L’J—LJ—CC—C——a00&-t’) Cl)•t’J1+INJ-1+H-1+1+1+1+,-‘—c9Obpooo-it4--.it00———_,——00 —U00CCDöo1+1+H-c1+1+1+E.C0,_1+1+00LItC‘JCUi4—L’JD———-0k)—C—00Ui1+1+°i+I+0CD1+,;D00-—a00—_,———l)t-J—CCC0CCCbCCbCCbbbb——t-j1’-.)——tJ•—000•‘-4——aCl)1+bo•1+1+,-C.JiItObobLbo—s__—%__—%__L’J——————‘—C:c0..1+L—a,_1+1+1+1+1+C-DU--_4.‘_——s__LJ-———HCCboCC099”‘cPC9’04C1+1+ i—,--’.p)1+1+CCD‘———‘-—%__%____H CD0) N.‘-0952500( 200I250Concentration (ng/mL) [GC-NPDIFigure 18: The correlation between concentrations of DPHM and[2H10JDPHM measuredusing the GC-MS method and a previously developed GC-NPD methodThe minimal detectable concentration of DPHM and[2H10]DPHM was 0.5 ng/mL (6.7pcg of the analyte at the detector). This corresponds to a signal to noise ratio (SIN) of> 3. Theminimal quantifiable concentration was 2.0 ng/mL (i.e., 27.6 pcg of the analyte at the detector).The minimal quantifiable concentration also fell within the acceptable limits of inter- and intraday variability (i.e., <20% relative standard deviation for the lowest concentration) (Shah et al.,1992).0 50 100 150 200963.1.2. Capillary Gas Chromatographic- Mass Spectrometric Analysis of DPMA and[2H10]DPMA3.1.2.1. Optimization of Mass Spectrometer and Gas ChromatographThe mass spectra of the tert-butyldimethylsilyl (TBDMS) derivatives of DPMA and[2H10IDPMA following GC-MSI.EI showed extensive fragmentation, resulting in numerous smallfragment ions with no molecular ion present at m/z 356 for DPMA and at mJz 366 for[2H10]DPMA (Figure 19). The prominent ions which also retained the stable isotope label for theTBDMS derivatives of DPMA and[2H10]DPMA were m/z 167 and 183, and m/z 177 and 193,respectively. Fragment ions of low intensity corresponding to [M-57r or the loss of the ten’butyl group from the derivatized DPMA and[2H10]DPMA (i.e., mlz 299 and 309) were alsoobserved (Figure 19). The fragmentation of the tert-butyldimethylsilyl derivative of the internalstandard, DPAA, resulted in a base fragment ion [M-57] of m/z 269, and a smaller fragment ionat m/z 165. SIM was used to optimize both the sensitivity and selectivity of this assay. Initiallyfragment ions m/z 183 and 193 were monitored for the quantitation of DPMA and[2H10]DPMA,respectively. However, the fragmentation of DPMA also yielded a small fragment at m/z 193which resulted in chromatographic interference when measuring[2H10JDPMA, particularly atlower concentrations. Next, the ion pair m/z 167 and 177 was selected for SIM quantitation. Theion chromatogram at m/z 177 resulted in a clean chromatogram at the retention time for[2H10]DPMA. On the other hand, the ion chromatogram for m/z 167 resulted in considerableinterference from co-extracted components in adult sheep urine, which could not be eliminatedeither through GC oven programming or sample clean-up using liquid-liquid extraction. Thus,the fragment ions mlz 183 and 177 were monitored for DPMA and [2HLOJDPMA, respectively.Although the fragment ion m/z 269 was much more prominent than others in the mass spectrumof the internal standard, DPAA, there was considerable interference in the ion chromatogram.Thus, the fragment ion m/z 165 was monitored for the quantitation of DPAA.97299 30963 193167 1 (I ‘H5H3‘77CH3/r251H—C S—CH3H—C2LdLLH5 L169173 57 173 575000000 1 7 177I 19)16) 45000004100000400000040000052500000250000020000000j 3000000c_I (30cO-c t 25000000 25000001 0ooooooo1 3000000150000015000001000000 153 1000000500000 5 500000_r4_I1L.;_l_i_..4_,i_.2092990 . 1, I - 20357 261 24604 576____________________________56 56 125 010 .._ 3)1 555 )7_______________________________________________________________—50 100 150 200 250 300 150 50 00 150 200 250 100flhJZ m/zFigure 19: The mass spectra of the TBDMS derivatives of DPMA and[2H10JDPMA and the mass fragment assignments following GC-MSwith electron impact ionization (70 eV) of the standards.98The optimization of the mass spectrometer tuning was achieved using the manual tuning optionto select the fragment ions m/z 100, 131, and 219 of PFTBA. This further enhanced thesensitivity of detection of DPMA and[2H10]DPMA to the level required for the assay. The dwelltime for SIM analysis was set a 125 milliseconds to provide at least 15 scans perchromatographic peak.As with the previous analytical method, two columns were examined for optimumchromatographic resolution between the analytes (i.e., DPMA,[2H10IDPMA, and DPAA) andendogenous co-extracted components. The Ultra-2 column provided optimum peak shape andresolution of the analytes from endogenous components. The peak shapes of DPMA and[2H10JDPMA were improved, and the peak width reduced by increasing the helium column headpressure from 7.5 to 15.0 P.S.I. (i.e., increasing linear gas velocity). Increasing the initial columntemperature from 150° C to 200° C resulted in shorter elution times for DPMA and[2H10]DPMA(i.e., 13.5 vs. 8.5 minutes); however, significant peak broadening and tailing was observed. Anoptimum initial column temperature of 150° C was thus chosen. Increasing the injectortemperature did not result in significant increases in the peak areas, but helped to overcome a“carry-over” phenomenon, which was noted following the injection of MTBSTFA derivatizedDPMA and[2H10]DPMA at lower injector temperatures. The injection of blank toluene wouldnot result in carry-over; however, the injection of toluene treated with MTBSTFA resulted in aghost-peak of DPMA and[2H10]DPMA. This problem was overcome by increasing the injectortemperature from 225 to 280° C. These GC-MS conditions resulted in ion chromatograms freefrom interference from co-eluting peaks in urine and plasma. The ion chromatograms of plasmaand of urine spiked with 250 ng/mL each of DPMA,[2H10]DPMA, and 400 ng/mL of DPAA andthe corresponding blank matrices can be seen in figure 20. It should be noted that the Y-axis forthe blank biological samples is amplified to better show the ion chromatograms of the extractedblank matrices.Abundonce200000PlasmaspikedIon:mhz165Abundance200000Urinespikedion:maIl165150000110000100000DPAA(IS.)0DPAA(1.S.)I1000005000050000__________________________________________________A_____________________________________________________________________________________010.5011.0011.0012.0012.0013.0010501100115012001250110050000000Time(minutes)0BlankPlasmaTime(minutes)110.5011.0011.5012.0012.5013.0010.5011.0011.5012.0012.0013.00Abundance200000Plasmaspikedat250.0ng/mLIon:mhz183Abundance200000Urinespikedat250.0.ng!mLIon:m1z183DPMA150000DPMA15000010000010000050000500000010.5011.0011.5012.0012.5013.0010.5011.0011.5012.0012.50-13.0050005000BlankUrineTime(minutes)PlasmaTime(minutes)0110.5011.0011.5012.0012.5013.0010.5011.0011.5012.0012.5013.00AbundanceoooooPlasmaspikedat250.0ng/mLIon:m7z177Abundance200000Urinespikedat250.0n/mLIon:m/z177150000100000100000150000(2{DPMA12H0}DPMA5000050000 C0In10.5011.0011.5012.0012.5013.0010.5011.0011.5012.0012.5013.0050005000rifleTime(minuteS)0LPlasmaTime(minutes)ankUI10011.5012.0012.5013.00105011.0011.5012.0012.0013.00Figure20:Ionchromatogramsof mlz165(internalstandard),183(DPMA),and177([2H10]DPMA)inblankplasmaandurine,andplasmaandurinespikedwith250.0ng/rnLofDPMA,250ng/mLof[2H10]DPMA, and400ng/mLoftheinternalstandardDPAA.l.S.-InternalStandardforanalysismethod*Note:They-axisscalingisincreasedintheblanktoshowmoreclearlythebase-lineof theionchromatograrn.1003.1.2.2. Optimization of Extraction and Derivatization Procedures for DPMA and[2H10JDPMA.Ethyl acetate and toluene resulted in similar relative extraction efficiencies for DPMAand[2H10]DPMA, but the ion chromatograms were cleaner following toluene extractioncompared to ethyl acetate. Although toluene resulted in greater emulsion formation followingmixing compared to ethyl acetate, toluene was subsequently used as the optimized extractionsolvent due to the ease of “cracking” the emulsion upon cooling. The mixing time wasoptimized to 20 minutes. DPMA and[2H10JDPMA were derivatized with both PFBBr orMTBSTFA to form PFB or TBDMS derivatives, respectively. The TBDMS derivatives providedbetter response using El mass spectrometry, while the PFB derivatives provided better responsesin NCI mass spectrometry. TBDMS derivatives were utilized because GC-MS/EI was to be usedfor analysis, and furthermore, the derivatization procedure was rapid and easier. No difference inpeak area response was noted with the different derivatization incubation times (i.e., 30, 60, and90 minutes) at 60° C, thus, a time of 60 minutes was chosen as optimal. In addition, withdiffering volumes of MTBSTFA (i.e., 10, 25, 50, and 100 jiL), the peak areas were equivalent,suggesting that the volume of derivatizing agent did not influence the sensitivity of the method.The volume ultimately chosen for the remainder of the assay development was 25 pL. Theoptimized extraction and derivatization procedure for DPMA and[2H10]DPMA is shown infigure 21.101Biological Sample-fetal or maternal plasma- fetal or maternal urineMake up to 1.0 mL with distilled water1Add internal standard (400 ng) DPAAAdd 0.4 mL 1.0 M HC1Add 5.0 mL Toluene1Mix for 20 minutes1Freeze for 10 minutes at -20°C1Centrifuge for 10 minutes at 3000 X gDiscard Aqueous layer Transfer organic layer to clean test tubeIEvaporate to dryness under a gentleN2 stream at 40 °C.IReconstitute with 200 i.iL dry toluerieAdd 25 jiL MTBSTFACap and vortex mix for one minute1Incubate at 60° C for one hourI1.0 tL for injectionFigure 21: The optimized extraction scheme for DPMA and[2H10]DPMA from ovineplasma and urine.1023.1.2.3. Calibration Curve for DPMA and[2H10]DPMAThe calibration curves for DPMA and[2H10]DPMA were linear over the concentrationrange examined (i.e., 2.5 to 250.0 ng/mL). Weighted linear regression was carried out using theweighting function of l/Y2 to reduce the bias of the interpolated concentrations at the low end ofthe assay. The resulting equations describe the linear regression for DPMA: Y=0.0088X +0.0009; r2 = 1.000, and{2H10]DPMA: Y=0.0095X + (-0.0013); r2 = 1.000 (Figure 22). Theseregression constants resulted in a -9% bias at 2.5 ng/mL and +1% at 250.0 ng/mL for DPMA,and a ÷4% bias at 2.5 ng/mL and a +1% bias at 250.0 ng/mL for{2H10]DPMA. The minimumquantifiable concentration of this analytical method was 2.5 ng/mL (i.e., 11 pcg at the detector),which corresponds to a signal-to-noise ratio of 15 for DPMA and 20 for[2H10]DPMA.• [2H10]DPMA o DPMA2.502.00 T•1.501.0060.50e00.00 p0 I I I0 50 100 150 200 250Amount Added (ng)Figure 22: Calibration curve for DPMA and[2H10IDPMA extracted from ovine plasma.1033.1.2.4. Extraction Recovery of DPMA and[2H10]DPMAThe mean (± S.D.) extraction recoveries at various concentrations of DPMA and[2H10]DPMA from plasma were 78 ± 5% and 86 ± 11% at 5.0 ng/mL, 78 ± 1% and 75 ± 2% at50.0 ng/mL, and 77 ± 2% and 74 ± 1% at 500.0 nglmL, respectively. In urine, the extractionefficiency for DPMA and[2H10]DPMA was 95 ± 6% and 99 ± 11% at 5.0 ng/mL, 73 ±9% and74± 14% at 50.0 ng/mL, and 79± 2% and 74 ± 2% at 500.0 ng/mL, respectively.3.1.2.5. Sample Stability AssessmentPlasma samples containing DPMA and[2H10JDPMA appeared to be stable when storedfrozen for up to a period of 6 months. Following three freeze-thaw cycles, the concentration ofDPMA and[2H10]DPMA did not differ significantly from the control values (Mann-Whitney Utest; P > 0.05). Moreover, the slopes of the linear regression of the measured concentration ofDPMA and[2H10]DPMA in plasma vs. time were not significantly different from zero (TwoSample T-test; P> 0.05) following bench-top storage for up to and including 24 hours. However,DPMA was labile when stored in an acidified sample matrix for a prolonged period of time. Thecalculated first order degradation half-life was 16.5 hours in water, 23.7 hours in blank plasma,and 33.6 hours in blank urine matrices (Figure 23). The area ratios of DPMA and{2H10]DPMA(i.e., DPMAJDPAA or[2H10]DPMA/DPAA) did not change for up to 96 hours of storage ofprepared samples on the auto-sampler tray at room temperature (22° C).104o [2H1OJDPMA - Water10000•• DPMA- Watero [2H10]DPMA- Urine• DPMA- Urine.2 o [2H10]DPMA- Plasma100 I0 4 8 12 16 20 24Time (hours)Figure 23: Degradation of DPMA in acidified (0.4 mL of 1.0 M HC1) water, plasma andurine matrices (i.e., pH < 1.2).3.2.1.6. Method Validation for DPMA and[2H10]DPMA AnalysisThe results of the intra- and inter-day variability studies for this analytical method areshown in table 3 and 4, respectively. The estimates of intra-day variability for DPMA and[2H10jDPMA were below 16% at the minimum quantifiable concentration of 2.5 ng/mL, andbelow 5% at all other concentrations investigated in plasma and urine (Table 3). The measuredinter-day variability for DPMA and[2H10jDPMA was below 10% at the minimum quantifiableconcentration of 2.5 nglmL and below 8% for all other points (Table 4).105Table 3: Intra-day variability of DPMAI[2H10]DPMA assay method in plasma and urine.Mean measured concentrations with standard deviation (SD) (n=4).Plasma UrineDPMA I [2H10]DPMA DPMA I[2H10JDPMA2.5 nglmLMean 2.35 2.24 2.19 2.31S.D. 0.23 0.06 0.35 0.13C.V. (%) 9.6 2.9 15.9 5.810.0 ng/mLMean 8.54 8.26 8.80 9.25S.D. 0.09 0.16 0.29 0.33C.V. (%) 1.0 1.9 3.3 3.650.0 ng/mLMean 44.1 42.4 47.3 48.0S.D. 0.4 0.7 1.0 1.2C.V. (%) 1.0 1.6 2.1 2.5250.0 ng/mLMean 235.4 219.7 251.5 249.1S.D. 2.2 3.5 11.7 11.7C.V. (%) 0.9 1.6 4.7 4.7Table 4: Inter-day variability of DPMAJ[2H10]DPMA assay method in plasma and urine.Mean measured concentrations with standard deviation (SD) (n=4).Plasma UrineDPMA I [H1o]DPMA DPMA I[2H10]DPMA2.5 ng/mLMean 2.30 2.39 2.25 2.51S.D. 0.05 0.19 0.08 0.18C.V. (%) 2.2 8.1 3.6 7.210.0 ng/mLMean 9.24 8.65 8.93 9.17S.D. 0.59 0.34 0.32 0.11C.V. (%) 6.4 3.9 3.6 1.250.0 ng/mLMean 46.5 45.5 47.3 47.8S.D. 2.5 2.9 0.2 0.5C.V. (%) 5.4 6.3 0.3 1.1250.0 ng/mLMean 250.8 247.8 253.0 253.1S.D. 11.2 19.1 2.3 4.2C.V. (%) 4.4 7.7 0.9 1.61063.2. Animal ExperimentationThe experimental details for all of the animal experiments conducted in this thesis areshown in Appendix 13.2.1. Disposition of DPH1’vl in Non-Pregnant Sheep.Initial experiments conducted to assess the renal contribution to the total body clearanceof DPHM were carried out in five non-pregnant ewes with a mean (± SEM) weight of 73.7 ± 1.6Kg (Appendix 1). Following a 100 mg IV bolus dose via the femoral vein of a non-pregnantsheep, the femoral arterial plasma concentrations of DPHM declined rapidly in a bi-exponentialfashion. The mean (± SEM) femoral arterial plasma concentrations following drugadministration are illustrated in figure 24. The mean total body clearance of DPHM in thesenon-pregnant sheep was 53.0 ± 10.4 mL/minute/Kg (Table 5). The mean (± SEM) cumulativerenal excretion of the drug is shown in figure 25. The renal clearance, calculated by dividing thecumulative amount of DPHM excreted in the urine by the AUC may be an underestimation,since an accurate estimate of ZXu is not always possible because urine was only collected for upto 12 hours in four of the five animals, and for 24 hours in the remaining ewe. In this latteranimal, if urine collection had been stopped at 12 hours, Xu would have been underestimatedby 7%; therefore, the values of renal clearance presented in table 5 likely fall within a 10%error. The average renal clearance was 0.36 ± 0.28 mL/minlKg, which represents approximately0.3% of the total body clearance of the drug.107Bile was not collected continuously, thus, it is not possible to estimate by direct meansthe biliary secretion of DPHM. The concentrations of DPHM in bile following a 100 mg IVfemoral venous bolus dose in E#248 and E#617 are shown in table 6.Table 5: Pharmacokinetic Parameters following a 100 mg IV bolus of DPHM via the femoralvein in non-pregnant ewes.E#543 E#248 E#3 16 E#105 E#617 Mean ± SEMParameteroc(min’) 0.095 0.590 0.182 0.033 0.040 0.22 ± 0.08B(min1) 0.010 0.018 0.015 0.010 0.011 0.012±0.001-T112 (mm) 7.3 1.2 3.8 21.0 17.3 10.1 ± 3.9B-T112 (mm) 69.3 38.2 46.2 69.3 63.0 57.3 ± 6.3CLT (mL/minlKg) 35 70 87 35 38 53.0 ± 10.7Vd (L/Kg) 4.34 2.89 5.00 2.25 2.47 3.4 ± 0.5MDRT (mm) 123.9 41.0 57.5 65.1 64.8 70.5 ± 14.0CLrenai (mL/minlKg) 0.10 0.03 0.17 1.45 0.03 0.36 ± 0.28% DOSE (Renal) 0.3 0.1 0.2 1.3 0.1 0.40 ± 0.23Table 6: Biliary concentrations of DPHM obtained from periodic collections (i.e., 10 minutecollections) of bile from two non-pregnant ewes following a 100 mg femoralvenous bolus dose of DPHM.E#617 E#248Time (mm) [Bile] ng/mL [Bile]/[Plasma] [Bile] ng/rnL [Bilej/[Plasma]15 922 1.7 579 2.830 492 1.6 319 1.945 302 1.3 170 1.060 190 1.0 126 1.2120 25 0.4 94 1.3180 nd 45 1.2108-JE 1000’rCCo _i_.-I-iooC.I1C.) •C .0C.)E 10001 I I I I0 60 120 180 240 300 360Time (minutes)Figure 24: Mean femoral arterial plasma concentrations (± SEM) of DPHM following a 100mg IV bolus via the femoral vein in adult non-pregnant sheep (n=5).— 1000’ 1.000)900’a)C. cnn. nort a)D.E 700 D600’ T 0.60T500’T—400’ 1 0.40 a)o I I I (0E I 0< 300’ II0.1 0, 200 I I 0.20111100o I I I I ooo0 120 240 360 480 600 720Time (minutes)Figure 25: The mean cumulative amount (± SEM) of DPHM excreted in the urine following a100 mg IV bolus dose of DPHM via the femoral vein in non-pregnant adult sheep(n=5).1093.2.2. Isotope Effect Studies3.2.2.1. Adult Non-Pregnant SheepControl experiments were conducted to assess the presence of any possible isotope effectsin the pharmacokinetic disposition of[2H10jDPHM. These experiments were conducted in 2non-pregnant ewes. Figure 26 shows a representative plot of the femoral arterial plasma drugconcentrations following equimolar doses of DPHM HC1 and[2H10]DPHM HC1 equivalent to100 mg of total DPHM free base (i.e., DPHM +[2H10]DPHM) in ewe 2169. The correspondingplot of the cumulative amount of DPHM and[2H10]DPHM excreted in urine is shown in figure27. The pharmacokinetic parameter estimates for the two animals are shown in table 7. In eachanimal there were no apparent differences between DPHM and[2H10]DPHM arterial plasmaconcentrations and the pharmacokinetic estimates. In addition, the representative plot showingthe plasma concentrations of the metabolites (i.e., DPMA and[2H10IDPMA) is shown for ewe2169 in figure 28, with the corresponding plot of the cumulative amount of these metabolitesexcreted in urine in figure 29. Thus, it would seem that the pharmacokinetic equivalence of[2H10]DPHM to DPHM also extends to this metabolic pathway. Overall, there do not appear tobe differences between[2H10jDPHM and DPHM in the parameters examined in this study.110Table 7: Pharmacokinetic parameters of DPHM and[2H10]DPHM following simultaneousIV bolus administration of equimolar amounts* of DPHM HC1 and[2H10JDPHMHC1 via the femoral vein in two non-pregnant ewes.EWE2167 EWE2169Parameter DPHM [2H10JDPHM DPHM [2H10JDPHMo (min’) 0.307 0.313 0.331 0.3313 (min’) 0.022 0.022 0.044 0.044xT112 (mm) 2.3 2.2 2.1 2.1I T112 (mm) 31.5 31.5 15.8 15.8CLT(mL/min) 4000 3900 2700 2700CLT (mLfminlKg) 60 60 40 40Vd88 (L) 176.7 172.9 68.2 68.6Vd (L/Kg) 2.49 2.44 0.97 0.98Vd (L) 186.5 179.6 61.8 62.1Vd(L/Kg) 2.63 2.53 0.88 0.89MDRT mm 43.6 44.1 24.3 24.9AUC0_co(ng*minJmL) 12499.6 12921.7 18252.8 18545.3AUC0—oo(DPHM)/ 0.97 1.02AUC0-co([2Hio]DPHM)Mean [Cp DPHM/Cp 0.99±0.04 0.98±0.07{2H10]DPHM]* Total dose (i.e., DPHM +[2H10]DPHM) is equivalent to total dose of 100 mg DPHM free base.Weight corrected parameters based on the weights at the time of arrival to research facility.111DPHM • [2H1OIDPHM-JE0 1000CC04-.Cu100Ca,0C0() SCu 10E00Cu 1 I• 0 60 120 180 240 300 360Time (minutes)Figure 26: A plot of the representative plasma concentrations of DPHM and[2H10]DPHM following an equimolar dose of DPHM HC1 and[2H10]DPHMHC1 administered simultaneously via the femoral vein to ewe 2169.(Isotope effect study).Total dose equivalent to 100 mg of DPHM (i.e., DPHM +[2H10]DPHM)free base.112—s— DPHM - -. -[2H1OJDPHM100 0.2090C a)- 80 0.16-D/*Ia.. 60 0.12 a,50Ui40 0.08 a,o U)E 0< 30a, T20 0.04io0 240 480 720 960 1200 1440Time (minutes)Figure 27: A representative plot of the cumulative amount of DPHM and[2H10]DPHM in urine following an equimolar dose of DPHM HC1 and10]DPHM HC1 administered simultaneously via the femoral vein to ewe2169. (Isotope effect study).Total dose equivalent to 100 mg of DPHM (i.e., DPHM +[2H10]DPHM)free base.-JEC)CC0.Icu.1-0Cci0C0()CuECl)cu0Cua)IFigure 28: A representative plot of the plasma concentrations of DPMA and[2H10]DPMA following an equimolar dose of DPHM HC1 and10]DPHM HC1 administered simultaneously via the femoral vein to ewe2169. (Isotope effect study).Total dose equivalent to 100 mg of DPHM (i.e., DPHM +[2H10]DPHM)free base.E DPMA113——-[2H10]DPMA100101—.0 120 240 360 480 600Time (minutes)720114—o---DPMA--• -[2H10]DPMA1000-.1200100..E 0 p I I I0 240 480 720 960 1200 1440Time (minutes)Figure 29: A representative plot of the cumulative amounts of DPMA and[2H19]DPMA in urine following an equimolar dose of DPHM HC1 and10]DPHM HC1 administered simultaneously via the femoral vein to ewe2169. (Isotope effect study).Total dose equivalent to 100 mg of DPHM (i.e., DPHM +[2H10]DPHM)free base.1153.2.2.2. Fetal Lambs3.2.2.2.1. Bolus StudiesIsotope effect studies were conducted on 5 fetal lambs following IV bolus administration(Appendix 1). The mean (± SEM) weight of the maternal sheep used in these experiments was72.8 ± 3.7 Kg, while the mean fetal weight was 2.3 ± 0.3 Kg. The mean gestation age was 130 ±1.8 days (Term 145 days). The total drug (DPHM +[2H10]DPHM) administered averaged 4.25 ±0.93 mg/Kg of fetal body weight. The femoral arterial plasma concentrations of DPHM and[2H10JDPHM in a representative fetal lamb (E#975) following a simultaneous administration ofequimolar amounts of DPHM and[2H10]DPHM are plotted in figure 30, while table 8 gives thepharmacokinetic parameters calculated for the two forms of the drug. No differences wereapparent between the plasma concentrations of DPHM and[2H10]DPHM in each animal. Inaddition, there were no differences between the AUC0ratios for the labeled and unlabeleddrug (Paired Sample T-test P>0.05). Further, no significant differences were noted between thepharmacokinetic parameters (i.e., o, f3, CLT, Vd, and MDRT) of DPHM and[2H10]DPHM(Paired Sample T-test, P>0.05). A representative plot showing the concentrations of DPHM and[2H10]DPHM for E#975 in amniotic and fetal tracheal fluids is shown in figure 31. These datasuggest that significant pharmacokinetic isotope effects do not exist for[2H10JDPHM in the fetallamb following bolus administration over a 1 minute interval.116Table 8: Pharmacokinetic parameters of DPHM and{2H10IDPHM following simultaneousadministration of equimolar amounts of DPI{M HCI and[2H10]DPHM HC1 via thefetal lateral tarsal vein (fetal isotope effect study).MeanSEM[H10]DPHME#207E#1 143E#975E#1 02E#1 124‘ 0.2500.0900.0360.004706541181467652180031322823827.32.6c. 13 AUC CLT Vd Vd MDRTDPHM (min’) (min1) (ng*minlmL) (mL/minlKg) (L/Kg) (L/Kg) (mm)E#207 0.195 0.038 1514 1820 45 48 24.6E#1 143 0.196 0.043 6825 300 6 7 20.6E#975 0.615 0.042 23091 100 3 3 25.1E#102 0.092 0.035 1618 1100 32 31 30.1E#1124 0.167 0.024 2275 600 24 27 35.9Mean 0.180 0.036 7154 800 23 25 27.6SEM 0.050 0.003 4216 313 8 9 3.00.1360.1400.3670.1060.1560.0360.0400.0390.0390.024227001571214119003001001100700457332275183322923.921.924.628.938.5values are reported as mean ± SEM*weight corrected values are based on fetal body weight at the time of the experiment.117DPHM • [2H10]DPHM-jE0) 1000•C0CuI.