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Analysis, pharmacokinetics, metabolism and pharmacodynamics of labetalol in pregnant and nonpregnant… Yeleswaram, Krishnaswamy 1992

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to the st. o • . d required.ANALYSIS, PHARMACOKINETICS, METABOLISM ANDPHARMACODYNAMICS OF LABETALOL IN PREGNANT ANDNONPREGNANT SHEEPByKRISHNASWAMY YELESWARAMB. Pharm., University of Madras, India, 1984.M. Pharm., Banaras Hindu University, India, 1987.A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYTHE FACULTY OF GRADUATE STUDIES(Faculty of Pharmaceutical Sciences)(Division of Biopharmaceutics and Pharmaceutics)We accept this thesis as conformingTHE UNIVERSITY OF BRITISH COLUMBIASeptember, 1992© KRISHNASWAMY YELESWARAMIn 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.(Signature)FACULTY OF PHARMACEUTICAL SCIENCESAnorminix of ^The University of British ColumbiaVancouver, CanadaDate^September, 1992DE-6 (2/88)ABSTRACTLabetalol, a combined a l and p-adrenoceptor blocker, is used as anantihypertensive, especially in the management of hypertensive disorders inpregnancy. While the antihypertensive efficacy of labetalol in pregnancy has beenassessed by a number of clinical trials, there is little information in the literatureabout the disposition or pharmacodynamics of labetalol in the in utero fetus.Hence, a detailed investigation into the maternal-fetal pharmacokinetics andpharmacodynamics of labetalol was undertaken. Studies in adult nonpregnantsheep were also conducted to complement the data obtained from pregnant sheep.The maternal-fetal pharmacokinetics and pharmacodynamics of labetalolwere studied in chronically instrumented pregnant sheep following a 100 mg i.vbolus. Labetalol was measured by a sensitive microbore high-performance liquidchromatographic assay with fluorescence detection, developed in this laboratory.The maternal total body clearance was 30.8 ± 3.83 inUmin/kg (mean ± SEM),elimination half-life was 2.79 ± 0.66 h and the volume of distribution at steady-state was 3.02 ± 0.18 LAT. Labetalol persisted in the amniotic and tracheal fluidsup to 24 h. The fetal exposure to labetalol, calculated as the ratio of fetal tomaternal area under the arterial plasma concentration-time curve, was equal to 14.4± 1.54%. Significant metabolic effects including maternal and fetal hyperglycemiaand lactic acidosis and a decrease in fetal oxygen content were observed,suggesting potent r32-agonist activity. The lactic acidosis was more pronounced inthe fetus than in the ewe.In adult nonpregnant sheep, a 100 mg i.v. bolus administration of labetalolcaused hyperglycemia, lactic acidosis, a significant increase in femoral blood flow,hypotension and tachycardia. The net output of lactate from the hind limb wasfound to be 6.25 ± 1.35 g (0.07 ± 0.015 mol). The metabolism of labetalol wasiiiiistudied using the urine and bile samples obtained in this study. The cumulativeurinary excretion of labetalol as unchanged drug, glucuronide and sulfate werefound to be 1.61 ± 0.38, 11.46 ± 2.83 and 1.47 ± 0.74% of the dose, respectively.A sensitive and specific gas chromatographic assay with mass selective detectionwas developed to identify and quantitate 3-amino-l-phenylbutane, an oxidativemetabolite of labetalol and a close structural analog of amphetamine. Using thisassay, the metabolite was identified in the bile and urine samples. The cumulativeurinary excretion of the oxidative metabolite was equivalent to 0.044 ± 0.016% oflabetalol dose. The exact role of this metabolite in the mediation of labetalolpharmacodynamics remains to be investigated.The vasodilation caused by labetalol in adult sheep, appears to be a directeffect of this drug, with little or no involvement of a-blockade or P-agonism. Inthe fetal hind limb, however, labetalol did not produce any consistent vasodilation.The pharmacokinetics and pharmacodynamics of dilevalol, the RR-isomerof labetalol, were studied in two adult nonpregnant sheep following a 25 mgintravenous bolus and the results suggest that most of the pharmacodynamics oflabetalol in sheep could be attributed to dilevalol.The pharmacokinetics, metabolism and pharmacodynamics of labetalol inthe fetus following exposure to clinically relevant labetalol concentrations werestudied after a 4 mg direct fetal i.v. bolus. The total body clearance (50.45 ± 1.37mL/min/kg), elimination half-life (4.35 ± 0.33 h) and apparent volume ofdistribution at steady-state (14.28 ± 0.95 L/kg) of labetalol in the fetal lamb weresignificantly higher than the corresponding values in the ewe following a 100 mgbolus. The calculated fetal transplacental clearance was 7.27 ± 2.11 mL/min/kgwhile the fetal nonplacental clearance was 43.18 ± 3.72 mL/min/kg. Glucuronideconjugates were present in the amniotic fluid samples, but sulphate conjugates and3-amino-l-phenylbutane, could not be identified in fetal plasma or amniotic fluidivsamples. Significant hyperglycemia and lactic acidosis were observed, but themagnitude of fetal lactic acidosis, calculated as the incremental increase in arteriallactate AUC (61.11 ± 15.59 mMh), was not significantly different from thatobserved following maternal administration (67.8 ± 7.26 mMh), despite a roughlyfour-fold higher fetal exposure to labetalol. The exact reasons for this discrepancyare not clear.VTABLE OF CONTENTSAbstract^ iiTable of Contents^ vList of Illustrations xiList of Tables xxList of Abbreviations^ xxiAcknowledgements xxivDedication^ xxv1. INTRODUCTION 11.1.^Labetalol^ 11.1.1.^Pharmacology 11.1.2.^Clinical Use 21.1.2.1. General^ 31.1.2.2. Hypertension in Pregnancy^ 31.1.3.^Fetal Effects 41.1.4. Methods of Analysis 61.1.5.^Pharmacokinetics^ 71.1.6.^Metabolism 91.1.7.^Placental Transfer 91.2.^Dilevalol^ 101.3.^Objectives and Rationale^ 111.3.1.^Specific Aims^ 122. EXPERIMENTAL 132.1.^Materials^ 132.1.1. Preparation of Standard Solutions and Buffers^ 152.2.^Equipment and Instrumentation^ 162.2.1. High-performance Liquid Chromatography^ 162.2.3. Gas Chromatography with Mass Selective Detection^172.2.3. Physiological Monitoring^ 172.2.4. General Experimental 182.3.^Development of a HPLC Assay with FluorescenceDetection for the Quantitation of Labetalol inBiological Fluids of Sheep^ 18v i2.3.1. Optimization of Mobile Phase^ 182.3.2. Optimization of Detection 192.3.3.^Optimization of Extraction 202.3.4. Procedures for the Analysis of Labetalol inBiological Fluids^ 202.3.4.1. HPLC Operating Conditions^ 202.3.4.2. Extraction from Biological Fluids 212.3.4.3. Preparation of Calibration Curve 222.3.4.4. Quantitation of Labetalol in Biological Fluids^222.3.5. Labetalol Assay Validation^ 222.3.5.1. Precision of Quantitation 222.3.5.2. Extraction Recovery Studies 232.3.5.3. Determination of Minimum Quantitation Limit^232.3.6. Analysis of Glucuronide and Sulphate Conjugatesof Labetalol^ 232.4.^Development of a GC-MSD Assay for the Identificationand Quantitation of 3-APB in Biological Fluids of Sheep^242.4.1. Optimization of GC Conditions^ 242.4.2. Optimization of Extraction and Derivatization^252.4.3. Optimization of Sensitivity 252.4.4. Procedures for the Analysis of 3-APB in BiologicalFluids^ 262.4.4.1. GC Operating Conditions^ 262.4.4.2. Extraction and Derivatization 262.4.4.3. Preparation of Calibration Curve 272.4.4.4. Quantitation of 3-APB in Biological Fluid Samples^282.4.5. Validation of 3-APB Assay^ 282.4.5.1. Precision of Quantitation 282.4.5.2. Extraction Recovery Studies 282.4.5.3. Determination of Minimum Quantitation Limit^292.4.6. Analysis of Glucuronide and Sulphate Conjugates of3-APB^ 292.5.^Standard Procedures for Sheep Experiments^ 292.5.1. Recording of Hemodynamic Parameters 292.5.2. Blood Gas Analysis^ 302.5.3. Glucose Measurement 302.5.4. Lactate Measurement 312.6.^Animal Preparation^ 312.6.1. General Maintenance 312.6.2. Pregnant Sheep Studies 32vii2.6.3. Nonpregnant Sheep Studies^ 362.7.^Experimental Protocols 362.7.1.^Maternal Bolus Studies 362.7.2. Bolus Studies in Nonpregnant Sheep^ 382.7.2.1. Labetalol Bolus^ 382.7.2.2. Dilevalol (RR-isomer of labetalol) Bolus 392.7.3. Infusion Studies in Nonpregnant Sheep 392.7.3.1. Norepinephrine (NE) Challenge^ 402.7.4. Intra-arterial Administration of Labetalol^ 402.7.4.1. Studies in Adult Nonpregnant Sheep 402.7.4.2. Studies in Fetal Sheep^ 412.7.5.^Fetal Bolus Studies 422.8.^Data Analysis 432.8.1. Pharmacokinetic Analysis^ 432.8.1.1. Selection of Weighting Factor 432.8.1.2. Pharmacokinetic Fitting 432.8.1.3. General Calculation of Pharmacokinetic Parameters^442.8.1.4. Calculation of Transplacental and NonplacentalClearances^ 462.8.2. Analysis of Hemodynamic and Metabolic Data^472.8.3.^Statistical Analysis 473.^RESULTS^ 483.1.^Development of a Microbore HPLC Assay withFluorescence Detection for the Quantitation of Labetalolin Biological Fluids^ 483.1.1. Optimization of Mobile Phase^ 483.1.2. Optimization of Detection of Labetalol^ 483.1.3. Optimization of Extraction of Labetalol 503.1.4. Extraction of Labetalol from Biological Fluids 503.1.5. Validation of Labetalol Assay^ 553.1.5.1. Precision of Quantitation 553.1.5.2. Extraction Recovery Studies 553.1.5.3. Minimum Quantitation Limit of Labetalol Assay^553.2.^Development of a GC-MSD Assay for theIdentification and Quantitation of 3-APB, an OxidativeMetabolite of Labetalol in the Biological Fluids of Sheep^593.2.1. Optimization of GC Conditions^ 593.2.2. Optimization of Extraction and Derivatization of 3-APB^59viii3.2.3. Derivatization and Extraction of 3-APB fromBiological Fluids^ 613.2.4. Validation of 3-APB Assay^ 663.2.4.1. Precision of Quantitation 663.2.4.2. Extraction Recovery Studies 663.2.4.3. Minimum Quantitation Limit of 3-APB Assay^ 663.3.^Maternal Bolus Studies^ 663.3.1. Experimental Details 683.3.2. Pharmacokinetics 683.3.3. Hemodynamic Effects^ 743.3.3. Metabolic Effects 743.3.3.1. Changes in Blood Gas Parameters^ 743.3.3.2. Blood Glucose and Lactate Levels 783.4.^Labetalol Bolus Studies in Nonpregnant Sheep^ 803.4.1. Pharmacokinetics^ 803.4.2. Metabolism 863.4.2.1. Conjugative Metabolism 863.4.2.2. Oxidative Metabolism^ 863.4.3. Hemodynamic Effects 923.4.4. Metabolic Effects 923.5.^Dilevalol Bolus Studies in Nonpregnant Sheep^ 1013.5.1.^Pharmacokinetics^ 1013.5.2. Metabolism 1013.5.3. Hemodynamic Effects 1063.5.4. Metabolic Effects^ 1063.6.^Labetalol Infusion in Nonpregnant Sheep^ 1133.6.1.^Disposition of Labetalol 1133.6.2. Hemodynamic Effects^ 1133.6.3. Metabolic Effects 1163.6.4. Intra-arterial Norepinephrine Studies^ 1163.7.^Intra-arterial Labetalol Studies 1223.7.1. Studies in Adult Nonpregnant Sheep^ 1223.7.2.^Studies in Fetal Sheep^ 1223.8.^Fetal Labetalol Bolus Studies 1223.8.1.^Experimental Details^ 1223.8.2.^Pharmacokinetics 1263.8.3.^Metabolism^ 130ix3.8.4. Hemodynamic Effects 1303.8.5. Metabolic Effects 1304. DISCUSSION 1424.1. Development of a HPLC Assay with FluorescenceDetection for the Quantitation of Labetalol in BiologicalFluids 1424.2. Development of a GC-MSD Assay for the Identificationand Quantitation of 3-APB, an Oxidative Metabolite ofLabetalol in Biological Fluids of Sheep 1444.3. Maternal Bolus Studies 1464.3.1. Pharmacokinetics 1464.3.2. Hemodynamic Effects 1494.3.3. Metabolic Effects 1504.4. Labetalol Bolus Studies in Nonpregnant Sheep 1574.4.1. Pharmacokinetics 1584.4.2. Metabolism 1584.4.3. Hemodynamic Effects 1604.4.4. Metabolic Effects 1614.5. Dilevalol Bolus Studies in Nonpregnant Sheep 1634.5.1. Pharmacokinetics 1644.5.2. Metabolism 1644.5.3. Hemodynamic Effects 1654.5.4. Metabolic Effects 1664.6. Labetalol Infusion in Nonpregnant Sheep 1674.6.1. Disposition 1674.6.2. Hemodynamic Effects 1684.6.3. Metabolic Effects 1694.6.4. Intra-arterial Norepinephrine Studies 1694.7. Intra-arterial Labetalol Studies 1704.7.1. Studies in Adult Nonpregnant Sheep 1704.7.2. Studies in Fetal Sheep 1714.8. Fetal Labetalol Bolus Studies 1724.8.1. Pharmacokinetics 1734.8.2. Metabolism 1764.8.3. Hemodynamic Effects 1774.8.4. Metabolic Effects 1775. SUMMARY AND CONCLUSIONS 1815.1. Quantitation of Labetalol in Biological Fluids 1815.2. Analysis of 3-amino-l-phenylbutane in BiologicalFluids 1815.3. Maternal Bolus Administration of Labetalol 1825.4. Labetalol Bolus in Adult Nonpregnant Sheep 1835.5. Dilevalol Bolus in Adult Nonpregnant Sheep 1845.6. Labetalol Infusion to Steady-state in Adult NonpregnantSheep 1845.7. Intra-arterial Administration of Labetalol 1845.8. Labetalol Bolus Administration in the Fetal Lamb 1856. REFERENCES 187xiLIST OF ILLUSTRATIONSFIG 1: Chemical structure of labetalol (* denotes chiral centre).