()Cu 10EC,)CuCu 10 120 240 360Time (minutes)Figure 30: A representative figure of the arterial plasma concentration of DPHM and[2H10jDPHM following a simultaneous IV bolus dose of DPHM and[210JDPHMvia the fetal lateral tarsal vein (E#975).118o DPHM —b--[2H10JDPHM —v- DPHM - -V- [2H10]DPHMAMN AMN TR TR1000--JE1000 120 240 360Time (minutes)Figure 31: A representative plot of the amniotic and fetal tracheal fluid concentrations ofDPHM and[2H10jDPHM following simultaneous administration of DPHMand[210JDPHM via the fetal lateral tarsal vein (E#975).AMN - amniotic fluid and TR - fetal tracheal fluid1193.2.2.2.2 Infusion StudyTwo experiments were conducted to examine the disposition of DPHM and[2H10JDPHMfollowing simultaneous infusion of equimolar doses of DPHM and[2H10JDPHM (60 JIg/minuteof each DPHM and[2H10]DPHM) via the fetal lateral tarsal vein for 90 minutes. Theseexperiments were conducted to check for differences in the disposition of the labeled drug in thefetal lamb following longer term drug administration. The concentrations of DPHM and[2H10]DPHM in fetal and maternal plasma, urine, and amniotic fluid, and, in addition, the plasmaconcentrations of DPMA and[2H10]DPMA were measured and are illustrated in figures 32-35,respectively. The mean steady-state concentrations of DPHM and[2H10]DPHM were 275.6 ±16.2and272.8± 15.7ng/mLforewe224l,and95.5± 1.1 and95.5± l.8ng/mLforewe2l8l,respectively (Figure 32). In addition, no apparent differences in the plasma concentrations ofDPHM and{2H10]DPHM were observed in maternal plasma, amniotic fluid, and fetal urine(Figure 32-34). DPMA and[2H10]DPMA steady-state concentrations were not achieved duringthe course of the infusion. However, there were no apparent differences between theconcentrations of the labeled and unlabeled form of the metabolite (Figure 35). Overall, the datado not indicate any significant isotope effect with labeled forms of DPHM and DPMA in thefetal lamb following a 90 minute infusion.120E#2181 E#2241__• DPHM-FA __O_[2Hio1DPHM..FAI_e_ DPHM-FA - - -[2H10JDPHM-FAL—. —DPHM-MA —L -[2H10]DPHMMAj-_A-_ DPHM-MA —1000Co4-ICu.4 IFj___________________lUllCa)C)C0C-)cu 10EU)cu0Cu 1 I30 60 90Time (minutes)Figure 32: Plasma concentrations of DPHM and[2H10]DPHM in fetal and maternal plasma inewes 2181 and 2241 following simultaneous infusions of 60 gig/mm of each DPHMand[H10]DPHM via the fetal lateral tarsal vein.* *Note: fetal arterial plasma concentrations of DPHM and[2H10jDPHM aresuperimposed.E#2181 E#2241 121a>Da>4-.a>0xwC0E—Time (minutes)Figure 33: Amount of DPHM and{2H10IDPHM in fetal urine in ewes 2181 and 2241following simultaneous infusions of 60 ig/min of each DPHM and[2H10]DPHM via the fetal lateral tarsal vein.E#21 81 E#2241DPHM I2HIOIDPHM DPHM [2H10 IDPHMFigure 34: Concentrations of DPHM and[2H10]DPHM in amniotic fluid in ewes 2181 and2241 following simultaneous infusions of 60 ig/min of each DPHM and[H10]DPHM via the fetal lateral tarsal vein.E[2HIQ1DPHM — DPHM (2H10 JDPHM5000400030002000-1000-030 45 60 75 9050-JEC) 40CC30cI-4-C20C000)0 1H [LI mu Fir [ir30 45 60 75 90Time (minutes)122E#2181 E#2241—--—DPMA _A_E2HioJDPMAj_o_DPMA- •-[2HQ]DPMA-J IEo 1OO—-z--0—Time (minutes)Figure 35: Concentrations of DPMA and[2H10]DPMA in fetal plasma in ewes 2181 and2241 following simultaneous infusions of 60 pg/mm of each DPHM and[H10]DPHM via the fetal lateral tarsal vein.1233.2.3. Hepatic First-Pass Metabolism Studies3.2.3.1. Non-Pregnant Sheep3.2.3.1.1. Mesenteric Bolus Administration in Normoxic ConditionsThe experimental details of this study are given in Appendix 1. The mean (± SEM)weight for the non-pregnant ewes used in first pass experiments was 70.2 ± 3.8 Kg. The meantotal dose administered (DPHM +[2H10]DPHM) was 1.64 ± 0.11 mg/Kg. The control (i.e., priorto experimentation) blood gas parameters were: pH, 7.43 ± 0.01; Po2, lii ± 8.6 mm Hg; andPco2, 41.3 ± 1.3 mm Hg, and experimental values were 7.43 ± 0.01, 107 ± 2.8 mm Hg, and 41.6± 0.1 mm Hg, respectively. There were no apparent differences in any of these parametersbetween the samples obtained during the control period and the samples obtained during theexperiments.In three of the ewes studied,[2H10JDPHM was injected via the mesenteric vein andDPHM was simultaneously administered via the femoral vein, while in the other two animals theorder was reversed to further minimize the likelihood of unmeasured isotope effects affecting ourdata (Appendix 1). A representative plot of the disposition of the two forms of the drugfollowing the simultaneous administration of DPHM via the mesenteric vein (portal vein) and[2H10]DPHM via the femoral vein (systemic) is shown in figure 36. The disposition of[2H10jDPHM following administration via the femoral vein showed a hi-exponential decline.The apparent hepatic first-pass effect of DPHM was extensive since the plasma concentrations ofDPHM following mesenteric venous administration were approximately ten fold lower than theplasma concentrations of the drug following femoral venous injection. Although the extractionfollowing mesenteric venous administration was extensive, in most cases the plasma levels were124high enough to permit pharmacokinetic modeling, and thus, the calculation of the AUC0.However, in one animal (E#989), DPHM could be detected in the plasma following mesentericadministration (i.e., at 5 and 10 minutes post dose), but the plasma levels were too low to permitpharmacokinetic assessment. The pharmacokinetic parameters are shown in table 9. Systemicbioavailability (F) averaged 0.068 (95% confidence interval: 0.026 <X 0.111) whichcorresponds to an extraction ratio (E=1-F) of 93.2 ± 1.4%.Table 9: Pharmacokinetic parameters obtained from non-pregnant sheep during a mesentericfirst-pass metabolism study following the simultaneous administration of DPHM or[2H10jDPHM via the femoral or mesenteric vein (n=5).Parameter E#139 E#1154 E#1158 E#989 E#102 Mean±SEMa. min’ 0.103 0.33 1 0.196 0.200 0.18 1 0.20 ± 0.0313 min’ 0.039 0.044 0.023 0.023 0.049 0.036 ± 0.005a.T112min 6.7 2.1 3.5 3.5 3.8 3.9±0.813 T112min 16.8 15.9 30.1 30.1 14.1 21.6±3.5CLT 58 55 46 80 87 65±7(mL/minlKg)Vd(L) 75.5 85.7 137.4 251.9 94.0 129 ±29(L/Kg) (1.2) (1.4) (2.1) (2.9) (1.3) (1.8 ±0.3)Vdg (L) 91.9 110.9 128.8 307.6 128.7 153.6 ±78.2(L/Kg) (1.5) (1.8) (2.0) (3.5) (1.8) (2.1±0.3)MDRT 20.4 24.3 45.7 35.9 14.4 28.1 ± 5.0(mm)F 0.042 0.095 0.096 N/A 0.040 0.068±0.014* *pharmaco]çjnetic parameters are calculated from the drug species administered via the femoralvein (i.e., DPHM or[2H10jDPHM).N/A - plasma levels following the mesenteric dose were too low to permit pharmacokineticanalysis.The weight corrected parameters are based on the weights at the time of arrival of the sheep atthe research facility125o DPHM • [2H10]DPHM-JED) 1000.lb..Z 100- •C .G)0C 0 •o .O °010 0 •E 0 •01 I I I0 120 240 360Time (minutes)Figure 36: A representative plot of the plasma concentrations of DPHM and[2H10]DPHM in a non-pregnant ewe (E#1 154) following simultaneousmesenteric (DPHM) and femoral venous([2H10]DPHM) administration.3.3.3.1.2. Mesenteric Bolus Administration during Mild HypoxemiaThe control (i.e., prior to experimentation) blood gas parameters measured in the non-pregnant animals were: pH, 7.43 ± 0.02; Po2, 123 ± 5.0 mm Hg; 02 saturation 100.9 ± 0.5%; andPco2, 38.7 ± 1.2 mm Hg, and experimental values were 7.44 ± 0.01, 62.4 ± 1.9 mm Hg, 82.9 ±1.1%, and 38.3 ± 1.0 mm Hg, respectively. Statistically significant differences were notedbetween control and test period for Po2 and 02 saturation (p<O.O5 Mann Whitney U -Test).Although the mean reduction in the Po2 and 02 saturation appeared to be similar for all animals,the initial (i.e., first 30 minutes following drug administration) reduction in these parameters wasgreatest in ewes 1154 and 1158 (Table 10).126Table 10: Initial (i.e., the first 30 minutes) and average percentage changes in blood gas statusfollowing the initiation of mild to moderate hypoxemia induced by nitrogen gasinfusion into the trachea of non-pregnant sheep.% Change Pco2 % Change pH % Change Po2 % Change02 SaturationEWE Initial Mean Initial Mean Initial Mean Initial Mean(SEM) (SEM) (SEM) (SEM)E#102 -3.7 10.0 -0.27 -1.0 -39.5 -42.0 -12.3 -17.3(3.8) (0.2) (7.6) (8.2)E#139 -0.3 3.3 -0.68 -0.5 -37.9 -41.5 -12.3 -17.3(2.0) (0.1) (4.2) (8.2)E#989 -12.4 -19.8 0.27 0.9 -45.2 -55.7 -7.7 -19.4(7.0) (0.3) (5.1) (5.9)E#1154 2.9 4.1 0.53 -0.7 -63.2 -55.2 -30.9 -20.8(9.9) (0.5) (3.7) (4.6)E#1158 8.3 5.7 0.26 -0.5 -58.8 -49.5 -25.6 -14.1(1.7) (0.2) (3.3) (2.9)The pharmacokinetic parameters calculated for the animals during hypoxemia are shown intable 11. There are no statistically significant differences between the pharmacokineticparameters calculated during the normoxic and hypoxemic periods (Mann-Whitney U test,P>0.05). Only in two animals was it possible to calculate the bioavailability, because in three ofthe animals the plasma concentrations of the drug species administered via the mesenteric veinwere too low to permit pharmacokinetic assessment. In the other two animals, namely, ewes1154 and 1158, a larger bioavailability was calculated (Table 11). However, these estimates arelikely to be inaccurate due to the difficulty of obtaining terminal elimination rate constants, andestimating the initial plasma concentrations (See figure 37).1272 • DPHM0 f2H10DPHME0) 1000CC04-0IC0 w. ••.o 00C 00 0C) 00 10 0 •E • •00• 1 I I I I I0 60 120 180 240 300 360Time (minutes)DPHM 0 [2H1OJDPHM-JE0) 1000CC000100 0C0 00 0o 0C) • 00 10E • 0Cl) 00 001 I I I0 60 120 180 240 300 360Time (minutes)Figure 37: Representative plots of the plasma concentrations of DPHM and[2H10jDPHM in non-pregnant ewes (E#1 158-above) and (E#102-below)following simultaneous mesenteric (DPHM) and femoral venous([2H10jDPHM) administration during mild hypoxemia.128Table 11: Pharmacokinetic parameters obtained from the non-pregnant mesenteric first-passmetabolism study following the simultaneous administration of DPHM or[2H10JDPHM via the femoral or mesenteric vein during mild hypoxemia (n=5).Parameter E#139 E#l 154 E#1 158 E#989 E#102 Mean±SEMx mind 0.2505 0.128 0.996 0.190 0.277 0.37 ± 0.1613 min’ 0.068 0.023 0.032 0.026 0.046 0.039 ± 0.009CLT 67 58 219 87 123 111 ± 29(mL/min/Kg)Vd (L) 97.9 85.9 244.0 237.8 160.7 165 ±33(L/Kg) (1.56) (1.36) (3.74) (2.74) (2.23) (2.33 ±0.43)Vd8 (L) 91.9 110.9 128.8 307.6 128.7 154±35(L/Kg) (1.47) (1.76) (1.97) (3.51) (1.79) (2.10±0.32)MDRT 23.4 23.5 17.1 31.7 18.1 22.8 ± 2.6(mm)F N/A 0.69 2.15 N/A N/A -**pharmacokinetic parameters are calcuiated from the drug species administered via the femoralvein (i.e., DPHM or[2H10]DPHM).N/A - plasma levels of mesenteric dose were too low to permit pharmacokinetic analysis.The weight corrected parameters are based on the weights of the sheep at the time of arrival atthe research facility3.2.3.2. Fetal LambsThe mean maternal weight (± SEM) of the pregnant ewes used in the fetal umbilicalvenous first-pass experiments was 79.4 ± 4.9 Kg, and the mean fetal weight was 2.3 ± 0.3 Kg.The mean maternal weight of the pregnant ewes used in the simultaneous umbilical/tarsal venousinfusion study was 74.5 ± 0.7 Kg, while the fetal weights were 2.0 ± 0.4 Kg. The mean fetalgestational age was 130 ± 1.8 days and 130 ± 2.7 days (term 145 days) for the fetal first-passbolus and infusion experiments, respectively. In some cases, pregnant animals were used in bothcontrol (isotope effect studies) and first-pass experiments (Appendix 1). A minimum wash-outperiod of 48 hours was allowed prior to a second experiment. In fetal lambs, prior toexperimentation, the measured blood gas values were: pH 7.31 ± 0.02; Po2 22.3 ± 1.7 mm Hg;Pco2 47.8 ± 0.9 mm Hg; 02 saturation 57.3 ± 5.9%, and hemoglobin concentration 10.5 ± 0.8g/dL. During experimentation these values were 7.32 ± 0.02, 23.1 ± 1.4 mm Hg, 48.1 ± 1.1 mm129Hg, 02 saturation 54.2 ± 4.9%, and hemoglobin concentration 10.3 ± 0.8 g/dL. The experimentalblood gas values remained unchanged from control values during the experiment.3.2.3.2.1. Umbilical Venous Bolus AdministrationIn the fetal experiment involving simultaneous drug injection via the umbilical and lateraltarsal veins, the combined drug dose (DPHM +[2H10]DPHM) averaged 3.80 ± 0.6 mg/Kg (n=6).In three of the experiments[2H10]DPHM was administered via the umbilical vein and DPHM viathe lateral tarsal vein, while in the other three fetal lambs, the order of injection was reversed. Arepresentative example of the arterial plasma drug concentration vs. time curve for one fetus(E#499) is illustrated in figure 38, while the pharmacokinetic parameters calculated using bothfemoral and carotid arterial plasma drug concentrations are given in table 12 and 13, respectively.The plasma concentrations of DPHM administered via the umbilical vein (test) were similar tothose observed following tarsal venous administration (control). Thus, in contrast to thesituation in adult sheep, a fetal hepatic first-pass effect following umbilical venous bolusadministration was not apparent. The systemic availability (F) of DPHM measured usingfemoral arterial blood following umbilical administration was 1.10 ± 0.08 (95% confidenceinterval 0.9<X<1.3), corresponding to an extraction (B) of-lO ± 8 %. There were no statisticallysignificant differences between the pharmacokinetic parameters calculated using either thefemoral or the carotid arterial reference sites (Paired Sample T-test, P>0.05). The availabilitymeasured from carotid arterial blood was 1.20 ± 0.11, and was not different from the valuemeasured using femoral arterial blood samples (Paired Sample T-test p>O.O5).130Table 12: Pharmacokinetic parameters calculated using femoral arterial plasma drugconcentrations during fetal umbilical first-pass metabolism experiments followingthe simultaneous administration of DPHM and[2H10]DPHM.Tarsal Venous Administration - Femoral Arterial ReferenceParameter E#989 E#208 E#499 E#l 143 E#543 E#975 Mean ± SEMcx min1 0.149 0.415 0.238 0.225 0.267 0.139 0.24±0.0413 min1 0.030 0.023 0.029 0.039 0.025 0.030 0.029 ± 0.003AUC 2436 2045 2828 2453 1636 10881 3713 ± 1579(ng*minlmL)CLT. 387 647 816 1063 879 263 700±100(mLlminlKg)Vd L 35.5 45.7 60.4 48.9 115.9 14.2 53 ± 15.2(L/Kg) (13.6) (24.8) (27.4) (26.1) (33.9) (8.1) (22±4.5)VdBL 33.7 52.9 62.5 51.6 122 15.5 56± 16.1(L/Kg) (12.9) (28.7) (28.4) (27.5) (35.6) (8.8) (24 ± 4.5)MDRT 34.7 37.8 33.1 24 38 30.2 33 ± 22(mm)F 0.91 0.93 1.27 1.36 0.95 1.15 1.10±0.08Umbilical Venous Administration - Femoral Arterial ReferenceParameter E#989 E#208 E#499 E#l 143 E#543 E#975 Mean ± SEMcx mill’ 0.163 0.253 0.281 0.437 0.254 0.131 0.25 ± 0.513 mill’ 0.032 0.022 0.029 0.040 0.025 0.029 0.030 ± 0.003AUC 2226 1907 3605 3335 1549 12465 4257 ± 1892(ng*mmnlmL)CLT 424 695 640 782 929 229 600 ± 130(mL/minlKg)Vd L 36.8 54.1 41.9 33.7 123 10.9 50±17(L/Kg) (14.1) (29.3) (19) (18) (35.9) (6.2) (20±9.9)VdBL 34.1 57.7 48.6 36.4 125.5 13.8 53± 17.4(L/Kg) (13.1) (31.3 (22.1) (19.4) (36.6) (7.8) (22 ±4.9)MDRT 32.8 41.7 29.2 22.5 38.2 26.5 32 ± 3.1(mm)*weight corrected values are based on fetal body weight at the time of the experiment.There were no statistical differences between the pharmacokinetic parameters calculated fromthe femoral arterial and carotid arterial reference sites (p>O.O5 paired sample T-test).131Table 13: Pharmacokinetic parameters calculated using carotid arterial plasma drugconcentrations during fetal umbilical first-pass metabolism experiments followingthe simultaneous administration of DPHM and[2H10]DPHM.Tarsal Venous Administration - Carotid Arterial ReferenceParameter E#989 E#208 E#499 E#l 143 E#543 E#975 Mean ± SEMX miii1 0.2122 0.7935 0.2183 0.852 0.0778 0.1744 0.39 ±0.1513 min’ 0.0363 0.0249 0.0330 0.0406 0.0198. 0.0325 0.031 ± 0.004AUC 2166 2827 2746 1827 1419 8998 3388 ± 1291(mL/minlKg)CLT 436 469 841 1428 1014 318 800±200(mL/min/Kg)Vd L 33.2 38.4 54.4 69 140.2 16.6 59 ± 19.6(L/Kg) (12.7) (20.8 (24.7) (36.8) (41) (9.4) (24±5.8)Vd L 31.2 34.8 56 65.9 175.6 1.7 61 ± 26.8(LIKg) (12) (18.8) (25.4) (35.2) (51.3) (1) (24±8)MDRT 28.7 43.9 28.9 25.2 39.9 29.2 33 ± 3.1(mm)F 0.98 1.01 1.54 1.44 1.18 1.03 1.20±0.10Umbilical Venous Administration - Carotid Arterial Reference.Parameter E#989 E#208 E#499 E#1 143 E#543 E#975 Mean ± SEMo min’ 0.053 0.8485 0.37 19 0.2336 0.1302 0.3023 0.32 ± 0.1313 min’ 0.022 1 0.0230 0.032 1 0.035 1 0.0 175 0.0332 0.027 ± 0.003AUC 2128 2804 4236 2622 1675 9254 3786 ± 1257(mL/minlKg)CLT. 443 472 545 995 859 309 600 ± 130(mL/minlKg)Vd L 29.7 36.7 26.7 47.6 97.2 15.1 42± 12.4(L/Kg) (11.4) (19.9) (12.1) (25.4) (28.4) (8.6) (18±3.6)VdL 52.3 38 37.4 53.1 167.9 16.3 61 ± 24.1(L/Kg) (20.1) (20.6) (17) (28.3) (49) (9.3) (24±6.3)MDRT 25.2 41.6 21.7 25 32.6 27.3 29±3.1(mm)*weight corrected values are based on fetal body weight at the time of the experiment.There were no statistical differences between the pharmacokinetic parameters calculated fromthe femoral arterial and carotid arterial reference sites (p>O.O5 paired sample T-test)132FA-DPHM •FA-[2H10JDPHM CA-DPHM A CA-[2H10JDPHM-JE 10000)C0100.1—iC)C0oE 10 iCl)0I1ci 1 I I I0 120 240 360Time (minutes)Figure 38: A representative figure showing the fetal femoral and carotid arterial plasmaconcentrations of DPHM and[2H10]DPHM following the simultaneousadministration of DPHM via the umbilical vein and{2H10]DPHM via the fetallateral tarsal vein.3.2.3.2.3. Umbilical Venous InfusionsFetal hepatic extraction was investigated with simultaneous 90 minute infusions ofDPHM and[2H10JDPHM via the umbilical and fetal lateral tarsal veins. The mean (± SEM)infusion rate for both DPHM and[2H10]DPHM was 57.2 ± 10.8 ig/min of each. The meanconcentrations of drug in the femoral and carotid arterial plasma following umbilical venousinfusion were 64.8 ± 13.2 ng/mL and 61.3 ± 11.9 ng/mL, respectively. The femoral and carotidarterial plasma concentrations following tarsal venous infusion were 66.6 ± 13.1 and 59.6 ± 11.9133nglmL. The total body clearances (CLT) measured from carotid and femoral arterial samples forboth routes of administration are shown in table 14. The differences between the total bodyclearances following tarsal venous and umbilical venous administration were 28.6 ± 44.8mL/minlKg, and -20.2 ± 53.9 mL/rninlKg measured from femoral and carotid arterial blood,respectively. Although the net hepatic clearance appeared to decrease when measured fromcarotid arterial blood as compared to femoral arterial blood, no statistically significantdifferences were noted (Table 14). During the 90 minute infusion period, there were statisticallysignificant differences between the femoral and carotid arterial plasma concentrations of lactateand glucose, oxygen saturation, and the partial pressure of oxygen (Table 15). All of theseparameters were higher in carotid arterial blood compared to the femoral arterial blood (SeeTable 15). In contrast, the concentrations of both forms of the drug where higher in FA than inCA (Table 15). The mean differences were 8.5 ± 2.1 ng/mL and 3.3 ± 1.3 ng/mL, for tarsal andumbilical venous drug infusion, respectively. Both mean differences were statistically differentfrom zero (2 Sample T-test, p<O.O5). Moreover, the difference measured following umbilicalvenous administration was lower than that measured following tarsal venous infusion.134Table 14: Clearances calculated following simultaneous steady-state umbilical and tarsalvenous infusions of DPHM or[2H10]DPHMEWE CL, (FA) CL (FA) CL (CA) CL (CA) CLCLt* CLCL1*‘mL/min/Kg) mL/min/Kg) mL/minlKg) mL/minlKg) (FA) (CA)E#1 142 447 645 582 691 191 118E#1242(2) 650 659 608 593 18.6 -3.8E#1242(1) 303 349 330 373 38.8 39.2E#1250 468 407 520 472 -61 -48E#2164 1158 1113 1341 1135 -44 -21Mean±SEM 605± 149 634± 135 676± 173 653± 132 28.6±44.8 -20.2±53.9* The difference is taken at each time sampling time point (i.e., 15, 30, 45, 60, 75, and 90minutes for each animal). These do not represent the difference of the mean values.TV and UV represent the route of administration (i.e., tarsal vein or umbilical vein) of DPHM or[2H10]DPHM.Table 15: Blood gas parameters during simultaneous umbilical venous and tarsal venousinfusion of DPHM or[2H10]DPHM to steady-state (n=5).Femoral artery Carotid arterypH 7.348 ± 0.008 7.356 ± 0.008Pco2 (mm Hg) 48.5 ± 1.0 47.4 ± 1.0*Po(mmHg) 20.3± 1.3 22.9± 1.4***Base Excess (megfL) 1.5 ± 0.5 1.6 ± 0.3HC03(meg/L) 26.3 ± 0.5 26.1 ± 0.4TCO2 27.6 ± 0.5 27.4 ± 0.4Hemoglobin (gIdL) 9.7 ± 0.8 9.6 ± 0.802 Saturation (%vlv) 47.2 ± 5.8 54.5 ± 8.0***[Lactate] mmol 1.4 ± 0.1 1.5 ± 0.l’[Glucose] mmol 0.9 ± 0.3 1.1 ± 0.5**[DPHM) uv (ng/mL) 64.8 ± 13.2 61.3 ± 11.9*[DPHMJ tv (ng/mL) 66.6 ± 13.1 59.6 ± 11.9*Data is presented as mean ± SEM* statistically significant difference p<0.05 [paired sample T-test]** statistically significant difference p<0.02 [paired sample T-test]statistically significant difference p<0.02 [paired sample T-testl1353.2.4. Paired Fetal-Maternal InfusionsThe five experiments involving simultaneous 6 hour infusions of DPHM and[2H10]DPHM to ewe and fetus, respectively, were carried out at 125-133 days gestation age (129± 1 days). Estimated fetal weight was 2.24 ± 0.06 Kg. In the control period the fetal descendingaortic values for pH, Po2, Pco2, 02 saturation, hemoglobin concentration, glucose concentration,and lactate concentration were 7.36 ± 0.02, 22.6 ± 1.65 mm Hg, 47.3 ± 0.5 m.m Hg, 55.3 ± 23.8%, 10.0 ± 0.3 gIdL, 0.98 ± 0.09 mM., and 0.70 ± 0.11 mM.3.2.4.1. Estimates of Trans-Placental and Non-Placental ClearancesThe average (± SEM) fetal and maternal femoral arterial plasma concentrations of DPHMand[2H10]DPHM in maternal and fetal plasma in five experiments involving 6 hour pairedmaternal (DPHM - 670 Jig/mm) and fetal (170 j.tg/min -{2H10]DPHM) infusions are illustrated infigure 39. The plasma concentrations reach a plateau at approximately 150 minutes. There wereno statistical differences between the plasma concentrations at 150 and 180, 240 and 270, and330 and 360 minutes (p>O.O5 repeated measures ANOVA), suggesting that steady-state wasachieved by 150 minutes. Thus, the mean steady-state concentrations were calculated from 150to 360 minutes, and are shown in table 16. The mean steady-state concentrations (± SEM) ofDPHM were 262.0 ± 24.2, 27.2 ± 4.4, and 26.6 ± 4.3 ng/mL in the maternal femoral (MA), fetalfemoral (FA), and fetal carotid (CA) arteries, respectively. The mean concentrations of[2H10JDPHM were 44.9 ± 7.4, 203.7 ± 29.9, 186.1 ± 27.0 ng/mL in the MA, FA, and CA,respectively. The above mean values were calculated excluding ewe 2181, since CA sampleswere not available for this animal (Table 16). The total FA, CA, and MA steady-stateconcentrations of DPHM (i.e., DPHM +[2H10]DPHM) were 282.4 ± 57.4 ng/mL { 231.0 ± 31.5136nglmL, excluding ewe 2181] (FA), 212.8 ± 25.9 ng/mL (CA), and 308.1 ± 25.8 ng/mL(maternal). There were statistically significant differences between the FA and CA plasmaconcentrations of[2H10]DPHM (Paired Sample T-test, p<O.O5). Although the meanconcentrations of DPHM appeared higher in FA compared to CA blood (Table 16), thesedifferences were not statistically significant for DPHM (maternally derived) and total DPHM(labeled + unlabeled) (Paired Sample T-test p>O.05). Following the cessation of the infusion,concentrations of DPHM and[2H10]DPHM in