^1FIG 2: Schematic diagram of ovine fetal hind limb to illustratethe position of the hind limb catheters and flowmeter.^35FIG 3: Effect of molarity of phosphate buffer in the mobilephase on the peak width and retention time of labetalolon a C-18 column.^ 49FIG 4: Absolute extraction recovery of labetalol with differentsolvents. A: Dichloromethane, B: Ethyl acetate, C:Diethyl ether and D: Toluene.^ 51FIG 5: HPLC Chromatograms obtained from blank sheepplasma following a two-step (A) and one-step (B)extraction.^ 52FIG 6: Optimized procedure used for the extraction oflabetalol from biological fluids.^ 53FIG 7: Superimposed HPLC chromatograms of blank andspiked biological fluids (1 - Labetalol and 2 - Internalstandard): A: Pregnant sheep plasma, B: Amnioticfluid, C: Fetal plasma and D: Fetal tracheal fluid.^54FIG 8: A typical calibration curve employed in the quantitationof labetalol (5 - 120 ng) in plasma (mean ± SD) (n=3).^56FIG 9: Extraction recovery of labetalol from sheep plasma asa function of amount added (mean ± SD) (n=3).^58FIG 10: HPLC chromatogram obtained from blank sheepplasma spiked with 0.5 ng labetalol (the lowestcalibration point). (1 - Labetalol and 2 - Internalstandard). Approximately 40 pg of labetalol wasactually injected. 60FIG 11: Effect of various reagents used for sample pHadjustment (prior to solvent extraction) on theabsolute recovery of 3-APB in urine. 62xiiFIG 12: Optimized procedure used for the extraction of3-APB from biological fluids.^ 63FIG 13: EI GC-MS following HFBA derivatization of standard3-APB and MPE (internal standard). A: Total IonChromatogram; B: Mass Spectrum of 3-APB Derivativeand C: Mass Spectrum of the MPE Derivative.^ 64FIG 14: 3-APB Derivative: Suggested m/z assignments for themass spectrum.^ 65FIG 15: Typical calibration curve following extraction of3-APB (0.5 - 1000 ng) from urine (mean ± SD; n=3).^67FIG 16: Representative plots of labetalol concentrations in twoexperiments following a 100 mg maternal intravenousbolus administration. (A - E# 109 and B - E# 201).(MA: Maternal arterial plasma, FA: Fetal arterial plasma,UT: Uterine venous plasma, UV: Umbilical venousplasma, AM: Amniotic fluid and TR: Tracheal fluid).^71FIG 17: Effect of a 100 mg i.v. labetalol bolus on maternal heartrate and arterial pressure (mean ± SEM) (n----6).^ 75FIG 18: Effect of a 100 mg i.v. labetalol bolus on fetal heart rateand arterial pressure (mean ± SEM) (n=7).^ 76FIG 19: Fetal and maternal arterial blood gas parameters (mean± SEM) before and after a 100 mg maternal intravenousbolus administration of labetalol. (MA: Maternal arterialblood and FA: Fetal arterial blood). Asterisks (*) denotesignificant difference from control values.^ 77FIG 20: Glucose and lactic acid concentrations (mean ± SEM)before and after a 100 mg maternal intravenous bolusadministration of labetalol. (MA: Maternal arterial blood,FA: Fetal arterial blood and AM: Amniotic fluid).^79FIG 21: Glucose and lactic acid concentrations (mean ± SEM)before and after a 20 mL control saline administration.(MA: Maternal arterial blood, FA: Fetal arterial bloodand AM: Amniotic fluid). 81FIG 22: Fetal and maternal arterial blood gas parameters (mean± SEM) following a 20 mL saline administration(control experiment). (MA: Maternal arterial bloodand FA: Fetal arterial blood).^ 82FIG 23: Disposition of labetalol in adult sheep plasma followinga 100 mg i.v. bolus (mean ± SEM).^ 83FIG 24: Cumulative urinary excretion of labetalol and itsconjugates (mean ± SEM). A: Cumulative amountexcreted and B: Percentage of labetalol dose recoveredin urine as free drug and conjugates.^ 87FIG 25: Concentrations of labetalol and its conjugates in adultsheep bile following a 100 mg i.v. bolus (E# 617).^88FIG 26: Total ion chromatogram (top panel) of urine sampleobtained from E#105 (nonpregnant) following a 100mg labetalol bolus. Asterisk denotes the 3-APB peakand the EI mass spectrum corresponding to that peak(bottom panel).^ 89FIG 27: Excretion of 3-APB in adult non-pregnant sheep urinefollowing a 100 mg labetalol administration.(mean ± SEM; n=5). A: Cumulative excretion over24 h; B: Excretion rate plot.^ 90FIG 28: Excretion of 3-APB conjugates following a 100 mglabetalol administration. A: Urine (amount) (E#105)and B: Bile (concentration) (E#248).^ 93FIG 29: Oxidative metabolism of labetalol.(Not exhaustive; dotted lines indicate potentialpathways; structure of amphetamine is included forthe sake of comparison; partly from Gal et al., 1988).^94FIG 30: Hemodynamic changes following labetalol bolus in adultnonpregnant sheep (mean ± SEM) (n=5). (Asterisksdenote significant difference from control values).^95FIG 31: Femoral arterial and venous blood gas changesfollowing labetalol bolus in adult nonpregnant sheep.(mean ± SEM) (n=5). (Asterisks denote significantdifference from control values).^ 97xivFIG 32: Effect of labetalol on oxygen homeostasis in adultnonpregnant sheep (mean ± SEM) (n=5). (Asterisksdenote significant difference from control values).^98FIG 33: Labetalol bolus in adult nonpregnant sheep: Femoralarterial and venous blood glucose and lactateconcentrations (mean ± SEM; n=5). Asterisks denotesignificant difference from control values.^ 99FIG 34: Changes in arterio-venous fluxes following labetaloladministration in adult nonpregnant sheep (mean ±SEM; n=5). Positive values indicate net uptake andnegative values indicate net release from the hind limb.Asterisks denote significant difference from controlvalues.^ 100FIG 35: Disposition of dilevalol in adult nonpregnant sheeparterial plasma following a 25 mg i.v. bolus.^ 102FIG 36: Cumulative urinary excretion of dilevalol asunconjugated, glucuronide and sulphate in an adultnonpregnant sheep (E#105).^ 104FIG 37: Concentrations of unconjugated (free) and conjugateddilevalol in bile of adult nonpregnant sheep followinga 25 mg i.v. bolus (E#105). 105FIG 38: Excretion of 3-APB and its conjugates following a 25mg dilevalol administration in an adult nonpregnantsheep (E#105). A: Urine (amount) and B: Bile(concentration).^ 107FIG 39: Hemodynamic changes in an adult nonpregnant sheep(E#105) following a 25 mg dilevalol bolus.