fetal and maternal plasma declined rapidly, withan apparent terminal elimination half-life in plasma of 77.5 ± 3.2 minutes and 52.3 ± 7.0 minutesin mother and fetus, respectively (Table 17). Both the corrected (i.e., corrected for loss of drugfrom fetus to mother or mother to fetus via the placenta) and the uncorrected volumes ofdistribution were larger in the fetus as compared to the mother (i.e., 27.1 ± 5.9 L/Kg and 9.2 ±1.7 L/Kg vs. 2.0 ± 0.24 L/Kg and 1.9 ± 0.23 L/Kg) (Table 18). As in previous studies, there wasaccumulation of DPHM (both labeled and unlabeled) in fetal lung and amniotic fluids (Figure40). The average drug concentration ratio between lung fluid and femoral arterial plasma was(4.0 ± 1.7) for DPHM and (4.5 ± 1.6) for[2H10]DPHM. During the infusion, the correspondingratios in amniotic fluid (i.e., amniotic fluid/FA) were 0.6 ± 0.2 and 0.8 ± 0.2 for labeled andunlabeled drug, respectively. Although the ratio is lower in amniotic fluid, the drug persistslonger in amniotic fluid than in fetal lung fluid.The non- and trans-placental clearance parameters calculated using fetal and maternalarterial plasma drug concentrations are shown in table 19. The weight normalized estimates ofCLfm (221 ± 32 mL/min/Kg) and CLfo (135 ± 26 mL/minlKg) were both significantly higherthan the corresponding maternal values (CLmf 53.7 ± 14.4 mL/min/Kg and CLmo 38 ± 2mL/minlKg) (Paired Sample T-test p<O.O5). The non-placental contribution to total body137clearance averaged (95.6 ± 1.0%) and (37.3 ± 4.9%) in ewe and fetus, respectively. The non- andtrans- placental clearances were also calculated using a mass balance approach (Table 20). Thesevalues are in good agreement with those calculated using the approach of Szeto et a!. (1982)(Table 19).Table 16: Mean (± SEM) maternal and fetal steady-state arterial plasma drug concentrationsfollowing simultaneous maternal/fetal infusions.Reference Site E#122Z E#2101 E#2177 E#218 1 E#2241(nglmL) (ng/mL) (ng/mL) (ng/mL) (ng/mL)FA(DPHM) 27.2 (1.2) 14.8 (3.2) 31.9 (1.7) 111.2 (5.6) 34.9 (1.6)FA([210]DPHM) 137.5 (2.4) 207.4 (29.7) 187.9 (6.9) 378.3 (10.2) 281.8 (8.9)CA (DPHM) 25.1 (1.4) 15.3 (2.6) 34.7 (1.9) N/A 31.4 (2.6)CA([210]DPHM) 125.2 (3.8) 177.8 (20.4) 184.9 (7.9) N/A 256.6 (5.0)UV(DPHM) 31.5 (3.2) N/A N/A 137.9(6.8) N/AUV([210]DPHM) 47.3 (3.2) N/A N/A 180.9 (7.6) N/AMA (DPHM) 262.1 (9.9) 223.1 (9.5) 232.6 (8.9) 241.5 (8.2) 330.0 (6.6)MA([210]DPHrvI) 40.6 (2.1) 30.5 (5.0) 42.8 (1.7) 38.8 (1.5) 65.5 (1.8)N/A - reference site could not be sampled due to catheter failure138Pharmacokinetic parameters calculated during paired maternal/fetal infusionsParameter E#122Z E#2101 E#2177 E#2181 E#2241 Mean±SEMAUCmm 95397 87278 88534 94710 128464 -(ng*minlmL)AUCff 50872 87989 83072 149359 109036 -(ng*minlmL)AUCfm 14089 12645 17516 15987 26466 -(ng*minImL)AUCrnf 10888 7684 16805 45697 14847 -(ng*minlmL)MDRT(M) 51.0 58.5 52.1 56.9 40.7 51.8 ± 3.1(mm)IVIDRT(F) 70.7 44.9 96.8 74.1 69.3 71.2±8.2(mm)a (M) (min’) 2.0 0.4 0.6 0.2 0.1 0.66 ± 0.35aT112(min) 0.4 1.7 1.2 4.3 5.3 2.6±0.9a (F) (min’) 0.3 0.4 11.2 0.54 0.43 2.56 ± 2.14*04 1 ± 0.05aT112(min) 2.8 1.7 0.10 1.3 1.6 1.8±0.3*15 ± 0.413 (M)(min’) 0.009 0.009 0.008 0.010 0.016 0.010 ± 0.00113T,2(min) 77.0 77.0 86.6 69.3 43.3 77.5±3.213 (F) (min’) 0.010 0.016 0.010 0.016 0.019 0.014 ± 0.00213T12(min) 64.3 43.3 69.3 43.3 36.5 52.3±7.0CLT(M) 2750 3000 2960 2770 2040 2704 ± 173(mL/min)CLT(F) 1300 750 790 440 610 778 ± 144(mL/min)CLT(M) 43.9 36.7 39.5 42.1 30.2 38.5 ± 2.4(mL/min/Kg)CLT(F) 575.2 364.8 352.0 202.2 250.0 348.9 ± 64.2(mL/mmnlKg)* The value of a calculated in E2177 was taken as an outlier and the value recalculatedexcluding this animal.Table 17:139Table 18: The uncorrected and corrected (i.e., for drug lost via the placenta) volumes ofdistribution of DPHM in maternal sheep and{2H10jDPHM in fetal sheep followinga simultaneous fetal infusion of[2H10]DPHM (170 tg/min) and maternal infusionof DPHM (670 Jtg/min).Parameter E#122Z E#2101 E#2177 E#2181 E#2241 Mean ± SEMMaternalVdss (L) 140.2 185.8 158.6 167.8 86.5 147.8 ± 17.0Vdss (L/Kg) 2.2 2.3 1.6 2.6 1.3 2.0 ± 0.2Vdss’(L) 132.0 182.6 151.6 154.7 83.8 140.9 ± 16.4Vdss’ (L/Kg) 2.1 2.3 1.6 2.4 1.2 1.9 ±0.2FetalVdss (L) 91.7 34.2 95.8 36.3 46.1 60.8 ± 13.6Vdss (L/Kg) 40.6 16.6 42.7 16.7 18.9 27.1 ± 5.9Vdss’ (L) 37.5 18.2 28.0 13.8 11.7 21.8 ±4.8Vdss’ (L/Kg) 13.6 8.8 12.5 6.3 4.8 9.2± 1.7Vdss’ Volume of distribution at steady-state corrected for fraction of maternal dose lost tothe fetus and the fraction of the fetal dose lost to the mother.L FA-DPHM —B—-FA-[2H10]DPHM —& MA-DPHM ——MA-[2H10JDPHM140-aFigure 39: Femoral arterial plasma concentrations of DPHM and[2H10]DPHM infetus and mother following simultaneous infusion of DPHM (670 pg/min)via the maternal femoral vein and[2H10]DPHM (170 j.Lg/min) via the fetallateral tarsal vein.-J20,CC04-CU4-CciC-)C00CU2Cl,CU0CU4-’10oo100•101InfusionN.-rN0NNI I I I I120 240 360 480 600Time (minutes)720141—e-- DPHM-AMN —. -[2H10JDPHM-AMN--€ - DPHM-TR - - [2H10]DPHM-TR10000 -1000 - S______\V__•__••-100 - .— -•0 120 240 360 480 600 720Figure 40: A representative plot of the concentrations of DPHM and[2H10]DPHMin amniotic fluid and fetal tracheal fluid following simultaneousinfusion of DPHM (670 pglmin) via the maternal femoral vein and[2H10]DPHM (170 ig/min) via the fetal lateral tarsal vein (E#2181).AMN - amniotic fluid and TR - fetal tracheal fluid142Table 19: Weight corrected trans- and non-placental clearances of DPHM in fetus andmother following a simultaneous infusion of DPHM to mother and[2H10]DPHMto the fetus (mL/minlKg).MeanSEMEWE39.92.3CA356.247.4CA53.714.4CA221.032.0CA38.02.0CA134.626.4CLmm CLff CLmf CLfm CLmo CLfoEWE FA FA FA FA FA FA(mL/minlKg) (mLlminlKg) (mL/min/Kg) (mL/min/Kg) (mLlminlKg) (mL/min/Kg)E#2177 40.7 441.1 60.1 308.8 38.9 129.2E#2101 38.9 370.6 26.9 173.4 38.2 197.3E#122z 43.4 471.9 47.6 280.2 41.3 191.7E#2181 44.8 225.0 105.6 139.3 41.3 85.7E#2241 31.5 272.6 28.2 203.5 30.4 69.1E#2 177E#2 101E# 1 22zE#2 181E#224140.935.843.4450.4427.4513.867.531.648.2315.7253.5305.538.835.041.3Mean 37.8 420.9 43.7 273.8 36.4 145.2SEM 2.7 46.7 9.1 22.3 2.4 29.731.3 292.1 27.5 220.7 30.3CA127.1173.9208.371.5Note: Carotid arterial samples were not available in ewe 2181.Weight corrected clearances (mL/minlKg) are normalized to maternal (CLmm and CLmo) orfetal (CLff, CLfm, CLmf, and CLfo) body weight estimated at the time of experimentation.Note: The CLmm is not equal to CLmo + CLmf because CLmf is weight corrected to fetalweight rather than maternal weight.143Table 20: Non weight corrected and weight corrected trans- and non-placental clearanceparameters of DPHM following simultaneous infusions of DPHM and[2H10]DPHM to mother and fetus, respectively. Calculated using the mass balanceapproach.EWE CLmf CLfm CLmo CLfo(mL/minlKg) (mLIminJKg) (mL/minlKg) (mL/minlKg)E#122z 69 344 44 257E#2101 34 220 37 154E#2177 70 291 39 83E#2181 105 144 41 72E#2241 29 212 30 47Mean 62 242 38.3 123SEM 14 34 2.4 38Weight corrected clearances (mL/minlKg) are normalized to maternal (CLmm and CLmo) orfetal (CLff, CLfm, CLmf, and CLfo) body weight.In the two animals which had umbilical arterial transit time flow probes and functionalumbilical venous catheters (ewes 122z and 2181), trans-placental clearances were calculatedusing the Fick method. The extraction ratios of[2H10JDPHM and DPHM were 0.64 ± 0.02 and -0.16 ± 0.05 in ewe 122Z, and 0.51 ± 0.01 and -0.23 ± 0.03 in ewe 2181, respectively. Thecorresponding values for CLmf were 31.6 ± 9.5 [ewe 122z] and 68.7 ± 7.4[ewe 2181)mL/minlKg, and for CLfm were 126.2 ± 4.4 [ewe 122z] and 153 ± 3.9 [ewe 2181) mL/minlKg.The values for CLmf calculated using the Fick method are lower in both animals (35 and 33% inewes 1 22z and 2181, respectively) compared to values calculated using the compartmentalmethod. The Fick estimate for CLfm was equivalent to the model estimate in ewe 2181, butunderestimated the model estimate by a factor of two in ewe 1 22z (Table 21). The ratio of fetalto maternal placental clearances (i.e., CLfmICLmf) from the Fick clearance estimates were 4.0144and 2.2 vs. the 2 compartment-open model estimates, which were 6.0 and 1.3 in ewes 122z and2181, respectively.Table 21: A comparison of model dependent estimates of trans-placental clearances (2compartment-open model), and model independent (Fick method) estimates oftrans-placental clearances.E#122Z E#218 1 Yoo et al. Mean (± SEM)(E#l 30)Extraction (M to F) -0.16 -0.23 -0.33 -0.24 ± 0.05Extraction (F to M) 0.64 0.51 0.65 0.60 ± 0.05Umbilical Arterial 195.4 294.9 a b2807 ± 39.1Flow (mL/minlKg)CLmf(Fick) 31.6 68.7 C926 64.3 ± 17.7CLfm(Fick) 126.2 153.0 C1824 153.9± 16.2CLmf (model) 47.6 105.6 d275 60.2 ± 23.4CLfm (model) 280.2 139.2 d1338 184.4 ± 47.9a - umbilical arterial blood flow was not measured in this experiment.b - mean blood flow includes data for an animal in which the umbilical venous catheter was notfunctioning, thus, extraction could not be calculated for this animalc - Fick clearance estimates are calculated using the extraction ratio from the previousexperiment and the mean umbilical arterial blood flow from the current experiment.d - model estimates obtained from an earlier experiment (Yoo et al., 1989)The clearance parameters were also calculated in four animals using the carotid arterialplasma drug concentrations rather than femoral arterial drug concentrations. Although thereappeared to be an overall increase in the mean values for CLff, CLfm, and CLfo using carotidarterial plasma concentrations compared to the clearance parameters calculated using the femoralarterial reference plasma drug concentrations, these differences were not statistically significant(p>O.O5 Paired sample T-test) (Table 19).1453.2.4.2. Metabolism of DPHM to DPMA in Mother and FetusA mean (± SEM) arterial plasma concentration vs. time plots of DPMA and[2H10JDPMAin maternal and fetal femoral arterial plasma samples are shown in figure 41 and 42. Althoughconcentrations of DPHM and[2H10]DPHM reached steady-state at 150 minutes from the start ofthe infusion, the concentrations of DPMA and[2H10JDPMA did not reach steady-state at thistime, and continued to increase for 30-120 minutes following the end of the infusion. At all timepoints during the infusion, the concentration of[2H10]DPMA was higher in the fetus than in themother, whereas the opposite was true in maternal plasma (i.e., DPMA was higher than[2H10]DPMA in maternal plasma). The peak concentration of DPMA in maternal and fetalplasma averaged 137.4 ± 18.5 and 92.8 ± 16.8 ng/mL, respectively, while the peak maternal andfetal plasma levels of[2H10]DPMA were 28.7 ± 4.3 and 135.0 ± 20.1 ng/mL (statisticallysignificant difference; Paired Sample T-test p<O.05), respectively. The time at which the peaklevels occurred post-infusion was 18.0 ± 7.3 minutes for both labeled and unlabeled DPMA inthe ewe, whereas in the fetus the value was 87.0 ± 14.5 minutes. Following the peak, themetabolite levels in the fetus declined much more slowly than in the ewe (Figure 41). The modelestimates for DPHM,[2H10jDPHM, DPMA, and[2H10]DPMA following simultaneous fitting ofthe parent drug and metabolite data are shown in table 22. The apparent elimination half-lives ofthe metabolite were 180.3 ± 13.0 mm (3.0 ± 0.2 hrs) and 911.4 ± 151.5 mm (15.2 ± 2.5 hrs) inmother and fetus, respectively. These values were significantly different (p<O.O5, Paired SampleT-test). The formation rate constant (Kf) could not be calculated because the volume ofdistribution of the metabolite was not known. The AUC ratio of DPMA to DPHM wassignificantly less than the AUC ratio of[2H10]DPMAJ[PHM in maternal plasma (i.e., 0.62146± 0.07 vs. 0.93 ± 0.12) [Paired Sample T-test, p<O.05]. Although amniotic and fetal lung fluidsamples were analyzed for the presence of the metabolite, it was never detected. The extractionof DPMA and[2H10]DPMA across the placenta was -0.07 ± 0.06 and -0.02 ± 0.01 for ewe 122zand -0.07 ± 0.01 and -0.09 ± 0.01 for ewe 2181, respectively.Table 22: Pharmacokinetic parameters calculated from the simultaneous fittingof DPHM and DPMA in maternal plasma and[2H10]DPHM and[2H10]DPMA in fetal plasma. (n=5).Parameter E#2241 I E#2177 I E#2181 I E#2101 E#122z Mean SEMmaternalK12(min) 0.0179 0.0619 0. 1343 0.3 128 0.324 0.170 0.06421(min’) 0.0204 0.0146 0.0276 0.1648 0.0413 0.054 0.028K10(min’) 0.0603 0.1576 0.1312 0.7086 0.3192 0.275 0.117Fm(min*L’) 0.0011 0.0005 0.0011 0.0014 0.0007 0.001 0.0002Vc(L) 33.6 18.4 20.9 4.3 9.5 17.3 5.1Km(min’) 0.0037 0.003 1 0.0038 0.0047 0.0043 0.0039 0.0003Ti12 (mm) 187.3 223.5 182.4 147.4 161.1 180.4 13.0(DPMA)K12(min’) 0.3814 0.1092 0.7442 0.8033 1.067 0.621 0.16921(min’) 0.0172 0.0221 0.0426 0.1603 0.0681 0.062 0.026K10(min’) 0.4290 0.0877 0.3211 0.1705 0.3196 0.266 0.061Fm (min*L4) 0.0012 0.0028 0.0029 0.0025 0.0027 0.0024 0.0003Vc(L) 1.4 9.8 1.4 4.5 3.8 4.2 1.5Km(min’) 0.0011 0.0006 0.0005 0.0010 0.0010 0.0008 0.0001T112 (mm) 630.0 1155.0 1386.0 693.0 693.0 911.4 151.9([H10]DPMA)fetal E#2241 E#2177 E#2 181 E#2 101 E# 1 22z Mean SEM147—-—FA-[2H10jDPMA —A—FA-DPMA —0- MA-[2H10JDPMA—.— MA-DPMA-JE 10000)CCInfusion I1 I I I I I I I0 360 720 1080 1440 1800 2160 2520 2880Time (minutes)Figure 41: Mean (± SEM) maternal and fetal femoral arterial plasma concentrationsof DPMA and[2H10]DPMA following simultaneous fetal and maternalinfusions of10]DPHM (170 ig/min) and DPHM (670 igImin),respectively.148—-—FA-[2HlD]DPMA —A--FA-DPMA —0- MA-[2H10]DPMA —.—MA-DPMA-JE 10000,CC0100-— — — ——- —:•1Ca)0 — —— — - — —C - -I-o 10- _-GZ10- — —i-Infusion10 120 240 360Time (minutes)Figure 42: Maternal and fetal femoral arterial plasma concentrations of DPMA and[2H10]DPMA following simultaneous fetal and maternal infusions of{10IDPHM and DPHM respectively (x-axis scaling is reduced to showinitial formation of metabolite).1493.2.4.3. Renal Clearance of DPHM,[2H10]DPHM, DPMA and[2H10]DPMAThe fetal and maternal urine flow data collected during and following the infusion areshown in table 23, while the renal clearances calculated for DPHM,[2H10]DPHM, DPMA, and[2H10]DPMA in both ewe and fetus are given in table 24. The cumulative excretion plot forDPHM and[2H10]DPHM in maternal and fetal urine clearly shows a plateau following theinfusion (Figure 43). The weight corrected renal clearance of DPHM was 200 fold less inmaternal sheep compared to fetal lambs (Table 24). The contribution of DPHM clearance to thematernal non-placental clearance was 0.02 ± 0.01%, while in the fetus the contribution was 2.3 ±0.4% (Table 24). The metabolites, DPMA and[2H10JDPMA, were both present in adult urineduring and following the infusion. In marked contrast to DPHM and[2H10]DPHM, thesemetabolites (DPMA and[2H10]DPMA) were present in only small quantities in fetal urine andoften were not detectable. The cumulative amount of DPMA and[2H10]DPMA excreted in fetalurine appears to increase following the cessation of fetal urine collection; therefore, Mu islikely somewhat underestimated in the fetal lamb. In addition, the excretion of the metabolitecontinues to increase in the ewe for some time following the infusion, and only appears to benear the plateau of the cumulative urinary excretion plot at the end of the experimental protocol(Figure 44). Thus, it appears that the renal clearance of DPMA is not entirely complete duringthe end of the experimental protocol. While an estimation of the renal clearance of DPMA inboth mother and fetus may have been underestimated, the weight corrected relative renalclearance of the DPMA was —50 fold greater in mother when compared to the fetus. Thepercentage of the dose excreted as DPMA was calculated based on the ratio of the doseadministered and the cumulative amount of DPMA (corrected for mass difference betweenparent drug and metabolite) excreted in the urine at time infinity (i.e., fraction of dose = DoseDPHMI(EMu0 DPMA*Mass Correction Factor). Although this estimate may underestimate thecontribution of the renal clearance of DPMA due to an underestimation of Mu0 DPMA, arough estimate suggests that around 1% of maternal dose can be accounted for by the renalelimination of DPMA.150151Table 23: Fetal and maternal urine flow during paired fetal and maternal infusions of DPHMand[2H10]DPHM, respectively.E# 1 22ZE#2101E#2177E#2 181E#2241Urine flow(mL/hr)75.7 ± 13.084.5 ± 11.744.3 ± 3.857.6 ± 3.135.4 ± 3.3Urine flow(mLlhr/Kg)26. 1± 8.612.0± 1.18.4 ± 0.917.9 ± 1.16.8 ± 0.8Urine flow(mLlhr/Kg)Urine flow(mLfhr)1.2 ± 0.21.0 ± 0.158.9 ± 19.4Maternal FetalMean ± SEM 59.5 ± 9.3 0.8 ± 0.1 31.6 ± 7.9 14.2 ± 3.50.6 ± 0. 124.7 ± 2.30.9 ±0.118.8 ± 2.10.5 ± 0. 138.9 ± 2.416.7 ± 2Values include the urine flow during the infusion and the post infusion sampling period.152Table 24: The renal clearances of DPHM and DPMA, and[2H10]DPHM and[2H10]DPMAin adult sheep and in fetal lambs.DPHM {2H10]DPHM [2H10]DPHM DPHMEwe CL renal CL renal % CLmo CL renal CL renal % CLfo(maternal) (maternal) (fetal) (fetal)mL/minlKg mL/min/Kg mL/rrnnlKg mL/minlKgE#2101 nd nd 0 1.43 1.80 0.7E#122Z 0.009 nd 0.023 4.09 3.64 2.1E#2177 0.023 nd 0.053 2.81 1.96 2.2E#2181 0.014 nd 0.031 1.31 1.85 1.5E#2241 0.001 nd 0.003 2.70 2.21 3.9Mean 0.012 0.020 2.47 2.29 2.1SEM 0.005 0.009 0.51 0.35 0.53DPMA [2H10]DPMA [2H10]DPMA DPMAEwe CL renal CL renal % Dose CL renal CL renal % Dose(maternal) (maternal) Excreted (fetal) (fetal) Excreted_____ mL/min/Kg mL/rnin/Kg mL/min/Kg mL/min/KgE#2101 1.98 2.02 1.01 nd nd ndE#122Z 1.00 1.08 0.97 0.008 nd 0.005E#2177 0.52 0.51 0.69 0.056 nd 0.050E#2l81 0.54 0.57 1.11 0.003 nd 0.003E#2241 0.42 0.63 0.95 0.005 nd 0.001Mean 0.89 0.96 0.95 0.0181 nd 0.014SEM 0.29 0.28 0.07 0.0128 — 0.011nd - intact drug or metabolite was below the minimal quantifiable concentration of the analysismethodThe % contribution to CLfo and CLmo for intact drug is based on the calculated weight correctedrenal clearance of intact drug (DPHM in mother and[2H10]DPHM. in fetus) and the weightcorrected maternal and fetal non-placental clearances calculated using the method of Szeto(1982).The % contribution of metabolite excretion to fetal and maternal dose is based on the weightcorrected total amount of metabolite excreted in fetal and maternal urine, and the dose of[2H10JDPHM and DPHM administered to mother and fetus, respectively.153—s—- DPHM - maternal1000 0.40Infusion9000) 800 0. 0.30D 700 0,.E 600x500 0.20 Liici400 00) 0> 3000.10.! 200DE 100________________________± ± -‘- -I() 0 I I 0.000 360 720 1080 1440 1800 2160 2520Time (minutes)—B——[2H10]DPHM - fetal1000 1.50Infusion9000)c 800 -0)D 700 a)600 C.)xw0) 500a)400 U)000__I I Ii> 300200 TjE 1000 360 720 1080 1440 1800 2160 2520Time (minutes)Figure 43: Mean (± SEM) cumulative amounts of DPHM in maternal urine and[2H10jDPHM in fetal urine following simultaneous fetal infusion of10]DPHM (170 pg/min) and maternal infusions of DPHM (670 jig/mm)(n=5). [total fetal dose = 66.2 mg and total maternal dose = 261.2 mgI.154• DPMA. maternalInfusion5000 1.00a)CD 40003000-a 0.502000-Ia)a)> 10000.00E 00 360 720 1080 1440 1800 2160 2520C-)Time (minutes)—e-—[2H10]DPMA50 0.80Infusiona)= 400.604-a)4- 30— x-ö.0 0.40 •LU4- 20 0a) 1 0.20> 10 IE 00 360 720 1080 1440 1800 2160 2520C)Time (minutes)Figure 44: Mean (± SEM) cumulative amounts of DPMA in maternal urine and[2H10jDPMA in fetal urine following simultaneous fetal and maternal infusionsof10]DPHM (170 .ig/min) and DPHM (670 .tg/min) respectively (n=5).[total fetal dose = 66.2 mg and total maternal dose = 261.2 mgI.1553.2.4.4. Fetal Effects Following Fetal and Maternal Paired Infusions of[2H10]DPHMand DPHM, Respectively.There were no consistent changes from the controls (i.e., 6 hours pre infusion) in fetal arterialpressure (52.4 ± 1.7 mm Hg), umbilical blood flow (280.7 ± 39.1 mLlminlKg; n=3), fetal urineflow (0.45 ± 0.10 mLlmin), and maternal and fetal urine pH (7.62 ± 0.07 and 6.78 ± 0.10,respectively) during the experiment. However, there was a reduction in fetal heart rate during thecourse of the infusion. The fetal heart rate increased following the end of the infusion (repeatedmeasures ANOVA p<O.O5) (Figure 46). In addition, a statistically significant increase inintermediate electrocortical activity was noted, with a corresponding reduction in high and lowvoltage states; however, decreases in high and low ECoG activity did not reach statisticalsignificance (Figure 45). There was also a trend for the reduction of fetal breathing movements,but these differences did not reach statistical significance (Figure 46).156100 Low ECOG80rT’Te 60 4:: VInfusion0 i I I I0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Time (hours)100Intermediate ECOG80*0 60E.Iz \*40 I *\ Ii201N. / infus n1!-‘—---i00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Time (hours)100•High ECOG800 60:TIT1TI0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Time (hours)Figure 45: Mean (± SEM) high, intermediate, and low electrocortical activity in fetalsheep following simultaneous fetal and maternal infusions of{2H10]DPHMand DPHM, respectively. (n=4)157100•Fetal Breathing Activity808) 60E4020Infusion0 i I I I I I I I I I I0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Time (hours)200 * * *Fetal Heart Rate19080(0.ii*II1TiT170a)a) 160 j150ifu1sina)z140 i p I I I I I I I0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Time (hours)Figure 46: Mean (± SEM) fetal heart rate and fetal breathing movements followingsimultaneous fetal and maternal infusions of[2HIOIDPHM and DPHM,respectively. (n=5)1583.3. In Vitro Studies3.3.1. Uptake of DPIIM in Blood CellsThe ratio of DPHM plasma concentration to DPHM BC concentration as a function of timeis shown in figure 47. The time required to reach equilibrium was rapid (i.e., <2.5 minutes). Theratio at equilibrium was close to 1, suggesting that the plasma concentration is a good reflection ofthe whole blood concentration of DPHM. The experiment investigating the effect of temperatureon DPHM uptake into BCs was to ensure that sample handling techniques (i.e., allowing thecollected samples to cool to room temperature prior to separating the BC and plasma fractions) didnot change the plasma DPHM concentration due to BC uptake. The ratio of DPHM in plasma toDPHM in BC was not significantly altered during when measured at either 25 or 39 °C.3.3.2. Plasma Protein Binding of DPMAAn equilibration time of 8 hours was required for the determination of the plasma proteinbinding of DPMA. No significant volume shifts were associated with this equilibrium time,while volume shifts did occur for longer dialysis times (i.e., 24 and 36 hours). Non-specificbinding of DPMA to the equilibrium dialysis cell and the membrane could not be detected. Themetabolite was highly bound in both maternal and fetal plasma, with the percent bound being99.4 ± 0.0 1% and 98.9 ± 0.07% for the five replicates, respectively (n=5). The free fraction ofDPMA in adult plasma was 0.006 ± 0.002, while the free fraction of DPMA in fetal plasma was0.010 ± 0.001.1591.50-I T I0 T415:Time (minutes)Figure 47: Time course for DPHM to distribute into blood cells3.3.3. Fetal and Adult Hepatic Microsomal Metabolism of DPHMThe metabolism of DPHM was examined using hepatic microsomes prepared from non-pregnant adult sheep and fetal lambs. The formation of N-demethyl DPHM was monitored using apreviously published analytical method (Abemethy and Greenblatt, 1983, and Blyden et al, 1986).This method was adapted for GC-MS application from GC-NPD. The fragment ions m/z 165 and167 were monitored for N-demethyl DPHM. Quantitation of N-demethyl DPFIM over aconcentration range of 2.5 to 250.0 ng/mL was not possible due to the non-linear nature of thecalibration curve above concentrations of 50.0 ng/mL. Thus, measurements of N-demethyl DPHMwere made within the linear range of the calibration curve (i.e., 2.5 to 50.0 ng/mL).The microsomal protein concentration and P450 content are presented in table 25 Thegestational ages of the fetuses used in this experiment were between 131 and 138 days (term = 145160days). Although both protein concentrations and P450 concentrations could be measured in adultmicrosomes, only the protein concentration could be measured in the fetal microsomes. This wasdue to both the contamination of the fetal microsomal preparation with hemoglobin and the lowamount of P450 present in the fetal microsomes.All incubations were carried out at 390 C. The incubation protocol was optimized forprotein concentration, substrate concentration, and time. Incubations were optimized for 2.0nmoles of P450. Because it was not possible to accurately estimate the P450 content in fetalmicrosomes, incubations were carried out with 1.0 mg of microsomal protein from either fetal oradult source. The amount of substrate used was 0.5 jimoles. No product (i.e., N-demethyl DPHM)was formed in incubations containing boiled microsomes, microsomal protein with no co-factors(i.e., NADH and NADPH), or no microsomal protein. Fetal and adult time course experiments areshown in figures 48 and 49. In adult microsomes, the amount of DPHM was found to decrease;however, the quantity of DPHM which was consumed did not correlate well with the N-demethylDPHM formed; that is, only 10% of the missing DPHM could be accounted for by N-demethylDPHM. In addition, N,N-didemethyl DPHM was not detected. Fetal microsomal incubationsresulted in less of a reduction in the amount of DPHM over time, and smaller amounts of Ndemethyl DPHM were formed. The concentrations of N-demethyl DPHM formed could not beaccurately estimated using the present chromatographic method due to the co-elution of the parentdrug (very high concentration) with the metabolite (very low concentration). Subsequentincubations in both fetal and adult microsomes were conducted for 90 minutes with 0.1 imoles ofsubstrate. These studies also demonstrated that the formation of N-demethyl DPHM was roughlyseven fold less (84% less) in hepatic microsomes prepared from fetal lambs compared to thosefrom adult sheep (Figure 50). However, a similar amount of DPMA was produced in both fetaland adult microsomes (Figure 51).161Table 25: Protein and Cytochrome P450 concentrations in fetal and adult microsomes.Animal # P450 content Protein Conc. P450/protein(nmollmL) (mg/mL) (nmol/mg)Adult #1 22.8 13.4 2.13Adult#2 28.5 11.4 2.50Adult#3 32.6 10.4 3.13Adult#4 15.5 10.1 1.53Adult#5 24.4 13.2 1.85Adult#6 14.8 8.5 1.74Fetus#230 na 3.92 naFetus#562 na 5.02 naFetus#1 143 na 5.21 na162—.--- DPHM—o— N-demethyl-DPHM600550-— 500 ITT ITE I20 -e- —-—0-——— —0-————Q0 I0 10 20 30 40 50 60Time (minutes)Figure 48: Time course of DPHM disappearance and N-demethyl DPHM production in adultmicrosomes.—.--- DPHM600-550-.500°}—}+I’ I400 135003002500 10 20 30 40 50 60Time (minutes)Figure 49: Time course of DPHM disappearance in fetal microsomes.(n=5)(n=3)-rMATERNAL16387.C600.0) 5EC00) -00)02C0FETALFigure 50: Production of N-demethyl DPHM in fetal and maternal microsomes following a 90minute incubation.87 (n=3) (n=5)a)4-000)00) 3ci)0 2E10FETAL MATERNALFigure 51: Production of DPMA in fetal and maternal microsomes following a 90 minuteincubation.1644. Discussion4.1. Development of a Gas Chromatographic-Mass Spectrometric Method for theSimultaneous Quantitation of DPHM and[2H10]DPHM from Biological FluidsObtained from Pregnant SheepThe simultaneous co-administration of stable isotope labeled and unlabeled drug offersseveral advantages over traditional experimental designs where only unlabeled drug is available,particularly in investigations of fetal/maternal pharmacokinetics (See Section 1.6.). In order toaddress many of the questions posed by previous studies examining DPHM disposition in theovine maternal/fetal unit, it was necessary to obtain a stable isotope labeled form of the drug. Asynthetic method for tritium labeled DPHM has been reported in the literature (Blackburn andOber, 1967); however, there are no reports describing the synthesis of a stable isotopicallylabeled form of the drug. Stable isotope labeling can be accomplished through a variety ofmeans, including deuterium-hydrogen exchange, chemical synthesis, derivatization, andbiosynthesis (Roncucci et al., 1976). Initial attempts to utilize deuterium exchange in thepresence of a catalyst, aluminum chloride, resulted in the formation of only very small quantitiesof[2H1]DPHM and[2H]DPHM. Therefore, this approach was abandoned. Chemical synthesisappeared to be a better alternative for the production of[2H10]DPHM, particularly due to the highisotopic purity of the inexpensive synthetic precursor (i.e.,[2H5]benzene > 99.7% isotopicpurity). The synthesis method reported in this thesis provided sufficient quantities of bothchemically and isotopically pure[2H10JDPHM for experimental and analytical use.The simultaneous administration of DPHM and[2H10]DPHM to chronically instrumentedpregnant sheep requires that there be an analytical method capable of differentiating betweenunlabeled and labeled drug when present together in a sample. Since the GC-NPD method used165in previous studies could not distinguish between{2H10]DPHM and DPHM (Yoo et at., 1986), anew method was required. The most readily available and powerful (sensitive and selective)detection technique for routine quantitation of STh compounds and their unlabeled counterparts ismass spectrometry (Chasseaud and Hawkins, 1990). To quantitate DPHM and[2H10]DPHMindependently in the same sample using mass spectrometry, the appropriate fragment ions mustbe selectively monitored. The ions selectively monitored should differentiate between labeledand unlabeled drug, be present in sufficient quantity (abundance) to provide the requiredsensitivity, and be free of undue chromatographic interference from co-eluting endogenouscomponents. Following GC-MSIEI, DPHM and[2H10]DPHM fragment extensively (Figure 8),as reported previously for DPHM (Chang et at., 1974). This limits the choice of fragment ionsfor SIM which meet the criterion outlined above. To avoid the extensive fragmentation,preliminary attempts were made to quantitate both DPHM and[2H10]DPHM using GC-MS withmethane positive chemical ionization (PCI). Methane PCI also resulted in extensivefragmentation of the compounds, and a further reduction in sensitivity; thus, El ionization wasused. The fragmentation of DPHM and[2H10]DPHM following GC-MSIEI results in a basefragment ion of m/z 58 with no apparent molecular ions. The previously reported GC-MSanalysis methods for DPHM all use the base fragment ion (m/z 58) for SIM (Carruthers et at.,1978, Rohdewald and Milsmann, 1986, Maurer and Pfleger, 1988, and Walters-Thompson andMason, 1992). Since the deuterium labels of[2H10]DPHM reside on the aromatic rings, the SIMof fragment m/z 58 could not provide the necessary differentiation between DPHM and[2H10JDPHM (Figure 8). The fragment ions which meet the criteria for SIM analysis of DPHMand[2H10]DPHM outlined above were determined to be m/z 165 (DPHM and orphenadrine) andm/z 173([2H10]DPHM).166The sensitivity of a quantitative method employing mass spectrometry can be enhancedby using SIM rather than scanning the entire mass range (as outlined above). In addition, thesensitivity can be further augmented by the appropriate tuning algorithm. Several tuningalgorithms were tested, including the auto, mid-mass, and manual tune. The manual-tune optionoptimized the mass spectrometer performance over the narrow mass range of m/z 100 to 219,rather than m/z 50 to 502 (auto-tune). This modification increased the sensitivity of theanalytical method by a factor of approximately 20 fold. The use of both SIM and the appropriatetuning algorithm has provided the necessary sensitivity for the analysis of DPHM and[2H1O]DPHM.A complication encountered during assay development resulted from the co-elution of Ndemethyl DPHM with DPHM on an Ultra-2 capillary column (i.e., liquid phase 5%phenylmethylsilicone). Previously, using GC-NPD, N-demethyl DPHM was not detected;however, due to a similar fragmentation pattern (m/z 165 was also prominent for N-demethylDPHM), N-demethyl DPHM now interfered with the quantitation of intact drug (Figure 9).Consequently, a capillary column with 5% phenylmethyl: 7% cyanopropylsilicone liquid phasecoating (DB-1701) was used. This column provided the necessary separation of DPHM and[2H10]DPHM and their respective N-demethylated metabolites. The chromatography of thecompounds was enhanced on this column through thermal trapping of analytes at the head of thecolumn by keeping the initial column temperature low (140° C), as outlined by Sandra (1989).This resulted in sharper, narrower, and more symmetrical chromatographic peaksThe narrower and sharper peaks produced by the enhanced efficiency of capillary GCrequires that an additional precaution be considered when conducting quantitative massspectrometry using SIM. Sufficient samples must be collected (scans/peak) over the time167window in which the chromatographic peak elutes, to ensure the accuracy of the peak shape andarea (Pettit, 1986, and Falkner, 1982). The dwell time, i.e., the time the mass spectrometerfocuses on one of the selected ions, should be adjusted so that at least 10-20 samples can becollected across the chromatographic peak of interest (Pettit, 1986, and Falkner, 1980). Thecurrent method uses a dwell time of 50 msec (—50 scans/peak) to ensure adequate sampling ofthe peaks of interest.Utilization of[2H10JDPHM can result in “isotope effects” (i.e., the differentiationbetween the labeled and unlabeled compound) in vivo, in vitro, and within the GC-MS (VanLangenhove, 1986). Isotope effects were evident within the GC-MS both with regard to theelution and the fragmentation of[2H10]DPHM.[2H10]DPHM was found to elute more rapidlythan DPHM (Figure 14). In addition, the abundance of the fragment m/z 165 (DPHM) wasgreater than the corresponding fragment m/z 173 for[2H10]DPHM (Figure 8). The lowerabundance of the m/z 173 fragment of[2H10]DPHM may be due to the breaking of a carbondeuterium bond involved in the formation of this fragment. This deuterium-carbon bond is morestable than the equivalent carbon-hydrogen bond, and thus, render the molecule less likely tofragment in this fashion (Van Langenhove, 1986, and McCloskey et al., 1967). Because thefragment abundances for the corresponding ions (i.e., mlz 165 and 173) were not equivalent, itwas necessary to simultaneously evaluate standard calibration curves for both DPHM and[2H10JDPHM.A common problem encountered during liquid-liquid extraction of tertiary amine analytesis non-specific binding to glassware (Smith and Stewart, 1981, and Jack, 1990). Numerousmethods have been employed to prevent this phenomenon, including the use of silanizedglassware. However, silanization is not always effective (Jack, 1980). The use of triethylamine168(TEA) in the extraction solvent has been suggested to prevent the binding of tertiary amines tonon-specific binding sites on glassware and other surfaces (Gupta and Molnar, 1979). Indeed,the use of 0.05 M TEA was found to aid significantly in the extraction recovery of DPHM and[2H10}DPHM (Figure 12), while the use of silanized glassware did not result in a further increasein the extraction recovery of the compounds.The validation of the analytical method involved estimation of intra- and inter-dayvariability. In addition, the method was cross-validated with a previously published method forthe quantitation of DPHM (Yoo et al., 1986). The estimates of intra-day variability for DPHMand[2H10]DPHM were below 17% at 2.0 ng/mL, and below 8% at all other concentrationsinvestigated in all three of the biological matrices tested (Table 1). The measured inter-dayvariability for the compounds was below 15% at 2.0 ng/mL, and below 10% for all other points(Table 2). These values fall within the acceptable guideline of ± 20% at the LOQ and ± 15% atother concentrations above the LOQ (Carr and Wahlichs, 1990, and Shah et at., 1992).Furthermore, the correlation between the concentrations measured using the GC-NPD method(Yoo et at., 1986) and the current method was excellent, suggesting that the two methods arehighly comparable. The results of these validation experiments and the experiments detailing thesample stability during storage and sample work-up suggest that the method developed is robust,and measurements of DPHM and[2H10]DPHM concentrations in the biological matricesexamined can be made with a high degree of confidence. The minimal quantifiableconcentration or the LOQ of the previously published GC-NPD used in the quantitation ofDPHM in biological fluids obtained from pregnant sheep was also 2.0 ng/mL; therefore, thismethod does not offer any advantage over the previously published method with regard tosensitivity (Yoo et at., 1986). Rather, the advantage of the current method is the ability to169simultaneously quantitate both DPHM and[2H10JDPHM when present together in a biologicalsample during one chromatographic run.4.2. Development of a Gas Chromatographic-Mass Spectrometric Method for theSimultaneous Quantitation of DPMA and[2H10]DPMA in Ovine Plasma and UrineA better understanding of the in vivo metabolism of the drug can be obtained through thestudy of the resulting metabolites following administration of the intact drug. Of the metabolitesidentified to date, unconjugated and various conjugates of DPMA have been shown to beprominent urinary metabolites of DPHM in dogs, rhesus monkeys, and humans (Drach andHowell, 1968, Drach eta!., 1970, Chang et al., 1974, Glazko et al., 1974, and Luo eta!., 1991).Furthermore, unconjugated DPMA has also been detected in plasma and urine of adult sheepfollowing the administration of DPHM, suggesting that a similar pathway is also functional insheep. The availability of[2H10]DPHM and an analytical method for the simultaneousquantitation of DPHM and[2H10]DPHM has facilitated the application of stable isotopetechniques to study the pharmacokinetics of DPHM in pregnant, non-pregnant, and fetal sheep.Thus, the development of an analytical method for the quantitation of DPMA and[2H10]DPMAwas pursued. Both DPMA and[2H10 ]DPMA were synthesized and purified. In addition, a GCMS/El analytical method using SIM was developed to simultaneously quantitate both[2H10]DPMA and DPMA.The derivatization of analytes for GC-MS quantitation can serve a variety of purposes.The derivatization of polar molecules can increase their volatility, and thus make them amenableto GC analysis. In addition, derivatization can improve instrument sensitivity for an analytethrough improved chromatography and fragmentation characteristics during GC-MS analysis170(Ahuja, 1976, and Pierce, 1993). Because DPMA and[2H10]DPMA contain polar carboxylicacid functional groups, these analytes required derivatization. Two derivatives were testedduring method development; namely, tert-butyldimethylsilyl (TBDMS) and pentafluorobenzyl(PFB) derivatives. Both types of derivatives of DPMA and[2H10]DPMA resulted in goodchromatography. The PFB derivatives did not offer any advantages over the TBDMS derivativesfor GC-MSIEI quantitation and were more difficult to prepare; therefore, the TBDMS derivativeswere utilized.The tert- butyldimethylsilyl (TBDMS) derivatives of DPMA and[2H10]DPMA underwentextensive fragmentation under El conditions (70 eV), with the majority of fragments resultingfrom the breakage of the ether linkage of DPMA and[2H10JDPMA (Figure 19). No molecularions were detected, and the characteristic fragments [M-57] (i.e., the loss of the tert- butylgroup) were present only in low abundance (Figure 19). Extensive fragmentation of thetrimethylsilyl derivative of DPMA was also noted by Chang et al.( 1974). Nevertheless, therewere a number of fragment ions which retained the stable isotope label and were of goodintensity for SIM quantitation of DPMA and[2H10]DPMA (m/z 167 and 183 - DPMA, and m/z177 and 193 -[2H10]DPMA). Initially, fragment ions m/z 183 and 193 were chosen for thedevelopment of the analytical method. However, it was discovered that the fragmentation ofDPMA also resulted in a small m/z 193 fragment ion of unknown origin (Figure 19). Thisfragment resulted in chromatographic interference in the m/z 193 ion chromatogram used toquantitate[2H10]DPMA. Although fragment ions m/z 167 and 177 gave good sensitivity andretained the stable isotope label, co-extracted components from adult urine co-eluted withDPMA in the m/z 167 ion chromatogram, resulting in the inability to reliably quantitate DPMAin urine. As a result, the fragment ions m/z 183 and 177 were chosen to quantitate DPMA and171[2H10}DPMA in plasma and urine. Despite the fact that this choice of fragment ions was lessoptimal than fragment ions from the corresponding fragments of unlabeled and labeledmetabolite (i.e., m/z 167 and 177, or 183 and 193), the assay method appeared to be free fromcomplications resulting from this choice of ions (i.e., non-linearity etc.). In addition, intra-dayvariability studies conducted in plasma using ions m/z 167 and 177, 183 and 193, or 183 and 177all gave similar results (unpublished results), suggesting that this choice of ions would beadequate for the quantitation of DPMA and{2H10JDPMA. Furthermore, the ion chromatograms(i.e., m/z 183 and 177) were free from chromatographic interference due to the elution ofendogenous components in ovine plasma and urine at the retention times of DPMA and[2H10]DPMA (Figure 20). Thus, despite the inability to use corresponding fragment ions forDPMA and[2H10]DPMA, fragment ions m/z 183 and 177 for SIM appeared to be suitablealternatives.The extraction recovery of DPMA and[2H10]DPMA from plasma appeared to be constantat different concentrations (i.e., 5.0, 50.0, and 500.0 ng/mL) of the analyte. However, while theextraction from plasma was constant, there was an unexplained concentration dependence ofanalyte recovery from urine. That is, the extraction recovery was 95 and 99% at 5.0 ng/mL ofDPMA and[2H10]DPMA, respectively, while at higher concentrations the average recoveries forthe compounds were 76% and 74% at 50.0 and 500.0 ng/mL, respectively. At the higherconcentrations, the extraction recovery in urine and plasma were comparable. The reason for thehigher recoveries from urine at the lower concentrations of DPMA and[2H10IDPMA is not clear.Poor sample stability either during storage and/or sample preparation can result inerroneous quantitation, which can lead to inaccurate interpretation of pharmacokinetic data. Thestability of DPMA and[2H10]DPMA was shown to be adequate in most circumstances; however,172the analyte was found to be labile in the presence of acid (Figure 23). The extraction conditionsemployed in this assay required the acidification of the plasma and urine biological matrices with1.0 M hydrochloric acid. Caution must be used during the extraction procedure to ensure thatexcess hydrochloric acid is not added, and further, that the extraction procedure is conductedrapidly. For our study, the addition of 400 jiL of 1.0 M HC1 was found to provide an adequatereduction in pH for plasma and urine (pH < 2) without significant loss of DPMA and[2H10]DPMA. Excess acid (> than 1.0 mL of 1.0 M HC1) was found to reduce the recovery ofDPMA and[2H10JDPMA and substantially increase the sample to sample variability. Stabilitystudies conducted under an acidic environment, similar to that encountered during samplepreparation, have shown that DPMA was stable during the course of an extraction. Themeasured half-lives for the degradation of DPMA in sample matrices would translate into thedegradation of 1.0%, 0.6%, and 0.4% of DPMA in water, plasma, and urine during the 30 minutetime interval required for the extraction procedure. Thus, despite the acid labile nature ofDPMA, degradation during a rapid extraction (less than 30 minutes) using the extraction methodoutlined above would appear to only minimally contribute to a decrease in the recovery ofDPMA and[2H10]DPMA.The results of the method validation experiments (i.e., intra-day and inter-day variabilitystudies) and the analyte stability experiments suggest that the newly established GC-MS assaymethod is robust, and that the concentrations of DPMA and[2H10]DPMA in ovine plasma andurine samples can be measured with a high degree of confidence.1734.3. Disposition of DPHM in Non-pregnant SheepThe pharmacokinetic parameters obtained during studies examining the disposition ofDPHM in non-pregnant sheep following a 100 mg IV bolus agree with the results obtainedpreviously during a dose ranging study of the drug in non-pregnant sheep (Yoo et al., 1990). Theclearance estimates in sheep (53 mL/minlKg) during the current study are greater than theclearances measured in humans (6-22 mL/minlKg) (Carruthers et al, 1978, Meredith et al., 1984,Blyden et a!., 1986, and Simons et a!., 1990). However, values for Vd8 appear to be similar inboth sheep (3.4 L/Kg) and humans (3.2-14.6 LIKg) (Carruthers et a!, 1978, Meredith et a!., 1984,Blyden et a!., 1986, and Simons et al., 1990). Unlike the previous studies examining DPHMdisposition in non-pregnant sheep, this experiment also detailed the renal clearance of the drug,and its contribution to the total body clearance. The renal clearance of DPHM was 0.36 ± 0.28mL/minlKg, which corresponds to only -.0.3% of the total body clearance of the drug. Theexcretion of unchanged DPHM in the urine in other species, including humans (<4%), rhesusmonkeys (-3%), dogs (-4%), rats (—1%), and rabbits (< 3%) is greater than in sheep (Albert eta!., 1975, Drach et al., 1970, and Parry and Calvert, 1982). These findings suggest that othernon-renal routes play an important role in the elimination of DPHM from sheep. In the presentstudy, cumulative bile collections were not possible; therefore, an accurate estimate of biliarysecretion of unchanged DPHM could not be calculated. The concentrations of unchangedDPHM in the bile from the passive collections (for 10 minute intervals at 15, 30, 45, 60, 90, 120,240, 360, and 720 minutes) were similar to the plasma concentrations of DPHM in the twoanimals in which bile was collected. Based on these concentrations and a literature value of theaverage bile flow in sheep (9.4 jiL/minlKg; Erlinger, 1982), a crude estimate of DPHM biliarysecretion can be made. This estimate suggests that approximately 100 jig, or 0.1%, of the174administered dose is secreted as intact drug in the bile. Thus, it appears that neither renal norbiliary elimination of intact DPHM can account for a significant portion of the administereddose, and thus, the total body clearance in sheep. It is, therefore, likely that hepatic or