^ 108FIG 40: Femoral arterial and venous blood gas changes in anadult nonpregnant sheep (E#105) following a 25 mgdilevalol bolus.^ 109FIG 41: Femoral arterial and venous blood glucose and lactateconcentrations in an adult nonpregnant sheep (E#105)following a 25 mg dilevalol bolus. 110xvFIG 42: Effect of dilevalol on oxygen homeostasis in an adultnonpregnant sheep (E#105).^ 111FIG 43: Changes in arterio-venous fluxes following dilevaloladministration in an adult nonpregnant sheep (E# 105).Positive values indicate net uptake and negative valuesindicate net release from the hind limb.^ 112FIG 44: Arterial plasma labetalol concentrations following acombined bolus (100 mg) and infusion (0.5 mg/min for6 h) in adult nonpregnant sheep (mean ± SEM).(Solid line indicates infusion period).^ 114FIG 45: Hemodynamic changes during and after continuousinfusion of labetalol in adult nonpregnant sheep (mean± SEM; n=5). Solid line denotes infusion period andasterisks denote significant difference from respectivecontrol values.^ 115FIG 46: Changes in femoral arterial and venous blood gasparameters during and after continuous infusion oflabetalol in adult nonpregnant sheep (mean ± SEM).Asterisks denote significant difference from controlvalues; solid line indicates infusion period.^ 117FIG 47: Oxygen homeostasis in adult nonpregnant sheepbefore, during and after continuous infusion oflabetalol (mean ± SEM). Asterisks denote significantdifference from control values; solid line indicatesinfusion period.^ 118FIG 48: Effect of labetalol on arterial and venous blood glucoseand lactate concentrations (mean ± SEM). (solid lineindicates infusion period; arterial and venous bloodlactate concentrations were significantly different fromcontrol between 30 min and 2 h post-infusion; arterialand venous glucose concentrations were significantbetween 3 h and 2 h post-infusion.).^ 119FIG 49: Hind limb arterio-venous glucose and lactate fluxbefore, during and after labetalol infusion in adultxvinonpregnant sheep (mean ± SEM). (Asterisks denotesignificant difference from control values; dashed lineindicates infusion period).^ 120FIG 50: Intra-arterial norepinephrine dose-response curvebefore and during labetalol infusion (mean ± SEM; n=4).^121FIG 51: A.: Intra-arterial labetalol dose-response relationship inadult nonpregnant sheep hind limb before and afterphentolamine administration (E# 543).B.: Intra-arterial labetalol dose-response relationship inadult nonpregnant sheep hind limb before and afterpropranolol administration (E# 105).^ 123FIG 52: Polygraph tracings showing the time course ofhemodynamic changes corresponding to intra-arterialadministration of A.: labetalol, B.: norepinephrine andC.: control saline. Vertical arrow on the time scalecorresponds to the time of injection.[1: Femoral Blood Flow (mL/min), 2: Mean ArterialPressure (mm Hg) and 3: Heart Rate (bpm)].^124FIG 53: Intra-arterial (HLA) injection of labetalol in the fetallamb (E#1154).^ 125FIG 54: Disposition of labetalol in pregnant sheep following a4 mg fetal intravenous bolus administration (mean ±SEM; n=5). (FA: Fetal Arterial Plasma; AM: AmnioticFluid; TR: Tracheal Fluid and MA: Maternal ArterialPlasma).^ 128FIG 55: Concentrations of labetalol and its glucuronideconjugate in the amniotic fluid following a 4 mg fetalintravenous bolus administration (mean ± SEM; n=5).^131FIG 56: Effect of a 4 mg fetal intravenous bolus on mean fetalheart rate and arterial pressure (mean ± SEM).Asterisk denotes significant difference.^ 132FIG 57: Effect of a 4 mg fetal intravenous bolus on mean fetalhind limb blood flow and vascular resistance (mean± SEM; n=5).^ 133xviiFIG 58: Effect of labetalol and control (saline) fetal bolusadministration on fetal arterial blood gas parameters(mean ± SEM; n=5). Asterisks denote significantdifference.^ 135FIG 59: Effect of a 4 mg fetal intravenous labetalol bolus onglucose concentrations (mean ± SEM; n=5). (FA:Fetal arterial blood; HV: Hind limb venous bloodand AM: Amniotic fluid).^ 136FIG 60: Effect of a 4 mg fetal intravenous labetalol bolus onlactic acid concentrations (mean ± SEM; n=5). (FA:Fetal arterial blood; HV: Hind limb venous blood andAM:Amniotic fluid). FA and HV concentrations werestatistically significant between 90 min and 12 h andAMN concentrations between 2-24 h. 137FIG 61: Glucose and lactate concentrations in maternal arterialblood following a 4 mg fetal intravenous bolus oflabetalol (mean ± SEM; n=5).^ 138FIG 62: Changes in glucose and lactate concentrations followingcontrol (saline) fetal intravenous bolus administration(mean ± SEM; n=5). (FA: Fetal arterial blood; HV:Hind limb venous blood; MA: Maternal arterial bloodand AM: Amniotic fluid).^ 139FIG 63: Effect of a 4 mg fetal intravenous bolus of labetalol onhind limb arterio-venous labetalol, glucose and lactatefluxes (mean ± SEM). Asterisks denote significantdifference from control values.^ 140xviiiLIST OF TABLESTable 1^Intra-sample variability in labetalol assay.^57Table 2 Experimental details for maternal bolus experiments.^69Table 3 Pre-experimental maternal and fetal blood gasparameters (mean ± SEM).^ 70Table 4 Labetalol pharmacokinetics following maternal bolus.^72Table 5 Comparison of the pharmacokinetics of labetalol inpregnant sheep with reported values in pregnantwomen.^ 84Table 6 Comparative pharmacokinetics of labetalol inpregnant and nonpregnant sheep.^ 85Table 7 Urinary excretion of 3-amino-l-phenylbutane.^91Table 8 Pharmacokinetics of dilevalol (RR-isomer oflabetalol) in adult nonpregnant sheep followinga 25 mg i.v. bolus administration.^ 103Table 9^Fetal bolus studies: experimental details. 127Table 10 Pharmacokinetics of labetalol in the fetal lamb andewe following a 4 mg fetal i.v. bolus (mean ± SEM).^129LIST OF ABBREVIATIONS3-APB:^3 -amino- 1 -phenylbutaneA: Exponential equation constanta:^Alpha, an exponential rate constantAUC: Area under the concentration vs time curveAUMC:^Area under the first moment curve (i.e. time vsconcentration*time)AM:^Amniotic fluidAUTOAN:^Computer program for pharmacokinetic modellingA-V: Arterio-venousB: Exponential equation constantBE:^Blood base excessB: Beta, an exponential rate constantbpm:^Beats per minute°C: Degree celsiusC: Arterial plasma concentration of labetalolCss:^Arterial plasma concentration of labetalol at steady-stateCt-last^Arterial plasma concentration of labetalol in the lastsample obtained or the last sample in which labetalolcould be quantitatedcAMP:^Cyclic adenosine monophosphateCI: Chemical ionizationCL:^Total body clearanceCLf: Fetal total body clearanceCLf.:^Fetal nonplacental clearancexixXXCLfp :^Fetal transplacental clearanceCLm : Maternal total body clearanceCLmn :^Maternal nonplacental clearanceCLmp : Maternal transplacental clearanceCO2 :^Carbon di-oxideCt : Concentration of labetalol at time tCV:^Coefficient of variationEC50 : Concentration that corresponds to 50% of maximalresponseEI:^Electron impact ionizationEM: Emission wavelengthEms :^Maximum pharmacological effecteV: Electron voltsEX:^Excitation wavelengthFA: Fetal arterialg:^gramg: acceleration due to gravityGC:^Gas chromatographyh: hour(s)Hb:^HemoglobinHFBA: Heptafluorobutyric anhydrideHg:^MercuryHLA: Hind limb artery (pudendo-epigastric artery)HLV:^Hind limb vein (pudendo-epigastric vein)HPLC: High performance liquid chromatographyHz:^Hertzi.d.: Internal diameterxxiI.U.:^International unitsi.v.: IntravenousJANA:^Computer program for pharmacokinetic modellingko: Infusion ratekg:^kilogramL: Liter[Lc]:^Concentration of lactic acid[Lc] t : Concentration of lactic acid at time tM: Molar (as a concentration term)MA:^Maternal arterialMANOVA:^Multivariate analysis of variancemeq: Milliequivalents14:^MicrogrampL: Microlitergm:^Micrometermg: milligrammin:^Minute(s)mL: millilitermm:^millimetersmmol: millimole(s)mol:^Mole(s)MPE: 1-methyl-2-phenoxyethylamineMS:^Mass SpectrometryMSD: Mass selective detectionmh:^Mass to charge ration: Number of samplesN:^Normal (as a concentration term)NAD:^Nicotinamide adenine dinucleotideNADH: Reduced form of nicotinamide adenine dinucleotideND:^Not detectableNE: Norepinephrineng:^nanogramnm: nanometerNONLIN:^Computer program for pharmacokinetic modellingNS: No sample02 :^Oxygenp: Probability factorP:^Exponential equation constantpCO2 : Partial pressure of carbon dioxidepg:^PicogrampH: Negative logarithm (base 10) of the hydrogen ionconcentrationTr:^Pi, an exponential rate constantp02 : Partial pressure of oxygenPFTBA:^PerfluorotributylaminePTFE: polytetrafluoroethylener:^Correlation coefficientr2 : Coefficient of determinationrpm:^revolutions per minutesec: secondsSCH:^Schering Corporation's compound, 5-124444-methylphenyl)-2-butylamino)-1-hydroxyethyl) salicylamidehydrochloride hemihydrateSEM:^Standard error of meanSIM:^Selected ion monitoring2.a . Sigma squared, notation for variancess:^(as a suffix) steady-statet-test: Student's t-test for statistical analysis-LA :^Half-lifeTEA: Triethylaminet-last:^Time at which the last sample was collectedtmax : Time corresponding to the maximum concentrationTR:^Tracheal fluidTRIS: Tris(hydroxymethyl)aminomethaneUT:^Uterine veinUV: Umbilical veinVD:^Apparent volume of distribution* Notation for product, i.e., multiplicationAcknowledgementsI would like to express my sincere appreciation and thanks to my researchsupervisors Drs. James E. Axelson and Dan W. Rurak, for their guidance,encouragement and support throughout my graduate training.Thanks to Drs. Frank S. Abbott, Graham H. McMorland and JoanneDouglas for their keen interest in the project and for the several valuablesuggestions. Special thanks to Dr. Wayne Riggs for his support and friendship,which helped me tide over the testing times.My sincere thanks to Mr. Eddie Kwan and Ms. Caroline Hall for theirexceptional technical assistance in sheep studies, which was often above andbeyond the call of duty.I would like to thank Dr. Timothy Stratton and Dr. Johnathan Berkowitz fortheir assistance with the statistical analysis.Many thanks are due to Mr. Ahmad Doroudian for his help in sampleanalysis and his friendship. I would like to thank Mr. George Tonn and Ms.Meike Tonn for their valuable friendship and for providing a home away fromhome. I was fortunate to enjoy the company of my colleagues in the laboratory,Dr. Sun Dong Yoo, Dr. Grace Chan, Dr. Matthew Wright, Dr. Andras Szeitz, Ms.Jing Wang, Ms. Judit Orbay, and Mr. John Kim. Special thanks to Dr. MatthewWright for his help in conducting experiments and helpful criticism during thepreparation of manuscripts.This work would not have been possible without the love, affection andmoral support of my family.Financial support in the form of studentship from the B.C. & Yukon Heartand University Graduate Fellowship are gratefully appreciated. This project wassupported by the Medical Research Council of Canada.xxivDedicationXXVThis thesis is dedicated to my beloved mother, Ms. Parvatham Yeleswaramand to the memory of my dad, Mr. Rajaram Yeleswaram.1. INTRODUCTION1.1. LabetalolLabetalol { 2-hydroxy-5- [1-hydroxy-2-(1-methy1-3-phenylpropylamino)ethyl] benzamide } hydrochloride (TrandateR) is a compound with two chiralcentres (Fig 1) and is marketed as a racemic mixture of all four isomers in roughlyequal proportions (Gold et al., 1982). Labetalol is a combined post-synaptic a land non-selective 0-adrenoceptor blocking agent (Farmer et al., 1972) and is usedas an antihypertensive.H 2N OCHOFIG 1: Chemical Structure of Labetalol (* denotes chiral centre).1.1.1. PharmacologyThe pharmacological effects of labetalol have been studied in vitro and invivo. In rabbit and rat aortic strips, rat vas deferens and guinea-pig isolated leftatrium, labetalol competitively inhibits both a and 0-adrenoceptors (Brittain andLevy, 1976). The in vitro a-blockade was 6-10 times less potent thanphentolamine; the 0-blockade was 1.5-3 times less potent than propranolol and initself, labetalol was 4-8 times more potent at p- than at a-adrenoceptors (Farmer et2al., 1972; Brittain and Levy, 1976). The competitive a and (3-adrenergicantagonism of labetalol was also demonstrated in barbitone-anesthetized dogs,pithed rats as well as in conscious, normotensive and hypertensive dogs andhypertensive rats (Farmer et al., 1972; Kennedy and Levy, 1975; Brittain andLevy, 1976). In anesthetized dogs following intravenous administration, labetalolwas about 7 times less potent than phentolamine in its a-blockade, about 4 timesless potent than propranolol in blocking cardiac