extrahepatic biotransformation of DPHM and/or non-reversible tissue binding plays a major role inDPHM elimination in this species.4.4. Isotope Effect StudiesThe application of stable isotope labeled drugs in analytical chemistry, pharmacology,pharmacokinetics, and drug metabolism has proven to be useful (Browne, 1986, Browne, 1990,Eichelbaum et al., 1982, Baillie, 1981, and Murphy and Sullivan, 1980). In pharmacokineticstudies, stable isotopically labeled drugs have often been co-administered with their unlabeledcounterparts to assess both absolute and relative bioavailability, drug distribution,biotransformation and excretion, chrono-pharmacokinetics, dose-dependent pharmacokinetics,and drug-drug interactions (Browne, 1990, and Murphy and Sullivan, 1980). However, tosuccessfully utilize a SIL drug in pharmacokinetic studies which involve the administration ofboth labeled and unlabeled drug, it is imperative that the SIL drug be “pharmacokineticallyequivalent” to its unlabeled counterpart (Baillie, 1981). In effect, the labeled drug must sharesimilar absorption, distribution, elimination, protein binding, and metabolism characteristics withthe unlabeled drug (Van Langenhove, 1986, and Eichelbaum et al., 1982). Instances can arisewhere the disposition of the labeled drug can be different from that of the unlabeled drug; this isreferred to as an “isotope effect” (Van Langenhove 1986). Use of deuterium labeled drug canresult in two types of isotope effects, namely, a kinetic effect and/or a physicochemical effect. Achemical bond between a heavy isotope and another atom will be stronger than the equivalent175bond between the light isotope and that atom. If breaking this bond is a rate limiting step in thebiotransformation and subsequent elimination of the compound, then the labeled drug may beeliminated slower than the unlabeled drug, yielding a kinetic isotope effect (Melander andSaunders, 1980). Physicochemical isotope effects can occur due to differences in pKa, lipidsolubility, and possibly plasma protein binding of the labeled compound due to incorporation ofdeuterium (Van Langenhove, 1986). The presence of an in vivo “isotope effect” during apharmacokinetic study involving the simultaneous administration of both labeled and unlabeleddrug can seriously limit the usefulness of a SIL drug. For pharmacokinetic studies, isotopeeffects must either be absent or non-significant (i.e., within the error limits of the measurementmethod) (Wolen, 1986, Browne, 1990, VandenHeuvel, 1986, and Van Langehove, 1986). Acommon method for assessing the presence or absence of an isotope effect involves thesimultaneous administration of equimolar doses of both labeled and unlabeled drug, andmeasurement of both the pharmacokinetic parameters to be studied and the concentrations oflabeled and unlabeled drug in the appropriate biological fluids (plasma, urine, etc.). (Eichelbaumet at., 1982, Browne, 1990, and Baillie, 1981). Therefore, prior to utilizing[2H10]DPHM on aroutine basis, isotope effect studies must be carried out.Three studies were conducted, namely, adult bolus, fetal bolus, and fetal infusion, inorder to rule out the presence of an isotope effect in the disposition of[2H10]DPHM. Nodifferences were evident between DPHM and[2H10]DPHM plasma concentrations, AUC ratios,and pharmacokinetic parameters calculated during these experiments. The plasma data suggestthat our experimental approach (the simultaneous administration of DPHM and[2H10IDPHM)can be used in pharmacokinetic studies conducted in the ovine fetal/maternal unit without theresults being confounded by the presence of an isotope effect. However, an additional precaution176of alternating the route of administration of DPHM and[2H10]DPHM was taken in all studiesexcept the simultaneous fetal/maternal infusions. This was to ensure that possible subtledifferences which may not have been detected by the experiments conducted above would notbias our results. It was also demonstrated that no differences were detected between theconcentrations of DPHM and[2H10JDPHM in adult ovine urine (adult bolus), fetal tracheal andamniotic fluids (fetal bolus studies), and fetal urine and amniotic fluid (fetal infusion studies).Furthermore, the analysis of DPMA and[2H10]DPMA in maternal plasma and urine (adult bolus)and in fetal plasma (fetal infusions) suggests that there is no isotope effect in the disposition ofthis metabolite. However, this must be independently confirmed via a simultaneousadministration of both DPMA and[2H10]DPMA.The pharmacokinetic parameter estimates for DPHM and[2H10]DPHM in the adult non-pregnant sheep correlate with the estimates reported by Yoo et al. (1990). However, followingfetal bolus administration of intact drug, the estimates of o and I are greater than the fetalparameters calculated following maternal bolus administration (Yoo et al., 1986). In addition,fetal CLT of DPHM following fetal administration (1.0 minute bolus and 90.0 minute infusion) is2-3 fold larger than that following a fetal infusion to steady-state (Yoo et al., 1993). The “sink”conditions in the ewe (i.e., a large constant concentration gradient from fetus to mother)following a fetal bolus could lead to increased drug transfer from fetus to mother, and could bepartially responsible for this observation. In the current study the weight corrected value for fetalCLT is almost 10 fold greater than the corresponding weight corrected estimate for CLT in adultsheep (Tables 5, 7, and 8). A similar observation has also been made for DPHM following fetaland maternal infusions to steady-state; however, the magnitude of the difference reported in thisstudy is much greater than that previously reported (10 fold vs. —3 fold) (Yoo et al., 1993). In177addition, the Vd in the fetus was also much larger than that observed in the ewe (22.3 ± 8.7L/Kg fetal weight vs. 1.8 L/Kg maternal weight). The reasons for the large differences betweenthe fetal and maternal weight corrected Vd and CLT are presently not well understood.However, it is possible that the large absolute maternal volume of distribution and clearance maybe contributing to the fetal estimate following fetal drug administration due to the rapid fetal tomaternal transfer of DPHM across the placenta.4.5. Hepatic First Pass Metabolism of DPHM in Adult and Fetal Sheep4.5.1. Hepatic First Pass Metabolism of DPHM in Adult Sheep During NormoxiaIn adult non-pregnant sheep, DPHM undergoes extensive first-pass metabolism followingmesenteric venous administration, with only 6.8 ± 3.1% availability. This means that 93.2 ±3.1% of the dose is eliminated prior to reaching the systemic circulation following mesentericvenous (portal) administration. From these data it appears that hepatic metabolism and/orhepatic uptake of DPHM is a major component of the total body clearance in non-pregnant ewes.Hepatic clearance (CLH) can be estimated using the equation CLH = QH*E, where QH is thehepatic blood flow and E is the extraction ratio across the liver (Gibaldi and Perrier, 1982, andGeorge and Shand, 1982). Because the concentration of DPHM in ovine blood cells was notsignificantly different than the drug concentration in plasma (See Section 3.4.1), an estimate oftotal hepatic blood flow was used to calculate the hepatic clearance of DPHM (Gibaldi andPerrier, 1982). Using a literature value for hepatic blood flow in adult non-pregnant sheep (QH)of 55 mL/min/Kg, the hepatic clearance of the drug is estimated to be -5 1 mL/min/Kg (Katz andBergmann, 1969). Since the measured total body clearance of DPHM is 65 mL/minlKg,178approximately 80% of the total body clearance of this compound can likely be attributed tohepatic uptake or metabolism. This data would appear to confirm the results obtained earlier innon-pregnant sheep, where renal excretion of intact drug did not contribute significantly to thetotal body clearance. The availability of DPHM measured in sheep is lower than that reported inhumans following oral administration (43-72%) (Albert et al. 1975, Carruthers et al., 1978,Spector et at., 1980, Meredith et al., 1984, and Blyden et al., 1986). However, in both species(humans and sheep) the hepatic clearance of the drug appears to contribute significantly to theobserved total body clearance (Meredith et al., 1984). The current data (i.e., the high hepaticextraction ratio - 90% of DPHM) would suggest that the hepatic clearance of DPI{M is high, orliver blood flow dependent (CLH QH) (Wilkinson and Shand, 1975). That is, it is likely thatchanges in liver blood flow could influence the hepatic clearance of DPHIvI following systemicadministration.4.5.2. Hepatic First-Pass Metabolism of DPHM in Adult Sheep during Mild HypoxemiaDrug elimination is a function of the delivery of the substrate to the organ of metabolismand the capability of that organ to eliminate the drug. Hypoxia has been demonstrated to alterboth of these functions (du Souich, 1978), and thus lead to changes in the pharmacokineticparameters. The current study was undertaken in non-pregnant ewes to investigate the influenceof acute mild to moderate hypoxemia on the pharmacokinetics and first-pass hepatic metabolismof DPHM. The infusion of N2 gas into the lung via a tracheal catheter in non-pregnant sheepresulted in significant reductions in Po2 and 02 saturation. All five animals experienced similarmean reductions in Po2 and 02 saturation (Po2 48.8 ± 3.1 % and 17.7 ± 1.1% 02 saturation)(Table 10). However, two ewes (1158 and 1154) experienced more severe initial reductions in179Po2 and 02 saturation (over the first 30 minutes) than the other three animals (60 vs. 40 %reduction in Po2, and 30 vs. 10% reduction in 02 saturation, respectively). Although there was atendency for CLT and Vd to increase during hypoxemia, these differences were not statisticallysignificant from values calculated during the normoxic period (Mann Whitney U-Test, p>O.05).The hepatic first-pass extraction of DPHM appeared to be extensive in three of the animalsfollowing mesenteric administration, as reported in the normoxic period. In the other two ewes alarge increase in bioavailability was noted (i.e., a 70 and 220 % increase; Table 11). The reasonfor the disparity within this study is unclear at present; however, the two animals in which thebioavailability increased were also the two animals which showed the greatest initial decrease inPo2 and 02 saturation (ewes 1154 and 1158). Since DPHM appears to be a high hepaticclearance drug (Section 4.5.1.), changes in both blood flow and hepatic intrinsic clearance mayinfluence the bioavailability, while only changes in hepatic blood flow will alter the systemicclearance (Wilkinson and Shand, 1975). Wilkinson and Shand (1975) have also pointed out thatfor a high clearance drug, a small decrease in the intrinsic clearance may result in a large increasein bioavailability, and that an increase in the hepatic blood flow will result only in aproportionate reduction in the bioavailability (i.e., F = QH/(QH + fub*CLjflt), where fb is thefraction unbound, and CL1 is the intrinsic clearance of the free drug by the liver; Wilkinson,1983). Thus, a reduction in hepatic intrinsic clearance of DPHM induced by the initialhypoxemic insult may partially explain the results obtained in the two animals in which largeincreases in the bioavailability were noted with no apparent changes in systemic clearance. Thereason similar changes do not occur in the other three animals in this study is not clear, but maybe due to initial reductions in Po2 and 02 saturation not reaching a critical threshold which wouldreduce the intrinsic clearance of DPHM. Such a phenomenon has been demonstrated for the180intrinsic clearance of propranolol in the isolated perfused rat liver. Reductions in hepatic oxygendelivery do not alter propranolol clearance until a threshold is reached, after which thepropranolol clearance decreases in a linear fashion with a reduction in 02 delivery (Elliot et al.,1993). Further, reductions in the total body clearance of lidocaine and indocyanine green (highclearance drugs) have been demonstrated in vivo with rabbits (Marleau et al., 1987). Thisreduction was speculated to be due to a reduction in the intrinsic clearance of both lidocaine andindocyanine green. However, in a subsequent study of mild to moderate hypoxia in dogs, areduction in lidocaine clearance could not be shown (du Souich et aL, 1992). It has beensuggested that the threshold for decreasing the intrinsic clearance of various drugs appears to behighly species- and substrate-specific, and in some cases may even occur during mild hypoxia(Angus et al., 1990, and Jones et al., 1989). Thus, it appears that a reduction in the intrinsicclearance of DPHM due to the initial hypoxemic insult may partially explain the increase inbioavailability in the two animals with the largest initial reduction in Po2 and 02 saturation.However, there was also a lag period in achieving the maximum plasma concentrations of drugin the systemic circulation following mesenteric bolus administration in the two ewes mostseverely affected by hypoxemia (1154 and 1158). The reason for this is not clear. Changes ingut and portal venous blood flow due to redistribution of cardiac output do not appear to occurduring mild to moderate hypoxia (du Souich et al., 1992). However, it is difficult to comparethe current study to previous studies, because in the current study hypoxemia was induced muchearlier (i.e., 15 minutes) relative to drug administration. Nesarajah et al. (1983) havedemonstrated that changes in blood flow distribution can occur immediately following acutesevere hypoxia, while changes in blood flow redistribution following prolonged mild to moderatehypoxia are unconmion. Therefore, it is possible that immediate changes in gut and liver blood181flow redistribution have influenced the bioavailability of DPHM in the current study. However,changes in hepatic tissue binding and or uptake/release of DPHM during the hypoxemic episodemay also contribute to this phenomenon. Jones et al. (1984) demonstrated an apparent release ofpropranolol from isolated rat livers into the perfusion media following 20 minutes of severehypoxia. Whether a similar phenomenon occurs with DPHM, resulting in a release of drugbound in the liver during mild to moderate hypoxemia in sheep, is presently not known. Thus,the exact reason for this increase in the bioavailability in two of the ewes in the current study isnot known; however, it is possible that changes in hepatic intrinsic clearance, hepatic blood flow,and hepatic binding may all have been contributing factors.4.5.3. Fetal Hepatic First-Pass Metabolism Following Umbilical Venous AdministrationTo examine the extent of the DPHM first-pass effect in the fetal lamb, we chose to employsimultaneous, paired injections of DPHM and[2H10]DPHM via the abdominal inferior cava (i.e.,tarsal vein) and umbilical vein. The latter route was utilized rather than the mesenteric veinbecause of the large proportion of total hepatic flow supplied by the umbilical vein in the fetus(Edelstone et al., 1978, and Bristow et al., 1981), and because this is the route by which drugs reachthe fetus from the mother.Unlike the situation in adults, where the majority of hepatic blood flow comes from theportal vein (—80%) and the hepatic arteries (-.20%) (Katz and Bergman, 1969), the umbilical veinprovides a substantial additional vascular input to the fetal liver. This vessel supplies —93% of theblood flowing to the left lobe and -.60% of the perfusion of the right and caudate lobes of the liverin the fetal lamb (Edelstone et al., 1978, Bristow et al., 1981, and Holzman, 1984). The umbilical182venous contribution to hepatic perfusion comprises —50% of total umbilical flow (the remaining—50% bypassing the liver via the ductus venosus) (Edelstone et al., 1978, Bristow et al., 1981, andHolzman, 1984) (Figure 52). Thus, if the fetal liver were functional in drug uptake and/orelimination, a portion of the drug in the umbilical venous blood which traverses the fetal liver couldbe removed in a fashion analogous to hepatic first-pass metabolism in adults following oral orintraperitoneal drug administration. Previous studies of lidocaine disposition in pregnant sheephave demonstrated the preferential distribution of drug present in umbilical venous blood to thefetal liver following maternal administration. It was shown that two minutes following a maternalbolus dose of the drug, the concentration in fetal liver was 7.5 fold greater than the fetal plasmalevel of lidocaine, whereas the drug levels in maternal plasma and liver tissue were similar (Finsteret al., 1971). The difference between the ewe and fetus does not appear to be due to preferentialaffinity of the fetal liver for the drug, since a subsequent study found that lidocaine concentrationsin fetal liver do not exceed those in plasma 4 hours following drug administration (Kennedy et al.,1990). Tn these studies of lidocaine, the impact of the initial fetal hepatic uptake of the drug on fetalsystemic levels was not assessed. However, such a mechanism could function in minimizing fetalexposure to maternally-derived drugs.An alternative approach to that employed in the current study to assess fetal hepatic druguptake would be via the Fick method. This method would require the measurement of drugconcentrations in fetal arterial and umbilical and hepatic venous blood coupled with measurementof liver blood flow. Due to the multiple vascular inputs to the fetal liver, this approach is morecomplicated than in the adult. However, it has been employed in fetal lambs in utero to studyhepatic oxygen and carbohydrate utilization (Bristow et al., 1981), and in exteriorized fetuses toassess propranolol uptake by the liver (Marshall et al., 1981, and Mihaly et al. 1982, see below).183However, the Fick method involves extensive surgical preparation of the fetus, which is not arequirement with our approach.Fetal Liver (Dorsal View)Figure 52: An anatomical sketch of the fetal liver in sheep. HV - hepatic vein, 1VC - inferior venacava, DV - ductus venosus, UV - umbilical vein.Both bolus drug injection and infusion to steady-state were employed to assess fetal hepaticDPHM uptake. The former protocol was similar to that employed in the adult sheep. However, inmarked contrast to the results in non-pregnant ewes, the availability of DPHM following umbilicaladministration in the fetus was 1.10 ± 0.07, a value not significantly different from 1. However, ifenzymes capable of metabolizing DPHM are functional in the fetal liver, but present only in smallquantities, the bolus administration of DPHM could result in a large amount of the drug beingrapidly presented to the liver, leading to saturation of the enzymes and limited fetal hepaticmetabolism of the drug. The simultaneous umbilical and tarsal venous infusions over 90 minuteswere used to examine this possibility, as this would result in an approximately 100 fold lowerHVCaudate LobeGall Bladder184concentration of drug being delivered to the liver in umbilical venous blood (—0.15 mg/mL vs.—12.5 mg/mL for the bolus 1 mm injection, assuming an umbilical blood flow of 400 mLlmin).With the infusion protocol, the difference between the clearances calculated from the tarsal venous(control) and umbilical infusion (test) would give the net hepatic clearance of DPHM with the latterroute of administration. However, the difference between the clearance values (28.7 ± 44.8 and -20.1 ± 53.9 mlJmin/Kg for FA and CA values, respectively) were not significantly different fromzero. As shown in Table 14, the inter-animal variation in the values was large. In two animals(E#1 142 and 1242#1), the difference between umbilical and tarsal venous clearance values wasconsistently positive, suggesting hepatic DPHM uptake. However, in another ewe (E#2 164), aconsistent negative value was obtained, while for the other two there was no consistent difference.The source of this large variability is not known; however, it could result from the subtraction oftwo large, variable estimates of fetal total body DPHM clearance to yield a much smaller value for“hepatic clearance’ with a much larger degree of variability. Overall, the results of bothexperimental approaches indicate that the liver of the fetal lamb does not function effectively in thepresystemic removal of DPHIVI from umbilical venous blood, and hence that the fetal equivalent ofa hepatic first-pass effect cannot be demonstrated.The lack of an obvious fetal first-pass effect for DPHM does not rule out the possibility thatthe fetal liver can metabolize the drug. One obvious complicating factor in the study design is that—50% of umbilical venous blood bypasses the liver via the ductus venosus (Edeistone et al., 1978,and Rudolph, 1985), and so is not exposed to hepatic drug uptake. However, even with this shunt,if fetal hepatic metabolic capacity for DPHM were similar to that observed in the adult, we wouldexpect to observe a systemic availability of —60% (i.e., the —50% of total drug administered bypassing the liver through the ductus venosus, plus —20% of the remaining drug that passes through185the liver). This is far outside the 95% confidence interval calculated for the availability in the fetus.Thus, it appears that the ability of the fetal liver to metabolize the drug is far less than in the adult.However, data from in vitro studies of hepatic microsomes obtained from fetal lambs in lategestation indicate conjugation of morphine and acetanilnophen at appreciable rates (Dvorchik et al.,1986, and Wang et al., 1986b). In addition, glucuronide conjugates of ritodrine, labetalol, andacetaminophen are formed by fetal lambs in utero, though with the former 2 drugs, the involvementof the fetal liver in the conjugation reactions has yet to be demonstrated (Yeleswaram et al., 1993,Wright et al., 1991, and Wang et al., 1986a). Other drug biotransformation reactions have alsobeen demonstrated in fetal hepatic microsomal preparations, including the N-dealkylation ofmethadone and meperidine, and the hydroxylation of benzo[a]pyrene and hexobarbital. However,these occur at rates lower than observed in the adult sheep (Dvorchik et al., 1986). N-dealkylationand deamination reactions are involved in DPHM biotransformation in adults of other species (dog,human, and rhesus monkey) (Drach et al., 1970, Chang et al., 1974, and Glazko et al., 1974). Ifthese pathways are not developed to the same degree in the fetal lamb, then this could explain theapparent low extraction of DPFIM across the fetal liver. It is not certain how these in vitro resultscorrelate with fetal hepatic elimination of drugs in vivo. However, Rane et al (1976) demonstratedthat data from in vitro experiments (Km and Vmax) could be used to estimate the intrinsicclearance, and thus the hepatic first-pass metabolism of various drugs in vivo in adults. If a similarsituation exists in the fetus, then the -85% lower rate of N-demethylation of DPHM in fetal hepaticmicrosomes is certainly consistent with a lower first-pass effect for the drug in the fetus ascompared to the adult (see Section 3.3.3.).There appears to have been only one other attempt to assess fetal hepatic drug uptake. Thisstudy was conducted utilizing the Fick method to examine propranolol disposition in anaesthetized186fetal and adult sheep. In the adult, the drug was extensively extracted by the liver following portalvenous administration (Marshall et al., 1981, and Mihaly et al., 1982). In contrast, with portalvenous administration of the drug in the fetus, systemic availability was —1 (Marshall et aL, 1981),a result similar to that which we observed with umbilical venous administration of DPHM. Withmaternal infusion of propranolol, and thus delivery of the drug to the fetus via the umbilical vein,fetal hepatic extraction was .