P i -receptors, 11-17 times lesspotent than propranolol in blocking vascular and bronchial 132-receptors and initself, 16 times more potent at cardiac P i than at vascular a-adrenoceptors(Kennedy and Levy, 1975; Brittain and Levy, 1976).Labetalol has also been shown to possess partial P2-agonist activity inisolated uterus of the rat (Carey and Whalley, 1979), isolated trachea of theguinea-pig (Carpenter, 1981) and innervated and denervated femoral vascular bedsof dogs (Baum et al., 1981). However, labetalol is devoid of any agonist activityat cardiac P i -receptors (Brittain and Levy, 1976).Brittain et al., 1981, studied the pharmacology of the individual isomers oflabetalol and found that the adrenergic activities of labetalol were not equallydistributed amongst its four stereoisomers. These authors and Gold et al., 1982,found that the RR-stereoisomer contributes to most of the P-adrenoceptor blockingactivity of labetalol and that this isomer was virtually devoid of any a-blockingactivity. The SR-stereoisomer, on the other hand, has most of the a-blockingactivity and shows insignificant 13-antagonism. The other two isomers, SS and RS,were found to have no significant a or P-adrenoceptor blocking activities (Brittainet al., 1981; Gold et al., 1982).1.1.2. Clinical Use3The results from the various clinical trials that were conducted to assess theantihypertensive efficacy of labetalol are summarized in this section. Theoutcome of clinical studies of labetalol in pregnancy is presented separately.1.1.2.1. GeneralLabetalol has been used in the management of systemic hypertension ofvarious etiologies, either alone or in combination with diuretics (Goa et al., 1989).Labetalol has been found effective in achieving target blood pressures in about65% of all hypertensive patients (Arinsoy and Oram, 1986). In a placebocontrolled trial, labetalol was found to cause significant decreases in both standingand supine systolic and diastolic blood pressures (Davidov et al., 1983). Theefficacy of labetalol has also been compared with that of other antihypertensiveagents in several clinical studies. The overall efficacy of labetalol in mild tosevere hypertension was found to be better than that of propranolol (Flamenbaumet al., 1985) and acebutolol (Thibonnier et al., 1980) and comparable to that ofmethyldopa (Frishman et al., 1983), atenolol (Nilsson et al., 1982), pindolol(Romo et al., 1984), prazosin (Gray et al., 1988), verapamil (Anavekar et al.,1982) and clonidine (Lilja et al., 1982).1.1.2.2. Hypertension in PregnancyA number of clinical studies have been conducted to determine the efficacyof labetalol in lowering hypertension in pregnancy without adversely affecting thefetus. Michael, 1979 and 1982, reported that orally administered labetalol (300-1200 mg p.o. daily for 1-19 wks) caused effective and sustained reduction insystolic and diastolic blood pressures and no maternal or fetal side effects. Pickles4et al., 1989, found that the fetal outcome in hypertensive patients treated withlabetalol was not different from that in the placebo treatment group. However,Mabie et al., 1987, who compared the efficacy and neonatal outcome of labetalolplus hospitalization with hospitalization alone, observed that labetalol treatmentwas associated with a higher frequency of fetal growth retardation. Theantihypertensive efficacy and perinatal safety of treatment with labetalol wasfound to be similar to that of methyldopa (Lamming and Symonds, 1979;Lamming et al., 1980; Plouin et al., 1988). In a comparative trial involvinglabetalol and hydralazine, the incidence of fetal distress was found to be less in thelabetalol group (Sibai et al., 1987). Lardoux et al., 1983, observed decreasedincidences of fetal growth retardation with labetalol treatment than with atenololtreatment. In summary, most of the clinical studies have found labetalol to be asafe and effective drug in the management of hypertension in pregnancy.Labetalol has also been used in the management of hypertensive crises inpregnancy. Satisfactory results with intravenous labetalol administration werereported by Garden et al., 1982; Michael, 1986; Ashe et al., 1987; and Mabie etal., 1987. Ashe et al., 1987, however, noted that the efficacy of labetalol inemergencies was less than that achieved with dihydralazine.1.1.3. Fetal EffectsMany of the conclusions about the effects of labetalol on the human fetuswere based on uteroplacental hemodynamics and/or neonatal follow-ups. Lunellet al., 1982 and Nylund et al., 1984, observed no significant changes inuteroplacental blood flow calculated using placental scintigraphy. Usingcombined real time and Doppler ultrasound techniques, Joupilla et al., 1986, foundthat maternal labetalol caused no changes in the blood flows in the intervillous5space, umbilical vein and fetal descending aorta. Umbilical artery flow velocitywaveforms and pulsatility index were studied by Harper and Murnaghan, 1991,using pulsed Doppler ultrasound. They found that labetalol increased theumbilical artery pulsatility index and attributed the increase to vasoconstriction inthe fetoplacental unit due to beta-blockade in the fetus. But, Pirnohen et al., 1991,found no significant changes in uterine artery, umbilical artery or fetal middlecerebral artery flow velocity waveforms. In a neonatal follow-up study,MacPherson et a/., 1986, monitored the systolic blood pressure, heart rate, palmarsweating, response to cold stress and blood glucose for 72 h postnatal in newbornswhose mothers had received labetalol and found that the only significantdifference in comparison to the control group was a transient hypotension at 2h. Itwas concluded that maternal labetalol does not cause significant sympatheticblockade in the newborn. Pickles et al., 1989, studied the fetal outcome followingmaternal labetalol in terms of birth weight, incidence of neonatal respiratorydistress syndrome, hypoglycemia and bradycardia and inferred that labetalol isapparently safe to the fetus. However, Sibai et al., 1987, found that labetalol wasassociated with a higher incidence of fetal growth retardation than untreatedcontrols and that the perinatal outcome was not improved despite reductions in thematernal blood