-.30% (Mihaly et al., 1982). However, the fetal right hepatic vein wassampled in these studies, and since the umbilical vein supplies only -60% of the flow to the rightlobe of the liver (Bristow et al., 1981), the observed extraction could have at least, in part, been dueto a dilutional effect resulting from the mixing of umbilical venous blood (high drug concentration)with blood from the portal vein (low drug concentration) in the right lobe of the liver. Also, theacute nature of these experiments could have affected fetal hepatic blood flow distribution viaanaesthetic effects or exteriorization of the fetus. More studies of fetal hepatic drug uptake andmetabolism are clearly warranted.Oxygen saturation and the concentrations of nutrients and endogenous metabolites havebeen noted to be higher in the carotid arterial (CA) blood than in femoral arterial (FA) blood(Charlton and Johengen, 1984, and Rudolph et al., 1991). A similar finding was also apparent inthe current study, in which statistically significant differences between FA and CA glucose andlactate concentrations, oxygen saturation, and oxygen partial pressure were noted. All weregreater in CA blood as compared to FA blood (See Table 15). The explanation for theseobservations primarily involves two factors: 1) the mixing in the thoracic vena cava of umbilicalvenous blood passing through the ductus venosus and inferior vena caval blood from the lowerbody, which results in concentrations of 02 that are substantially higher than in superior venacaval blood; and 2) there is preferential shunting of inferior vena caval blood through the187foramen ovale to the left heart, and therefore, a lesser degree of dilution of this blood withdeoxygenated pulmonary venous blood than is the case in the right heart, where the more highlyoxygenated inferior vena caval blood mixes with the superior vena caval return (Dawes, 1968,Rudolph, 1985, and Teitel et al., 1982). An additional factor may be the incomplete mixing ofductus venosus blood with abdominal inferior caval blood in the thoracic vena cava, andpreferential streaming of the former through the ductus venous. Evidence for this phenomenonhas come from studies demonstrating the preferential distribution to the fetal upper body ofradioactive microspheres injected via the umbilical vein (Edelstone and Rudolph, 1979). It hasbeen suggested that a similar distributional phenomenon may occur with drugs infused directlyinto the fetus (Rudolph, 1985). If this were to occur with maternally derived blood (i.e., via theumbilical vein), there could be increased delivery of drug to the fetal heart and brain. This wouldbe particularly significant for agents which affect CNS or cardiac function. Differences betweenFA and CA plasma concentrations of DPHM were not apparent following bolus administration;however, this is not unexpected since rapid mixing of blood occurs due to rapid fetal circulationtimes (2-4 seconds) (Power and Longo, 1975). However, a statistically significant difference wasnoted between the FA and CA plasma concentrations of DPHM following tarsal venous andumbilical venous infusions. Unlike the situation with 02, Po2, glucose, and lactate, the druglevel in the descending aorta was higher both with tarsal and umbilical venous infusions(although with the latter route of infusion the concentration difference between FA and CA wasless than the former route). An explanation for these results may involve the fact that the higher02 concentration in CA blood appears primarily due to the lesser degree of dilution of inferiorvena caval blood in the left heart, and hence, ascending aorta compared to the right heart anddescending aorta. In the fetal lamb, inferior vena caval blood comprises —77% of venous return188to the left heart, with the remainder being supplied by the lungs and coronary circulation,whereas in the right heart —66% of venous return in supplied by the inferior vena cava, with therest coming from the upper body via the superior vena cava (Teital et at., 1982). Thus, theconcentration of 02, glucose, and other placentally derived substances of intermediarymetabolism in the inferior vena cava are diluted to a lesser degree in the left heart, resulting inhigher concentrations in the ascending aorta than in the right heart and descending aorta (—90%of right ventricular output is delivered into the descending aorta via the ductus arteriosus,Rudolph, 1985, Teitel et at., 1982). With DPHM at steady-state, there would likely be minimalnet uptake of drug by the tissues of the upper body, particularly by the skin, muscle, and bone.Thus, DPHM levels in the superior vena caval blood should not be much different from those inarterial blood. Therefore, there should be minimal dilution of the drug delivered to the rightheart via the inferior vena cava. However, we have previously found that DPHM is taken up bythe lung in the fetal lamb, and hence the pulmonary venous drug concentration is lower than inthe pulmonary artery (Rurak et al., 1991). Therefore, in the left heart there would be mixing ofinferior vena caval blood with pulmonary venous blood that had a lower drug concentration. Thenet result would be higher DPHM levels in descending aorta compared to the ascending aorta,which is the result obtained. The observation for a lower FA-CA DPHM concentrationdifference with umbilical venous infusions could be due to the preferential streaming of ductusvenosus blood to the left heart and upper body, thereby counteracting the dilutional effect of themixing of pulmonary venous and inferior vena cava blood in the left heart.4.6. Paired Maternal/Fetal Infusions of DPHM and[2H10]DPHM4.6.1. Fetal Behavioral Effects Following Simultaneous Maternal/Fetal Infusions ofDPHM and[2H10]DPRM189There appear to be dose-related differences in the pattern of adverse effects reported withDPHM in humans. At lower circulating levels of DPHM (40-80 ng/mL) CNS depression hasbeen reported, while at higher drug levels (6-22 X normal) CNS stimulation was observed(Douglas, 1985). Although a direct comparison of the effects of DPHM in the post- and the prenatal period is complicated by differences in the sleep pattern, the constellation of the drugeffects of DPHM in the fetal lamb also appear to vary with plasma concentrations of the drug(Rurak et al., 1988). At low fetal plasma levels following maternal infusion of DPHM (i.e., 36.3ng/mL) there are sedative-like effects (a reduction in low voltage ECoG and a decrease in fetalbreathing movements), while at higher concentrations (i.e., 448 ng/mL) there was a decline in theamount of low voltage ECoG accompanied by a marked increase in the intermediate ECoGvoltage pattern. In addition, initial transient declines in fetal arterial Po2 and pH associated withtachycardia and vigorous fetal breathing were noted (Rurak et al., 1988). The effects observed inthe current study appear to be in between the effects noted in the previous studies. There was amarked increase in the intermediate ECoG voltage pattern, similar to that following fetal druginfusion in the previous study (Figure 45), accompanied by a tendency for fetal breathingmovements to decrease during the infusion (not statistically significant), similar to the resultsobtained from the previous maternal infusion of DPHM (Figure 46). The “intermediate” natureof the fetal effects in the present study may be due to the plasma concentrations of DPHM [282.4ng/mL (238.2 ng/mL[2H10]DPHM + 45.1 ng/mL DPHM)], which fall between the two plasmaconcentration ranges from the previous study. These data, taken with the previous results,suggest that there may be a relationship between the plasma concentration of DPHM and theconstellation of fetal effects observed. However, whether there is a clear pharmacodynaniic190relationship between fetal behavioral effects and fetal DPHM plasma concentrations requiresfurther study, preferably including measurement of brain tissue or extracellular fluid drug levels.In a previous study, a transient increase in the fetal heart rate was noted immediatelyfollowing the start of a fetal infusion of DPHM. This phenomenon was attributed to theanticholinergic effects of DPRM observed at higher doses (Rurak et al., 1988, and Douglas,1985). A similar finding was not observed in the present study; rather, a decrease in the fetalheart rate during the course of the fetal-maternal infusions was noted. The reasons for this arenot presently clear, but again it may relate to differences in the fetal circulating level of the drug,particularly during the initial infusion period.4.6.2. Trans- and Non-Placental Clearances of DPI{M in the Maternal/Fetal Unit.In the current study there was a tendency for the mean fetal steady-state plasmaconcentrations to be less than those observed by Yoo et al. (1993), despite the same fetalinfusion rates. As a result, the clearance estimates based on the fetal steady-state arterial plasmaconcentrations appeared to be greater than those estimated by Yoo et al. (1993) (Table 26).191Table 26: Comparison between total, trans-, and non-placental clearances from a previousstudy (Yoo et al., 1993) using time separated maternal and fetal infusions, and thecurrent study using simultaneous fetal and maternal infusions of[2H10]DPHM andDPHM.Parameter Yoo et al. (1993) Current Study(mLlminlKg) (mLlminlKg)CLmma 44.3 ± 9.8 40.0 ± 5.2CLff 223.9 ± 95.8 356.0 ± 106.1CLmf 41.0 ± 24.1 54.0 ± 32.4CLfm 124.4± 60.9 221.0±71.6CLmoa 38.0 ± 4.5 43.2 ± 9.5CLfo 99.5 ± 36.8 135.0 ± 59.1a- calculated based on maternal weightValues are shown as mean (± S.D.) for comparison purposes.A statistical comparison between the two estimates is difficult due to the large inter-animalvariability in both the steady-state plasma concentrations and the resulting estimates of the non-and trans-placental clearances. The sample size required to show a statistically significantdifference between two means of a predetermined magnitude (e.g., 10, 20, etc %) associated withthe current level of variability can be calculated (Zar, 1984). To detect a 10% statisticallysignificant difference between the clearance values calculated in the present study to thosecalculated by Yoo et al. (1993) would require a sample size of 105 animals for CLmm and 392animals for CLff. Therefore, with only 5 the animals in the current study, no meaningfulconclusions can be drawn from the differences between our present data and that of Yoo et al.(1993).In the previous study of Yoo et al. (1993), the wash-out period between the timeseparated infusions was 48 hours, due to the short half-life of DPHM in the ovine maternal/fetalunit (i.e., 60 minutes). Since the fetus grows at a rate of —3-5% per day (Kong et al., 1975), it192is unlikely that fetal growth and differentiation of fetal organ systems during the wash-out periodwould result in detectable differences in DPHM disposition between the maternal and fetalinfusions. However, this may not be the case for drugs which have a long disposition half-life,where in the course of a longer wash-out period between the time separated fetal and maternalinfusions, detectable time dependent changes in drug disposition could occur, necessitating theuse of simultaneous maternallfetal infusions. In addition, simultaneous maternal and fetalinfusions should provide more precise (less variable) estimates of the placental and non-placentalclearances compared to time-separated infusions due to the elimination time dependent effects offetal growth and development, and to a reduction in the inter-day variability within amaternallfetal pair. In the present study there was a trend (though not statistically significant; F-test p>.O.O5) for a reduction in the variability of the clearance estimates CLmm and CLmo.Simultaneous infusions of labeled and unlabeled drug offer additional benefits in that thelikelihood of successful experiments is substantially increased (two separate experimentsconducted at two different times on the same animal are not required). In addition, such pairedexperiments allow for a greater experimental utilization of the pregnant animals, since moreexperiments (following the appropriate wash-out period) can be conducted with a preparationover the 1-3 week experimental window available in each animal.The trans- and non-placental clearances for the maternal-fetal unit are calculated byequations derived from a 2 compartment-open model (Figure 6). The assumptions of this modelare that transfer and elimination must be first-order processes, no drug must be present at timeinfinity (i.e., drug must be entirely eliminated by CLmo and/or CLfo), and elimination andtransfer occur from the central compartment in both mother and fetus. The approach used bySzeto et al. (1982) and Szeto (1982) to arrive at solutions for the trans- and non-placental193clearances of DPHM require that steady-state plasma concentrations be achieved, so that there isno net flux between drug present in the central and the peripheral compartments in mother andfetus. Another approach, which retains the same model structure as the Szeto approach but doesnot require steady-state plasma concentrations to be achieved, is the mass balance approach(Ebling and Jusko, 1986). The assumptions for the 2 compartment-open model using the massbalance approach are similar to those above, but in addition, it is assumed that elimination ofdrug does not occur from both maternal and fetal distributional compartments radiating off thecentral compartments (Ebling and Jusko, 1986). This model is “model-independent” with regardto tissue distribution, thus, the clearance estimates obtained from this model cannot provideinformation about the drug in the peripheral compartments. The estimates obtained from thisapproach correlate well with the estimates obtained from the Szeto approach (Table 19 and 20).With both approaches, measurable quantities of drug must be present in maternal plasmafollowing fetal administration, and vice versa. If an infusion, rather than a bolus, is required toachieve measurable drug concentrations when using the mass balance approach, post-infusionsampling required to calculate the AUCs prolongs the experiment, and increases the total volumeof fetal blood sampled. This would limit the usefulness of this approach.A potential limitation of the 2 compartment-open model for fetal/maternal drugdisposition is that it neglects the placenta as an organ of drug elimination. Rather, the placenta isconsidered to be only an organ of maternal-fetal exchange. If drug elimination did occur in thematernal andlor fetal compartment of the placenta, this would be calculated in the estimates ofCLmo and CLfo, respectively (Szeto et al., 1982). We tested for placental DPHM metabolismutilizing the Fick method to estimate trans-placental clearance values. Traditionally, the Fickmethod has only been used to measure the uni-directional clearance of substances from fetus to194ewe (Wang et at., 1986a). However, with simultaneous infusions of DPHM and[2H10]DPHM,bi-directional placental clearances can be calculated. In the two animals, where both umbilicalvenous blood samples were collected and the umbilical blood flow was measured, the modelindependent estimates of both CLfm and CLmf were calculated following simultaneousfetal/maternal infusions of DPHM and[2H10]DPHM. The placental extraction of DPHM and[2H10JDPHM across the placenta measured in this experiment was similar to an estimate obtainedby Yoo et at. (1989) (Table 21). The placental clearance estimates for CLmf were approximately30% lower than the model derived estimates. While the estimates of CLfm were in goodagreement for ewe 2181, in ewe 1 22Z the Fick derived estimate underestimated the modelderived parameter by a factor of two. However, based solely on the mean extraction ratios fromthis experiment and that of Yoo et at. (1989), and the mean umbilical blood flow obtained in thisexperiment, estimates for CLmf (67.4 mL/minlKg) and CLfm (168.4 mL/minlKg) werecalculated (Table 21). These estimates correlate well with the CLmf, but underestimate theCLfm by 24% in our study. A proportion of the difference between the model derived and theFick estimates of trans-placental clearance may result from placentally derived blood mixingwith blood returning from the fetal membranes (-6% of total umbilical blood, Makowski et at.,1968a) in the umbilical vein. Furthermore, this difference is unlikely to be due to placentalmetabolism of the drug. Placental DPHM metabolism (on the fetal side of the placenta) shouldresult in a higher Fick estimate of CLfm, since the estimate would be determined in part by a valueof umbilical DPHM extraction that included drug loss due to both fetal to maternal transfer andplacental metabolism. Therefore, as discussed above, with the model derived estimates of fetalDPHM clearances, placental drug metabolism would be included in the estimate of CLfo. Overall,it appears the model independent estimates of trans-placental clearances, obtained by the Fick195method, are similar to those obtained with the 2 compartment-open model. Thus, significantplacental metabolism of DPHM in pregnant sheep appears unlikely. In a previous study, theplacental clearance of acetaminophen estimated using the Fick method was also found tocorrelate with the clearance values calculated using the 2 compartment-open model (Wang et al.,1 986a). This may be significant because the magnitude of the fetal placental and non-placentalclearances of DPHM (CLfm 221 mL/minlKg and CLfo 135 mL/minlKg) are substantiallydifferent from acetaminophen (CLfm 31 mL/min/Kg and CLfo 11 mL/min/Kg) (Wang et al.,1986a). Thus, the 2 compartment-open model appears to provide good estimates for at least twodrugs which show quite different disposition characteristics, and it also suggests that themetabolism of both drugs by the placenta is minimal.The model derived estimates of CLfm can also be compared to empirically derivedestimates of CLfm. The fraction of the fetal dose([2H10]DPHM) which is required to producematernal steady-state plasma concentrations of the compound can be calculated from thematernal total body clearance (CLmm) and the maternal steady-state plasma concentrations of[2H1OJDPHM (fraction of fetal dose CLnhIfl * Cb55maternai[2H10]DPHM I KOi’etai[2H10]DPHM).If the model derived estimates for CLfm are accurate, then the fraction of drug transferred acrossthe placenta from the fetus to the mother (CLfm*AUCffI(CLfm + CLfo)*AUCff) estimatedusing model derived estimates of CLfm and CLfo should equal the empirical estimates of thefraction of the dose required to produce the observed maternal steady-state plasma concentrationsof[2H10]DPHM. The model derived estimate (69 ± 11%) correlates well with the empiricalestimate (70 ±7%). This evidence appears to further substantiate the validity of the 2compartment-open model for DPHM disposition in the fetallmaternal unit.196Previously, a significant difference between the model derived estimates of CLfm andCLmf of DPHM was noted, with CLfm being three fold greater than CLmf. A similar findingwas observed in the current investigation, with the difference between the model derivedestimates for CLfm and CLmf being approximately four fold higher when estimated using the 2compartment-open model. A similar difference was noted when calculations for numerous otherdrugs including morphine, methadone, and metoclopramide were made using this model (Szetoet al., 1982b, and Riggs et al., 1990). This phenomenon does not appear to be due to modelintroduced bias, since the difference between CLfm and CLmf calculated using the modelindependent method (i.e., the Pick method) was 4.0 and 2.2 fold in ewes 122Z and 2181,respectively. Yoo et al. (1993) demonstrated, for various drugs studied, that the magnitude ofthe difference between CLfm and CLmf correlated with the value of CLfm of the drug. Thereason for this observation is not clear. However, more studies using drugs with varieddisposition characteristics are required to examine this phenomenon.The apparent volume of distribution of DPHM following fetal drug administration (fetalbolus or fetal infusion) is very large (Tables 12, 13, and 18), and can exceed the maternal weightcorrected volume of distribution by 12-13 fold. It is clear that, despite the differences in the bodycomposition between the fetal and maternal sheep, these apparent volumes of distribution areoverestimated. The reason for this overestimation is not entirely clear, but may be due to theapparent volume of distribution measured following fetal drug administration being composed ofboth the fetal apparent volume of distribution and a maternal component. By definition, theapparent volume of distribution of is “a proportionality constant relating drug concentration inthe blood or plasma to the amount of drug in the body” (Gibaldi and Perrier, 1982). It is clearthat the amount of drug in the fetal body at any particular time does not correlate well with the197dose administered, since a large portion of the dose administered to the fetus is transferred acrossthe placenta to the mother (-P60-70% of the fetal dose in the case of DPHM). If the volume ofdistribution is corrected for this loss across the placenta (CLmrn*AUCfm), the volume ofdistribution is reduced by — 66% (i.e., from 27.1 L/Kg to 9.2 L/Kg). In order for this correctedvolume of distribution to be valid, negligible placental metabolism must be assumed; asdiscussed above, this assumption is probably valid for DPHM. This postulated mechanism forthe higher Vd8 estimates for DPHM in the fetus is supported by the results obtained with otherdrugs. For example, the apparent volume of distribution of ritodrine, a drug with poor placentalpermeability (AUC fetal/AUC maternal 0.03) is not statistically different in the mother (10.1L/Kg) and the fetus (8.5 LIKg) following maternal and fetal bolus administrations, respectively(Wright, 1992). Labetalol, a drug with a placental permeability greater than ritodrine, but lessthan DPHM (AUC fetal/AUC maternal 0.13), has a greater fetal than maternal volume ofdistribution (14.3 L/Kg vs. 3.0 L/Kg, respectively). The magnitude of the difference between thefetal and maternal Vd of labetalol is larger than that of ritodrine, but less than DPHM(Yeleswaram, 1992). From these limited data, it appears that placental permeability may play arole in the fetal volume of distribution measured following fetal drug administration. The fetalweight corrected Vd following correction for the proportion of the dose which is lost for thefetus to the mother is also substantially larger than the weight corrected estimate in