pressure.Information on the fetal effects of labetalol from animal studies is also quitelimited. Nicholas et al., 1978, observed that labetalol increased the amount ofalveolar surfactant in fetal rabbit lungs, possibly through its partial f3 2-agonism.Ahokas et a/., 1989, found that while labetalol effectively lowered blood pressure,it was not associated with any decrease in placental perfusion, in the hypertensiveterm-pregnant rat. Mohan et al., 1990, studied the hemodynamics of varyingdoses of labetalol in normotensive and hypertensive pregnant ewes and found thatthe fetal mean arterial pressure and heart rate did not change significantly.6Eisenach et al., 1991, investigated the effect of labetalol in pregnant sheep duringacute hypertension produced by norepinephrine as well as the degree of alpha andbeta-adrenergic blockade in the mother and fetus, determined by isoproterenol andphenylephrine challenges. Those authors found that labetalol effectively decreasesthe elevated arterial pressure, overcomes the decrease in uterine blood flow as wellas improves the fetal acidemia and hypoxemia induced by noradrenaline. Also,the adrenergic blockade was found to be significantly less in the fetus than in themother (Eisenach et al., 1991).1.1.4. Methods of AnalysisA number of HPLC assay procedures have been reported for thedetermination of labetalol in human plasma. Methods involving ultra-violetdetection (Dusci and Hackett, 1979; Woodman and Johnson, 1981; Hidalgo andMuir, 1984) in general, were limited in application due to lack of sufficientsensitivity to detect concentrations below 20 ng/mL. Hence, electrochemical andfluorescence detection systems were used in an attempt to improve the lowerlimits of detection. Wang et al., 1985, and Abernethy et al., 1986, employedelectrochemical detection using the inherent redox behaviour of labetalol andimproved the sensitivity to about 5 ng/mL. HPLC assays with fluorescencedetection were reported by Meredith et al., 1981, Oosterhuis et al, 1981, andOstrovska et al., 1988, with minimum detection limit in the range of 2-4 ng(amount injected). Alton et al., 1984, used a macroporous co-polymer (PRP-1),which withstands alkaline pH conditions as the stationary phase instead of theconventional reverse-phases (C-18 or C-8) which have a pH tolerance range of2.5-7.5. They used pH 9.5 carbonate buffer and acetonitrile as the mobile phasesince it has been shown that the optimum pH for the fluorescence detection of7labetalol is between 9-10 (Oosterhuis et al., 1981). However, the limit ofdetection was only marginally improved (1.5 ng injected). Luke et al., 1987reported an improved method, which employs PRP-1 column as well as post-column addition of ammonium hydroxide to adjust the pH of the eluent to 11.0and obtained a minimum detection limit of about 0.45 ng. A thermospray massspectrometric method for the determination of labetalol was also reported (Lant etal., 1987), but the authors noted deterioration in sensitivity with repeatedinjections, thus limiting the applications of that method.It should be mentioned that the assay procedures discussed above areachiral and measure total, i.e., racemic labetalol. There has been no validatedreport to date on the quantitation of the individual isomers of labetalol. Lalonde etal., 1990, attempted to study the stereoselectivity in the disposition of labetalol inhumans, using a HPLC assay with oc i -acid glycoprotein chiral stationary phase.However, the details of the assay conditions provided were sketchy and there wasno mention of any validation studies.1.1.5. PharmacokineticsLabetalol undergoes rapid absorption following oral administration, withpeak plasma concentrations reached within 1 to 2 h after drug administration(Abernethy et al., 1985; McNeil et al., 1979). Like other P-blockers, labetalolundergoes significant first-pass metabolism (McNeil and Louis, 1984). Further,considerable inter-patient variability in the extent of first-pass metabolism andhence, the oral bioavailability (11 to 86%) has been observed (McNeil et al.,1979). Daneshmand and Roberts (1982), have shown that food increases thebioavailability of labetalol by about 11%.The protein binding of labetalol in human plasma was reported to be about50% (Martin et al., 1976). Studies in rats, rabbits and dogs have shown thatlabetalol undergoes extensive distribution into the lung, liver and kidney, withlittle being present in brain tissue (Martin et al., 1976). The reported estimates ofapparent volume of distribution of labetalol in humans are in the range of 2.5-15.5L/kg (Goa et al., 1989).The plasma clearance of labetalol following intravenous administration inhypertensive patients was found to be in the range of 10 to 20 mL/min./kg(Abernethy et al., 1987; McNeil et al., 1982; Nyberg et al., 1982). The reportedestimates of elimination half-life following oral or intravenous administration innormotensive or hypertensive subjects have ranged from 3 to 8 h (Goa et al.,1989).The phannacokinetics of labetalol is essentially unchanged by pregnancy(Rubin et al., 1983). A decrease in clearance and increases in elimination half-life, bioavailability and peak plasma concentrations were observed in elderlysubjects as compared to young subjects (Abernethy et al., 1985). Decreasedclearance was also observed in patients with chronic liver diseases (Homeida etal., 1978; Daneshmand et al., 1982), but the pharmacokinetics of labetalol wasunchanged in patients with chronic renal diseases (Wood et al., 1982).Evidence of stereoselectivity in the disposition of labetalol in humans wasreported by Lalonde et al., 1990, who used a stereoselective assay to measure theproportions of the individual stereoisomers in steady-state plasma samples,obtained following labetalol administration. They found that the percentage ofRR-isomer at steady-state was significantly lower than that of the other threeisomers. Further, co-administration of cimetidine, a hepatic enzyme inhibitor, wasfound to cause a significant increase in the steady-state concentrations of all theisomers except the RR-isomer. These results, although based on an assay whose8validity is not clearly established, may suggest stereoselectivity in the dispositionand