the ewe. Ifthe placenta is included (0.3-0.4 Kg), the weight corrected fetal Vd is further reduced.Although the placenta in terms of mass is relatively small, binding of DPHM to the placenta maybe substantial. This is so because in the human and other species, the placenta has been shown tobe rich in monoamine oxidase-type A (MAO-A) (Tan et al., 1991). The concentration of MAOA in human placenta is almost two-fold greater than in the brain. (O’Carroll et al, 1989).198Furthermore, DPHM has been shown to bind extensively to MAO in rat lung and rat lungmitochondria (Yoshida et al., 1989, and 1990). If the binding of DPHM to MAO in the ovineplacenta is similar to that reported in rat lung, the placenta may contribute significantly to theestimates of fetalAs in the studies employing simultaneous 90 minute fetal infusions of labeled andunlabeled DPHM (section 4.5.3), drug concentrations were measured in both the fetal carotid andfemoral arteries during the paired maternal-fetal drug infusions to check for preferential drugdistribution to the upper body. As in the earlier experiments, the reverse situation was found with[2H10JDPITM, the form of drug infused via the fetal tarsal vein. The mean FA-CA concentrationdifference of 6.7 ± 2.8 ng/mL was significantly different from 0, and similar to the value obtainedin the 90 mm infusion studies (8.5 ± 2.1 nglmL). However, with DPHM (the drug administered tothe ewe, and hence reaching the fetus via the umbilical vein) the FA-CA gradient of 1.1 ± 1.1ng/mL was not significantly different from 0. This is different from the results obtained withumbilical venous drug infusion (where there was a positive FA-CA difference), although this wassignificantly lower than the value obtained with DPHM infusion via the tarsal vein (section 4.5.3).As discussed previously, the higher FA drug concentration with inferior vena caval administrationmay be due to a minimal dilution of the drug delivered to the right heart in the inferior vena cava(via mixing with superior vena caval blood), compared to the situation with oxygen and othermetabolic substrates which have lower concentrations in the blood returning from fetal tissues. Incontrast, in the left heart there would be mixing of inferior vena caval blood with pulmonaryvenous blood that had a lower drug concentration, thereby resulting in higher[2H10]DPHM levelsin descending aorta compared to the ascending aorta. The lack of a significant FA-CA DPI-IIVIconcentration difference for maternally infused DPHM could be due to a greater degree of199preferential streaming of ductus venosus blood to the left heart than occurred during the 90 minuteumbilical drug infusions (section 4.5.3). The former result is most relevant to the normal situationwith fetal drug exposure, i.e., maternally administered drug reaching the fetus via the umbilicalvein. The findings suggest that preferential distribution of DPHM to the fetal brain, heart and otherupper body structures does not occur. However, studies of other drugs are warranted to determinewhether the results obtained with DPHM are generally applicable to a range of xenobiotics. In thisregard it would be interesting to study drugs, such as valproic acid, which do not accumulate infetal lung fluid and are probably not taken up by the lung (unpublished results; Mr. John Gordon).This would allow additional proof for the proposed mechanism outlined above for the positive FACA DPHM concentration difference, since according to this mechanism we would expect aminimal or no difference between the FA and CA concentrations of a drug not subject to significantuptake by the fetal lung.4.6.3. Fetal and Maternal Renal Clearances of[2H10]DPHM and DPHMThe renal elimination of drugs and other substances is a function of three processes:glomerular filtration, renal secretion, and tubular reabsorption (Roland and Tozer, 1989).Glomerular filtration involves the filtration of plasma water and its dissolved components (<4nm) across the capillary endothelium and the tubular epithelium (Ganong, 1985). Thus, for themost part it is the free or unbound drug in plasma which is filtered by the glomerulus (i.e.,CLrenal = fub*GFR, where fub is the fraction unbound in plasma and GFR is the glomerularfiltration rate). If renal clearance exceeds the predicted clearance based on the filtration of theunbound drug, active tubular renal secretion of the solute is inferred (Roland and Tozer, 1989).Specific active renal secretion pathways exist for both acidic and basic compounds (Ganong,2001985). Finally, the solute or drug in urine can be reabsorbed from the renal tubule back into theblood (Roland and Tozer, 1989).The renal clearance of DPHM in the pregnant ewe is 0.0 12 ± 0.009 mL/rninlKg. Thisvalue is significantly and markedly less than the reported glomerular filtration rate (GFR) insheep, which is reported to be 2.4 mL/min/Kg (Hill and Lumbers, 1988). This suggests that aportion of filtered and/or the secreted load of DPHM (based on plasma protein binding of 86% insheep; Yoo et al., 1993) is reabsorbed in the renal tubule. The renal clearance of DPHM in adultsheep contributes less than 0.1% to total maternal body clearance of the drug. This is similar tothe findings following bolus administration (Section 3.3.1.). In contrast, the fetal renal excretionof DPHM is 2.44 mL/minlKg, which exceeds literature values for fetal GFR (1.03 ±mL/min!Kg)(Hill and Lumbers, 1988). This suggests that DPHM is being filtered and secretedby the kidney of the fetal lamb in late gestation. Our observations for the fetal renal clearance ofDPHM are similar to previous studies conducted with other organic cations such as cimetidine,ranitidine, meperidine, and tetraethylammonium (Mihaly et al., 1983, Czuba et at, 1990, Szeto etal, 1979, and Elboume et at, 1990). Meperidine has a renal clearance of 9.3 ± 2.2 mL/min.,which exceeds the rate of inulin clearance (i.e., 3.3 mL/min ± 1.4 mL/min), inferring thatmeperidine undergoes renal secretion (Szeto et a!., 1979). Likewise, the renal excretion ofcimetidine and ranitidine in fetuses at 140 days gestation also exceeded literature values for GFR(i.e., 12 mL/min and 7.8 mL/min for cimetidine and ranitidine, respectively) (Mihaly et a!., 1983,and Czuba et at, 1990). In contrast, the renal clearance of lidocaine is low (i.e., 1.5 mL/minlKg)and is below the reported renal glomerular filtration rate (Morishima, et at., 1979). Thus, theindirect evidence for most of the basic drugs examined thus far (meperidine, cimetidine,201ranitidine, and DPHM) suggests that renal secretion of organic cations is functional in the lategestational fetal lamb.The reason for the discrepancy between the maternal and the fetal renal clearances ofDPHM is not yet known. However, renal reabsorption may play a role in accounting for thegreater renal clearance of DPHM in the fetal lamb than that in the mother. The pH of the fetalurine is 6.78, compared to the pH in the maternal urine of 7.62. Because DPHM is a weaklybasic drug with a pKa of 9.0, the fraction of the compound ionized fetal urine will be greater thanin maternal urine, 99.4 vs. 95.6 %, respectively. The lesser degree of ionization in the maternalurine will facilitate greater renal reabsorption of DPHM in the mother compared to the fetus,resulting in a greater amount of the drug excreted in the fetal urine (Roland and Tozer, 1989, andGanong, 1983). In addition, the greater urine flow in the fetus (—14.2 mL/hrfKg, Table 23)compared to the mother (—0.8 mLlhr/Kg) may also facilitate the increased fetal renal clearanceof DPHM by reducing the reabsorption of the drug from the urine (Roland and Tozer, 1989).4.6.4. Disposition of DPMA and[2H10]DPMA in the Ovine Fetal/Maternal Unit followingSimultaneous Maternal/Fetal InfusionsIn humans, monkeys, and dogs, DPHM is thought to be metabolized via two sequentialN-demethylation steps, followed by deamination (See section 1.7.3). The deaminatedmetabolite, DPMA, and its conjugated counterpart (glycine or glutamate conjugates) are majorurinary metabolites in several species (Chang et al, 1974, Drach and Howell, 1968, Drach andHowell, 1970A, Drach et al., 1970, and Glazko et al., 1974). DPMA was present in the urineand plasma of non-pregnant ewes following DPHM administration (Section 3.3.3.2.1.). DPMAand[2H10]DPMA were also present in both maternal and fetal plasma following the simultaneous202maternal and fetal infusions of DPHM and[2H10]DPHM, respectively (Figure 41 and 42). It isimportant to remember that following the maternal administration of drug, the presence ofmetabolites in the fetal circulation may not be due to fetal drug metabolism, but could result frommaternally derived metabolites which traverse the placenta (Wang et al, 1985). However, in thepresent study the formation of DPMA appears to occur in both mother and fetus. During thesimultaneous infusion of DPHM and[2H10]DPHM to mother and fetus, respectively, theconcentration of DPMA is greater in the maternal than in the fetal arterial plasma, while theconcentration of[2H10]DPMA is higher in the fetal than in maternal arterial plasma. If thismetabolite were formed by the mother and transferred to the fetus, the ratio of DPMA to[2H10]DPMA in fetal plasma should be similar to the ratio in maternal plasma; this is clearly notthe case (Figure 41 and 42). Furthermore, it is unlikely that[2H10JDPMA is selectivelytransferred from maternal plasma to fetal plasma because this would require a 20 fold greateruptake of[2H10JDPMA, compared to DPMA. This is not likely to occur since no isotope effectwas evident from the plasma concentrations of DPMA and[2H10]DPMA following simultaneousfetal infusions of DPHM and[2H10]DPHM (Figure 35). Moreover, as discussed below, theavailable data suggests limited placental permeability of DPMA in sheep. Thus, our datastrongly suggests that the metabolism of DPHIVI to DPMA occurs in the fetus as well as in themother. The source of this metabolite in the fetus and mother and its quantitative importance arecurrently unknown.The peak plasma concentrations of DPMA and{2H10]DPMA occur much later in the fetalplasma in comparison to the maternal plasma. This may be a function of the longer apparentelimination half-life of DPMA in the fetus (911.1 ± 151.9 mm) compared to the mother (180.4 ±13.0 min)(Houston, 1982). The elimination rate of the metabolite was calculated based on a 1203compartment model due to constraints imposed by the sparse data (Figure 7). If the metabolitedisplays 2 or more exponential disposition phases, the model derived estimate of the eliminationrate will likely be overestimated (T112 will be underestimated). Therefore, these values must beconsidered to be “apparent” estimates. The apparent elimination half-life of the metabolite issubstantially greater than that of the parent drug, which in mother and fetus are 70.5 ± 6.9 and51.8 ± 7.2 minutes, respectively. In most cases, drug metabolism forms more water solublemetabolites, and thus biotransformation expedites the removal of the drug and the resultingmetabolites from the body. However, the formation of DPMA appears to impede the eliminationof the DPHM body load (i.e., DPHM and metabolites). The extended half-life of DPMA in thefetal plasma, compared to the maternal plasma, suggests that the elimination pathways for thismetabolite are not as developed in the fetus as they are in the ewe (see Section 4.6.5.).Furthermore, the persistence of DPMA in the fetal plasma suggests that DPMA does not readilytransfer across the placenta from fetal blood to maternal blood, compared to the parent drug(DPHM). The possibility of minimal placental transfer is further substantiated by the zeroextraction of DPMA by the placenta in the two ewes with UV catheters. However, the largerratio ofAUC([2H10]DPMA) I AUC([2H10JDPHM) vs. AUC(DPMA) I AUC(DPHM) in maternalplasma suggests that at least a portion of the metabolite that is formed in the fetus is transferredacross the placenta to the mother; however, the permeability appears to be substantially less thanDPHM (Figure 41).. If this were not the case, then the above ratios should be equivalent.Mechanisms which are involved in placental transfer of drugs may include simple diffusion,facilitated diffusion, active transport, pinocytosis, and bulk flow (Reynolds and Knott, 1989).For most drugs, simple diffusion is the dominant mechanism of transport, and therefore, bloodflow and placental permeability are important in determining the rate of transfer. Placental204transfer is a function of numerous factors, including fetal and maternal plasma protein binding,lipophilicity, molecular size, pKa, blood flow, and placental morphology (Reynolds and Knott,1989). Unlike humans, which possess a hemochorial placenta, the ovine placenta(epithelialchorial) is much less permeable to polar and higher molecular weight drugs (Rurak etat., 1991). The reason for the limited placental permeability of DPMA is not known, but mayresult from the large degree of protein binding, and a possible reduction in the lipophilicity ofthis metabolite compared to the parent drug. The pharmacological and/or toxicologicalimplications of the prolonged persistence of DPMA in the fetal circulation are unknown at thistime.4.6.5. Fetal and Maternal Renal Clearance of DPMA and[2H10]DPMAThere are significant differences in the extent of the renal excretion of DPMA in adultand fetal sheep. The renal clearance of DPMA was 0.9 ± 0.6 mL/min/Kg in the ewe (whichcorresponds to approximately 1% of the total dose), while the renal clearance of DPMA in thefetus was 0.02 ± 0.01 mL/min/Kg (- 0.02 % of the total dose). The difference in the renalclearance of DPMA was almost 30 fold greater in the ewe compared to the fetal lamb on aweight corrected basis. In the ewe, filtration of unbound DPMA by the glomerulus would resultin a renal clearance of —0.03 mLlmin/Kg, assuming that only the unbound fraction is filtered andno further reabsorption of DPMA occurs from the renal tubule. This value is 30 times less thanthe measured renal clearance of this metabolite, suggesting that renal secretion of this compoundoccurs in the adult. In the fetus, if only unbound drug were filtered, the estimate of renalclearance would be 0.020 mLlmin/Kg, which is in reasonable agreement with the observed renalclearance in the fetal lamb (0.018 mL/min/Kg). Despite the fact that the fetal renal clearance205may be underestimated due to the underestimation of the fetal Mu, it appears that this acidicmetabolite is efficiently secreted by the adult but not the fetal kidney. A similar phenomenon infetal lambs has also been observed for other organic anions, such as para-aminohippurate,acetaminophen conjugates (sulfate and glucuronide), and morphine-3-glucuronide (Elbourne etal., 1990, Wang et al., 1986a, and Olsen et al., 1988). There are also preliminary data suggestingminimal renal excretion of valproic acid and indomethacin in fetal lambs [unpublished results].These data suggest that while pathways for organic cation renal secretion are developed in thefetal lamb (see Section 4.6.3.), similar pathways for organic anions are not functional. Thiscontrasts the situation observed in fetal pigs, where the excretion of PAH exceeds the inulinclearance by a factor of almost 4 (Alt et al., 1984), and in neonatal dogs in which the PAH renalsecretion pathway is functional (Rennick et al., 1961, and Bond et al., 1976). However, in therabbit the pathway develops largely during the post-natal period, and is due primarily to a postnatal increase in the intracellular content of lignin, a protein responsible for binding andintracellular storage of organic anions (Cole et al., 1978). The overall results suggest that thereare species differences in perinatal development of renal secretatory pathways for organic anions.The low renal clearance of DPMA in the fetus may help to explain the lack of measurablequantities in the anmiotic fluid, since fetal urine is a major component of amniotic fluid(Battaglia and Meshia, 1986). However, renal secretion is not the only route of delivery fordrugs and metabolites to the amniotic fluid. Szeto et al. (1979) demonstrated that meperidineappears in amniotic fluid despite ligation of the urethra and urachus, suggesting that drugs maybe transported across the allantoic and amniotic membranes. In addition, Olsen et al. (1988)demonstrated that morphine-3-glucuronide accumulates in amniotic fluid despite completedrainage of the fetal bladder during the initial portion of the experiment in one animal. However,206a portion of the metabolite delivered to the amniotic fluid may have resulted from incompletedrainage of urine from the fetal bladder by the catheter due to nonligation of the urethra andurachus (Olsen et al., 1988). The reason that DPMA does not appear to traverse the fetalmembranes may be due to the very high plasma protein binding of this metabolite in fetal plasma(see Section 3.3.2.). In contrast, the plasma protein binding of morphine-3-glucuronide isessentially 0%, thus, it is unlikely that the transport of this conjugate is impeded by plasmaprotein binding (Olsen et al., 1988). Therefore, the lack of DPMA in the amniotic fluid mayresult from low quantities of the metabolite delivered to the amniotic fluid via the fetal urine, andpossible impeded transfer across the fetal membranes.4.7. Plasma Protein Binding of DPMAThe plasma protein binding of DPMA in this study is extensive (- 99%) in plasmaobtained from fetal and maternal sheep. DPMA is also highly bound (-97%) to human serumalbumin (Drach et al., 1970); thus, presumably DPMA also binds to fetal and maternal albuminin sheep. Although significant differences have been noted between fetal and maternal plasmaprotein binding of basic drugs thought to bind to c- 1-acid glycoprotein, acidic drugs which areknown to bind to albumin show similar degrees of binding in humans (Hill and Abramson, 1988,Kremer et al., 1988, and Vaini et al., 1991). The differences in the binding of acidic and basicdrugs in humans could result from differences in the plasma concentrations of fetal and maternalproteins. In humans, the concentrations of a-i-acid glycoprotein are substantially lower in thefetus, while concentrations of albumin are similar to maternal levels (Nau and Krauer, 1986, Hilland Abramson, 1988, and Wood and Wood, 1981). Similar findings regarding the plasma207protein binding of drugs have also been reported in sheep. The binding of basic drugs appears todiffer in maternal plasma and fetal ovine plasma (130-140 days gestation), for example,meperidine 75 vs. 58%, propranolol 86 vs. 50%, metoclopramide 49 vs. 39%, methadone 37 vs.24%, lidocaine 43 vs. 27%, and DPHM 86 vs. 72% (Szeto et a!., 1982c, Morgan et at., 1988,Riggs et at., 1988, Szeto et at., 1981, Kennedy et at., 1990, and Yoo et at., 1993). However, asdemonstrated by the similar degree of binding of DPMA, acidic drugs appear to show similarbinding to maternal and fetal plasma proteins in sheep: indomethacin 97.6 vs. 98.5%,acetylsalicylic acid 73 vs. 76%, and acetaminophen 12.5 vs. 9.3% (Anderson et at., 1980,Anderson et at., 1980a, and Wang et at., 1986a). This data suggests that sheep may be similar tohumans with regard to the fetal and maternal concentrations of various plasma proteins (ovinealbumin and ct- 1-acid glycoprotein); therefore, differences in fetal and maternal plasma proteinbinding could be due to the same factor in both species.4.8. In Vitro Metabolism of DPI{M in Hepatic Microsomes Prepared from Fetal andAdult Sheep.Fetal drug metabolism appears to depend on species and gestational age (Juchau, 1990).In vitro drug metabolism data from small animals such as mice, rats and guinea pigs suggest alack, or a diminished capacity for drug metabolism (Juchau, 1990, Sandberg et at., 1993, andKeunzig et at., 1974). However, evidence does exist suggesting that some of these pathways aredeveloped in humans and higher primates by mid gestation. The presence of a fetal CytochromeP450-3A related isoform in human fetal microsomes has been detected, and is thought to beactive in the metabolism of cocaine, dextromethorphan, and ethylmorphine (Krauer and Dayer,1991, Ladona et at., 1989, Ladona et at., 1991, and Jacqz-Aigrain and Cresteil, 1992). In208addition, there appear to be differences in the ontogeny of various fetal drug metabolizingpathways and the expression of various fetal Cytochrome P450 isoforms in humans (Juchau,1990). From the limited data available in fetal sheep, it appears that some phase II metabolicpathways (different isoforms of glucuronyl transferase) develop prior to phase I pathways(Dvorchik et al., 1986, and Wang et al., 1986b). For example, efficient in vitro hepaticmicrosomal conjugation of morphine and acetaminophen occur in the fetal lamb (Dvorchik et al.,1986, and Wang et al., 1986a), and there is evidence for the in utero fetal formation ofglucuronide conjugates of ritodrine, labetalol, and acetaminophen in sheep (Yeleswaram et al.,1992, Wright et al., 1992, and Wang et al., 1986b). However, N-dealkylation (i.e., Phase I) ofmethadone and meperidine, and the hydroxylation of benzo[a]pyrene and hexobarbital in lategestational fetal lambs occur at low rates compared to adult sheep (Dvorchik et al., 1986). In thecurrent study, the quantities of N-demethyl DPHM (per mg of microsomal protein) formedfollowing a 90 minute microsomal incubation were less in the fetal microsomes compared to theadult microsomes (-86% less). This appears to correlate with results obtained for the Ndealkylation of methadone and meperidine (Dvorchik et al., 1986). The reason for thediminished capacity of oxidative metabolic pathways for these substrates is not clear, but it maybe due a to decreased amount of the required enzymes in the fetal lamb (Cytochrome P450isoforms). The content of Cytochrome P450 in microsomes prepared from the livers of fetallambs is —10 fold lower than those prepared from maternal sheep (Dvorchik et al., 1986). In thecurrent study, the concentration of total Cytochrome P450 could not be measured in fetalmicrosomal preparations, while it could be estimated in preparations from maternal liver. Thismay have been due to both contamination of the fetal microsomal preparation with hemoglobin,and likely, the low concentration of the enzyme in the fetal microsomes. An interesting finding209in the current study is that the deamination of DPHM to form the acidic metabolite DPMAappears to proceed at equal rates in hepatic microsomes prepared from both fetal and adult non-pregnant sheep. In addition to providing direct evidence for the fetal formation of thismetabolite, these results suggest that the functional in vitro capacity of this metabolic oxidativepathway is essentially equivalent to that observed in adult sheep. Recently, it has beendemonstrated in our laboratory by Mr. Sanjeev Kumar that DPHM appears to be metabolized infetal microsomal suspensions in the absence of the co-factors required for Cytochrome P450mediated reactions (NAPH and NADPH). Furthermore, this reaction is completely blocked bythe addition of pargyline (a non-specific monoamine oxidase blocker) (Dostert et al., 1989).Although monoamine oxidases (ED 1.4.3.4.: MAO) are known to be involved in the metabolismof endogenous biogenic amines, they have only recently been shown to be involved in thebiotransformation of several xenobiotics (Benedetti et al., 1988). Since DPHM binds to MAOs,it is possible that the drug is also metabolized by this group of enzymes (Yoshida et al., 1989,and Yoshida et al., 1990). Thus, it is possible that the metabolism of DPHM in both mother andfetus may occur by enzyme systems other than Cytochrome P450, and that this pathway may beas efficient in fetal lambs as in the ewe. However, this hypothesis requires further study.2105. Summary and Conclusions5.1. Synthesis of[2H10]DPHM, and Simultaneous Analysis of DPHM and[2H10]DPHMin Biological Fluids Obtained From Pregnant SheepThe synthesis of[2H10]DPHM and the development of a selective and sensitive GC-MSmethod for the simultaneous quantitation of DPHM and[2H10]DPHM in biological fluidsobtained from maternal and fetal sheep has been reported (Tonn et al., 1992).[H10JDPHM wassynthesized, and purified, and both its structure and the purity were verified. Biological sampleswere prepared for analysis using liquid-liquid extraction. The addition of TEA enhanced therecovery of DPHM and[2H10]DPHM by approximately 4 fold to yield an almost completerecovery (—100%) from the biological matrices examined. The method employed GC-MS in theelectron impact ionization mode with SIM of fragment ions m/z 165 for DPHM and orphenadrine(i.e., internal standard), and m/z 173 for[2H10]DPHM. The LOQ of DPHM and[2H10]DPHMfrom a 1.0 mL sample was 2.0 ng/mL in fetal and maternal plasma, fetal tracheal fluid, andamniotic fluid. The method was validated from 2.0 ng/mL to 200.0 ng/mL for both DPHM and[2H10]DPHM in plasma, fetal tracheal fluid, and amniotic fluid.5.2. Synthesis of[2H10]DPMA, and Simultaneous Analysis of DPMA and[2H10]DPMA inBiological Fluids Obtained from the Ovine Fetal/Maternal UnitDPMA, a major urinary metabolite of DPHM in monkeys, dogs, and humans, wasdetected in the plasma and urine of sheep following an intravenous bolus of DPHM. In studiesutilizing simultaneous administrations of both DPHM and[2H10]DPHM, measurement of bothDPMA and[2H10JDPMA is required to study the disposition of this metabolite in thefetal/maternal unit.[2H10JDPMA was synthesized, characterized, and purified. The GC-MS211analysis method for DPMA and[2H10]DPMA utilized a single step liquid-liquid extractionprocedure with toluene for sample cleanup (Tonn et al., 1995). DPMA was found to degradeunder acidic conditions similar to the ones employed during the extraction of this analyte fromaqueous samples. However, during the time required for extraction, degradation would result inthe loss of only 1% of the analyte. Following extraction, the samples were derivatized with Nmethyl-N-Qert-butyldimethylsilyl) trifluoroacetamide. A 1.0 jiL aliquot of the prepared samplewas injected into the GC-MS operated in the El ionization mode and quantitated using SIM. Oneion was monitored for each compound, namely, m/z 165 for the internal standard, diphenylaceticacid, m/z 183 for DPMA, and m/z 177 for[2H10]DPMA. The ion chromatograms were free fromchromatographic peaks co-eluting with the compound of interest. The calibration curve waslinear from 2.5 ng/mL (LOQ) to 250.0 ng/mL in both urine and plasma. The intra-day and inter-day variabilities of this assay method were below 20% at the LOQ and below 10% at all otherconcentrations.5.3. Lack of Isotope Effects Following Fetal and Maternal[2H10]DPHM AdministrationPharmacokinetic studies employing the simultaneous administration of both DPHM and[2H10]DPHM requires that[2H10]DPHM has the same dispositional characteristics as DPHM.That is,[2H10}DPHM should not demonstrate any measurable isotope effects. Isotope effectswere not noted following equimolar intravenous doses of DPHM and[2H10JDPHM in two non-pregnant ewes. This equivalence between DPHM and[2H10]DPHM was also apparent in thecumulative amounts of DPHM and[2H10]DPHM excreted in the urine. In addition, arterialplasma concentrations of DPMA and[2H10]DPMA, and the cumulative amounts of thesemetabolites excreted in the urine following DPHM and[2H10]DPHM administration were212comparable. Similarly, experiments conducted following bolus administration of DPHM and[2H10JDPHM to fetal lambs demonstrated equivalent arterial plasma concentrations of DPHMand[2H10]DPHM. This equivalence was also demonstrated in amniotic and tracheal fluid, sincelevels of DPHM and[2H10jDPHM in these fluids were essentially equal. The pharmacokineticparameters estimated showed that AUC, CLT, Vd8 Vd, T112, and MDRT were equivalent forlabeled and unlabeled drug. Following simultaneous fetal tarsal venous infusions of DPHM and[2H10]DPHM, the concentrations of DPHM and[2H10]DPHM in fetal arterial plasma, amnioticfluid, and fetal urine were equivalent. In addition, the fetal arterial plasma concentration of themetabolites DPMA and[2H10]DPMA were also similar. Overall, these data suggest that there areno isotope effects for the disposition of[2H10]DPHM in maternal and fetal sheep.5.4. Hepatic First-Pass Metabolism of DPHM in Adult and Fetal SheepThe administration of a 100 mg IV bolus of DPHM demonstrated that less than 1% of thedose is recovered as intact DPHM in urine. In addition, preliminary studies examining DPHMconcentration in bile suggests that biliary excretion of DPHM is also not a major route ofelimination, accounting for approximately 0.1% of the administered dose. These data indicatethat DPHM is eliminated in adult sheep largely by hepatic or extrahepatic biotransformation. Toinvestigate the possibility of hepatic uptake and/or biotransformation of DPHM in adult sheepand in fetal lambs, stable isotope techniques were utilized to investigate the hepatic first-passmetabolism of the drug. In adult sheep, following a total administered dose of 1.6 ± 0.3 mg/Kg,extensive presystemic elimination was noted, with 93.2 ± 3.2 % of the mesenteric dose beingeliminated prior to reaching the systemic circulation. A similar experiment conducted duringmild to moderate hypoxemic conditions in adult non-pregnant sheep showed that while the213extensive first-pass metabolism in three ewes remained essentially unchanged in the other twoewes the presystemic elimination decreased. These two ewes also experienced the most severeinitial drop in Po2 and 02 saturation from normoxic levels. Other pharmacokinetic parameters,such as CLT, and T112 appeared unaffected by hypoxemia.Unlike adult sheep, fetal lambs receive a large portion of their hepatic perfusion fromumbilical venous blood. Hence, in order to conduct a similar hepatic first-pass experiment infetal lambs, the labeled and unlabeled drugs were administered simultaneously via the umbilicalvein (fetal liver) and the lateral tarsal vein (systemic circulation). These experimentsdemonstrated that the fetal liver is not very efficient at extracting DPHIVI following umbilicalvenous bolus administration, since following an average dose of 3.8 ± 1.5 mg/Kg, the hepaticextraction was -10% ± 18%. A portion of the umbilical venous blood is shunted through thefetal liver via the ductus venosus (-50%). However, an extraction of DPHM from the remainingportion of umbilical venous blood that perfuses the fetal liver similar to that observed in the adultwould result in ‘-40-45% of the umbilical venous dose being eliminated prior to reaching thesystemic circulation. This was far greater than the value actually determined. To rule outsaturation of fetal drug metabolizing enzymes due to rapid drug administration, simultaneousumbilical and tarsal venous infusions of DPHM and[2H10]DPHM were employed. Thisexperiment also demonstrated that the extraction of DPHM by the fetal liver following umbilicalvenous administration was minimal. The reason for this is not clear but may be due to adeficiency of the necessary drug metabolizing enzymes or the presence of reduced quantities ofthe required enzyme systems responsible for DPHM metabolism. These data also suggest thatthe fetal hepatic clearance does not contribute significantly towards the observed fetal nonplacental clearance, while data from adult animals suggests that a significant portion of the adult214non-placental clearance may be due to hepatic elimination. However, some contribution of thefetal liver toward the fetal total non-placental clearance of DPHM cannot be ruled out.The concentrations of oxygen and other metabolic substrates delivered to the fetus via theumbilical vein are higher in the ascending aorta than the descending aorta. A similarphenomenon has been suggested for drugs but never tested. In our studies significant differenceswere noted between femoral and carotid arterial concentrations of glucose, lactate, and oxygen(all being higher in carotid arterial sample). In contrast to the case with 02 and othermetabolites, the concentrations of DPHM were significantly higher in FA than in CA bloodfollowing both tarsal and umbilical venous administration, although the FA-CA difference wasless with the latter route.5.5. Simultaneous Maternal/Fetal Infusions of DPHM and[2H10JDPHMFollowing simultaneous fetal and maternal infusions of DPHM and[2H10]DPHM,significant fetal effects were observed. There was a decrease in the intermediate voltage ECoGpattern, and an initial decrease in fetal heart rate. Fetal breathing movements tended to decreaseduring the infusion, but this did not reach statistical significance. The pattern of fetal effectsobserved appear to be intermediate to the fetal effects observed previously during separate maternaland fetal infusions of the drug. This observation appears to correlate with the intermediate fetalplasma concentrations seen in this study, compared to the previous investigation. However, aprecise pharmacodynamic relationship between the constellation of fetal effects elicited by DPHMand the fetal plasma concentration of the drug remains to be defined.In the current study, the placental and non-placental clearances of DPHM were estimatedin pregnant sheep using simultaneous maternal and fetal infusions of DPHM and[2H10]DPHM,215respectively. The fetal and maternal steady-state plasma concentrations were used to calculatethe trans-placental and non-placental clearances of DPHM using a 2 compartment-openpharmacokinetic model. The estimates of the trans-placental clearances (CLmf and CLfm) andfetal and maternal non-placental clearances (CLfo and CLmo) agree with the estimates obtainedpreviously through the use of time-separated infusions of DPHM to the fetus and mother.However, due to the large inter-animal variabilities, meaningful statistical inferences between thetwo studies were not possible.A potential limitation of the 2 compartment-open model employed in this study is that itneglects the placenta as an organ of drug elimination and would attribute any drug metabolism bythe placenta as either maternal or fetal non-placental clearance. To check this possibility forDPHM during the paired maternal-fetal drug infusions, maternal and fetal placental clearances werealso estimated using the model independent Fick Method. Overall, these estimates agreed with theplacental clearance values determined using the 2 compartment-open model, suggesting thatsignificant placental metabolism of DPHM does not occur, and that the latter experimentalapproach is valid for the drug.The measured fetal renal clearance of[2H10IDPHM and DPHM in this study was 2.5 ±0.5 mL/minlKg. This was greater than literature values of fetal GFR, inferring that[2H10]DPHMand DPHM are secreted by the fetal kidney. In the ewe, the renal clearance of DPHM was muchlower (0.012 ± 0.005 mL/minlKg) than reported values for GFR in adult sheep. The disparitybetween fetal and maternal renal clearances may be due to enhanced reabsorption of DPHM fromthe renal proximal tubule in the adult compared to the fetal kidney, possibly due to differences inurinary pH and urine flow rates.216Simultaneous maternal/fetal infusions of DPHM and[2H10JDPHM have provided strongevidence for the production of the deaminated metabolite of DPHM, DPMA, in both mother andfetus. DPMA and[2H10]DPMA have a much longer apparent elimination half-life in the fetus(mean ± SEM; 15.2 ± 2.5 hrs) compared to the ewe (3.0 ± 0.2 hrs). In addition, DPMA bindsextensively to plasma proteins in both fetus and ewe, with greater than 99.4 and 98.9% binding,respectively. The reason for the persistence of DPMA and[2H10]DPMA in fetal blood could bedue to both poorly developed elimination pathways for this metabolite and the limited placentalpermeability of the compound.There were substantial differences between adult and fetal sheep in terms of the renalclearance of DPHM and DPMA. Unlike DPHM and[2H10JDPHM, the fetal renal clearance ofDPMA and[2H10]DPMA (i.e., 0.02 ± 0.01 mL/min/Kg) was well below the reported fetal GFR.However, in the ewe the renal clearance of these compounds was substantially greater (0.9 ± 0.03mL/minlKg) than reported values for GFR. Thus, these data suggest that the fetal kidney canefficiently secrete DPHM (organic base), but not DPMA (organic acid). Similar findings havebeen noted for other organic bases (i.e., meperidine, ranitidine, and cimetidine) and organic acids(i.e., para-aminohippurate, indomethacin, and vaiproic acid). Unlike DPHM, DPMA and[2H10]DPMA could not be measured in amniotic and fetal tracheal fluid. The lack of thismetabolite in amniotic fluid was not surprising since fetal urine, which contributes largely to thecomposition of amniotic fluid, contains very minimal amounts of[2H1o1DPMA and DPMA, andfurther, the high degree of plasma protein binding could preclude[2H10]DPMA and DPMA fromdiffusing across the fetal membranes into amniotic fluid.2175.6. Fetal and Maternal Hepatic Microsomal Metabolism of DPHMThe in vitro metabolism of DPHM was assessed in hepatic microsomes prepared fromfetal lambs and adult sheep. Hepatic microsomal incubations of DPHM resulted in the Ndemethylation of DPHM in fetal and adult preparations. The amount of this metabolite formedin fetal microsomes was 84% less than that observed in adult microsomal preparations. Thedeaminated metabolite, DPMA, was observed following both fetal and maternal microsomalincubations, and the amounts formed were similar in both fetal and maternal microsomalincubations. This finding, in addition to demonstrating similar rates of this metabolic pathwayfor DPHM, provides more direct evidence for the fetal formation of DPMA.5.7. Global SummaryIn the current study, the measured fetal renal clearance of DPHM contributed only -2% tothe observed fetal non-placental clearance. Further, a previous study has shown that thepulmonary extraction of DPHM is minimal (8 ± 6 %), and since the fetal lung only receives asmall portion of the fetal cardiac output (< 5%), fetal pulmonary clearance likely contributes-8% towards the fetal non-placental clearance (Yoo, 1989). Thus, the total non-placentalclearance of DPHM measured by direct methods (i.e., pulmonary and renal clearance) canaccount for about 10% of the observed model derived non-placental clearance estimate. If thefetal liver were to contribute the remainder of the non-placental clearance, then the extraction ofDPHM from umbilical venous blood (i.e., the portion of umbilical venous blood that is notshunted past the fetal liver via the ductus venosus, —100 mL/min/Kg) across the fetal liver wouldhave to be nearly complete. Since fetal hepatic first-pass uptake of DPHM was not consistently218detected following umbilical venous bolus and infusions of DPHM, fetal hepatic elimination ofDPHM clearly does not explain the remaining portion of the fetal non-placental clearance.However, even though the fetal liver does not appear to account for a large portion of the fetalnon-placental clearance, the demonstration of fetal hepatic in vitro metabolism of DPHM (i.e.,fetal formation of N-demethyl DPHM and DPMA) suggests the fetal liver may be capable ofmetabolizing DPHIVI, and thus still contribute a portion of the fetal non-placental clearance.While the low renal clearance of intact DPHM is similar both in fetus and mother, there appearsto be a large difference in the hepatic uptake and/or metabolism of DPI{M between mother andfetus. This suggests that the pathways for the non-placental elimination of DPHM differ inmother and fetus.It is still not known which fetal organ(s) are primarily responsible for the observed fetalnon-placental clearance. Acetaminophen exhibited similar characteristics since only 38% of theCLfo has been accounted for, even though a much larger portion (-98%) of the maternal non-placental clearance could be explained by maternal renal elimination and conjugation reactions.An analogous situation was also observed for ritodrine, where only -22% of the fetal dose couldbe accounted for by glucuronide conjugation. Since we can only account for a small portion offetal drug elimination, our understanding of fetal non-placental metabolism and drug dispositionremains limited and requires substantial further study. Assessment of the quantitative role ofDPMA, which is formed both in utero by the fetal lamb and in vitro by fetal hepatic microsomes,may account for a further portion of the fetal non-placental clearance. It would also be prudent toinvestigate the role of other identified and as of yet unidentified metabolites of DPHM towardsthe fetal and maternal non-placental clearances of this drug.2195.8. ConclusionsThe conclusions which can be drawn from the experiments carried out in this thesis are:1. There are no isotope effects which would interfere with the interpretation of the resultsfrom experiments conducted using simultaneous administrations of both DPHM and[2H10]DPHM in non-pregnant, pregnant, and fetal sheep.2. There is a considerable hepatic first-pass drug uptake in adult sheep following mesentericadministration of DPHM.3. There is no significant fetal first-pass effect following umbilical venous bolus DPHMadministration and infusion, suggesting that this mechanism does not play a major role inreducing the fetal exposure to maternally derived DPHM. However, due to the variabilityassociated with these results and the in vitro data from the fetal hepatic microsomalincubations, fetal hepatic elimination of DPHM cannot be completely ruled out.4. Fetal renal clearance of DPHM in both maternal and fetal lambs cannot account for alarge portion of the measured non-placental fetal (-.2%) or maternal clearance (-.0.1%).5. While the fetal kidney appears to be able to readily excrete DPHM (i.e., renal clearance isgreater than literature values of fetal GFR), this is not the case for DPMA.220A6. The metabolism of DPHM to DPMA occurs both in vitro (hepatic microsomes) and invivo by fetal lambs and adult maternal sheep.7. DPMA persists longer in fetal sheep than in the mother.8. 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Sci 82: 145, 1993.Yoshida, H., Kamiya, A., Okumura, K., and Hori, R.: Contribution of monoamineoxidase (MAO) to the binding of tertiary basic drugs in lung mitochondria.Pharm. Res. 6:877-882, 1989.Yoshida, H., Okumura, K., and Hori, R.: Contribution of monoamine oxidase (MAO)to the binding of tertiary basic drugs in isolated perfused rat lung. Pharm. Res.7:398-401, 1990.Zar, J. H.: Biostatistical Analysis, Second Edition. N.J.: Prentice-Hall, Inc., 1984.2Appendix 1: Experimental animal data and experimental detailsEWE MW (kg) FW (kg) GA (days) No. of Fetal Administration DoseNo. fetuses Sex Route (ug)A. Non-pregnant bolus_administration543 76.4 na na na na DPHM - MV 100000248 80.0 na na na na DPHM - MV 100000316 78.2 na na na na DPHM-MV 100000105 60.1 na na na na DPI{M - MV 100000617 73.6 na na na na DPHM - MV 100000B. non-pregnant control (isotope effect) studi2169 70.3 na na na na [2H10]DPHM-MV 50300DPHM-MV2167 69.4 na na na na [210]DPHM-MV 50300DPHM-MVC. Non-pregnant mesenteric first-pass study (normoxic and hypoxic)139 62.6 na na na na [2H10]DPHM-Mes 50300DPHM-MV1154 63.1 na na na na 210jDPHM-MV 50300DPHM-Mes1158 65.3 na na na na [10JDPHM-Mes 60200DPHM-MV989 87.6 na na na na [210]DPHM-MV 50300DPHM-Mes102 72.1 na na na na 10]DPHM-Mes 60200DPHM-MVa4oEWE MW (kg) FW (kg) GA (days) No. of Fetal Administration DoseNo. fetuses Sex Route (ug)D. fetal control (isotope effect) study207 68.1 1.7 133 1 m [2H10JDPHM-TV 4600DPHM-TV1143 75.5 2.3 129 2 f [210jDPHM-TV 4700DPHM-TV975 85.3 2.0 138 2 f [210JDPHM-TV 5000DPHM-TV102 72.1 2.2 124 [210]DPHM-TV 3700DPHM-TV1124 63.1 3.5 132 1 f [210]DPHM-TV 5100DPHM-TVE. fetal umbilical first-pass study989 69.4 2.6 129 2 m [2H10]DPHM-UV 2500DPHM-TV208 91.2 1.8 124 2 f [210]DPHM-TV 2400DPHM-UV499 85.3 2.2 127 3 m [210]DPHM-TV 5100DPHM-UV1143 75.5 1.9 127 2 f [210JDPHM-TV 4900DPHM-UV543 102.1 3.4 128 2 m [210]DPHM-UV 4900DPHM-TV975 85.3 1.8 135 2 m [210]DPHM-UV 5000DPRM-TVMW-maternal weight, FW-fetal weight at time of experiment, GA-fetal gestational age at time of experiment. na -not applicable. MV-maternal femoral vein, Mes-maternal mesenteric vein, TV-fetal lateraltarsal vein, UV-common umbilical vein.F. Simultaneous fetal umbilical and tarsal venous infusion study1142 74.4 1.5 132 2 f [2H10]DPHM-UV 60.8DPHM-TV 60.61250 73.9 3.2 138 2 m [210jDPHIVI-TV 55.8DPHM-UV 55.31242(1) 73.5 1.8 123 2 m [210]DPHM-UV 60.5DPHM-TV 60.61242(2) 73.5 2.6 134 2 m [210]DPHM-UV 60.3DPHM-TV 60.62164 77.1 1.0* 125 2 m [210]DPHM-TV 75.4DPHM-UV 79.2MW-maternal weight, FW-fetal weight at time of experiment, GA-fetal gestational age at time of experiment. na -not applicable. MV-maternal femoral vein, Mes-maternal mesenteric vein, TV-fetal lateral tarsal vein, UVcommon umbilical vein. *Ewe died in utero weight determiniation based on time of death1L4EWE MW (kg) FW (kg) GA (days) No. of Fetal Administration DoseNo. fetuses Sex Route ug/minG. Paired simultaneous_maternal/fetal_infusions2101 81.6 2.056* 133 1 f [2H10IDPHM-TV 170DPHM-MV 670122z 62.6 2.260* 127 2 m [210]DPHM-TV 170DPHM-MV 6702177 74.9 2.244* 128 1 m [210}DPHM-TV 170DPHM-MV 6702181 65.8 2.176* 131 2 f [210]DPHM-TV 170DPHM-MV 6702241 67.6 2.440* 125 2 f [210]DPHM-TV 170DPHM-MV 670H. Fetal Hepatic Microsomal Preparations230 76.3 2.08 131 3 f562 65.9 2.70 138 2 f1143 74.5 2.40 131 3 fAPPENDIX 2‘H-NMR (400 MHz) of Diphenhydramine HCL in D207,1/!.iPPMAPPENDIX 2 (Cont.)‘H-NMR (400 MHz) of[210]Diphenhydramine HCL in D206.5 6.0 5.5 5.0 4.54.0 3.5 3.0PPMAPPENDIX 2 (Cont.)‘H-NMR (200 MHz) of[210]Diphenhylmethoxyacetic acid in CDCL3Note TMS was used as an internal stardard in this sampleOCI2OS. 114AU PROS:X00 AUDATE 12- 10—93SF 200.132ST 80.001 3450 000SI 32768TO 32768SW 4000.000HZ/PT .244P14 0.0PD 0.0AG 4.096RG 40NS 32TE 298FW 500002 3140.000OP 63L 20LB 300GB .100CX 25,00CV 17.00Fl 1l.500PF2 —1.0002HZ/C14 100.063PP14/CM .500SR 2341.60ZLPI114 A TI/1.0.Q 4 ‘-a i.e ..oZLSAPPENDIX 2 (Cont.)‘H-NMR (200 MHz) of Diphenhylmethoxyacetic acid in CDCL3P8005 HI FT RUN IN ±CL3B€R1441205. 107AU PROS:X00 AUDATE 12—5—93SF 200 13250 80.001 3450.000SI 32768TO 32768SW 4000.000HZ/PT .244PM 0.0RD 0.0AG 4.096PG 16NS 32TE 298FM 500002 3140.000OP 63L PDLB .300GB .100CX 25.00CT 17.00Fl 11.500PP2 —1.000PHZ/CM 100.063PPM/CH.OO000B’OO 60 5O 4O 30 20 O O1. I .> z U) S CID 0‘.3 U-AmnioticCavityAllan-jCavityibilicalVeinBladderFemoralArtery

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