hepatic elimination of labetalol.1.1.6. MetabolismMass balance studies with 3H and 14C labelled labetalol were conducted byMartin et al., 1976 in rats, rabbits, guinea-pigs and humans and they found thatless than 5% of an oral dose of labetalol was excreted unchanged in the urine.About 20% of the dose was recovered as glucuronide and in humans, the majormetabolite was an unidentified conjugate. No sulfate conjugates were found inany of the species studied (Martin et al., 1976). Recently, Gal et al., 1988,reported that labetalol undergoes oxidative metabolism via N-dealkylation to form3-amino-l-phenylbutane and 3-amino-1-(4-hydroxyphenyl) butane. Thesecompounds, which are structural analogs of amphetamine, were identified in theurine samples of three patients on labetalol therapy. The fraction of dose thatundergoes N-dealkylation, however, was not known.1.1.7. Placental TransferThere has been no report in the literature that has attempted to quantitateplacental transfer and fetal exposure to labetalol following maternal administrationin any species. Martin et al., 1976, used whole body autoradiography followingadministration of 14C-labelled labetalol in pregnant rats and rabbits and found thatthe total radioactivity in the fetus was less than 1% of the maternal levels. Thereare no published data in any animal species regarding disposition andpharmacokinetics of labetalol in the in utero fetus following maternal or fetaladministration. In humans, the cord blood/ maternal venous labetalol9concentration ratio at the time of delivery averages 0.5 (range: 0.2 to 1.0)(Michael, 1979; Nylund et al., 1984; Rogers et al., 1990), suggesting significantfetal exposure to the drug. However, a single point estimate obtained at the timeof delivery is of limited value and could even be misleading since it changesconstantly with respect to the time of drug administration (Anderson et al.,1980b). The concentrations of labetalol in amniotic fluid and breast milk wereless than that in maternal plasma (Michael 1979; Lunell et al., 1985).1.2. DilevalolDilevalol is the RR-stereoisomer of labetalol and has 3 to 6 times theactivity of labetalol in blocking p-adrenoceptors, but only about 10% of a l -blocking activity of labetalol (Sybertz et al., 1981). The relative selectivity ofdilevalol for p i - vs ocradrenoceptors was around 500:1 in rats (Gold et al., 1982)and 400:1 in anesthetized dogs (Brittain et al., 1982). The partial agonist activityof dilevalol is mostly at the 02-receptor subtype and is about 7 times more potentthan labetalol (Sybertz et al., 1981). The antihypertensive effect of dilevalol isbelieved to be caused primarily through a reduction in peripheral vascularresistance (Clifton et al., 1988). The hypotension caused by dilevalol may beaccompanied by tachycardia (Baum et al., 1981; Baum and Sybertz, 1983).Intravenous dilevalol administration results in an increase in norepinephrine andepinephrine levels (Grossman et al., 1989).Dilevalol is rapidly and completely absorbed after oral administration butthe bioavailability is only about 11 to 30% (Kramer et al., 1988). The apparentvolume of distribution of dilevalol of about 25 L/kg is higher than that of labetalol(Chrisp and Goa, 1990). About 73-77% of the drug was bound to plasma proteins(Sugeno et al., 1987a). The reported total body clearance of dilevalol in humansI 01 1was 23 mUmin/kg and the elimination half-life was around 8 to 12 h (Kramer etal., 1988). While about 1.25% of the dose was excreted unchanged in urine, about80% of the dose was recovered as glucuronide conjugates (Sugeno et al., 1987a).Only about 0.03% of the maternal dose was transferred to the fetus in studiesconducted in pregnant rats (Sugeno et al., 1987b).Dilevalol was withdrawn from the North American market by itsmanufacturers (Allen Hanburys) in 1991 because of suspected hepatotoxicity.1.3. Objectives and RationaleAlthough a number of clinical studies have been conducted to assess theefficacy of labetalol in pregnancy, there is no information in the literatureregarding the in utero fetal exposure to maternal labetalol as well as the fetaleffects of labetalol. The primary objectives of this study were to characterize thematernal-fetal pharmacokinetics, metabolism and pharmacodynamics of labetalolin the chronically instrumented pregnant sheep. The rationale for these objectiveswas that labetalol may cause adrenergic blockade in the fetus if it undergoessubstantial placental transfer.Fetal exposure to drugs and pharmacological/toxicological effects of theseagents in the fetus cannot be accurately determined in humans due to technical andethical constraints. The chronically instrumented pregnant sheep has beenextensively used as a model to understand developmental physiology andpharmacology since the fetal physiology and biochemistry in sheep are similar tothat of the human fetus (Comline and Silver, 1974; Van Petten et al., 1978).Further, the rigorous maternal and fetal blood sampling that can be accomplishedin this model cannot be reproduced in small animal models like guinea-pig or ratsdue to the physical size and limited blood volumes in those animals.1.3.1. Specific AimsThe specific aims of the project at its outset were:1. To develop a sensitive and selective assay method for the quantitation oflabetalol in various biological fluids. The assay should be sensitive enough toquantitate a few nanograms of labetalol present in a few hundred microlitersof biological fluid.2. To determine the maternal pharmacokinetics, placental transfer and extent offetal exposure and maternal-fetal pharmacodynamics following maternalintravenous bolus administration.3. To determine the fetal total body clearance and placental and nonplacentalcontributions to fetal total body clearance of labetalol.4. To study the oxidative and conjugative metabolism of labetalol in both fetusand ewe.122. EXPERIMENTAL2.1. MaterialsThe reference standards, chemicals, reagents and other materials used arelisted below, along with information on purity (where applicable) and the source.Unless otherwise specified, the materials were used without further purificationor modification. Labetalol hydrochloride (>99% purity) (lot # 85F-0253)(Sigma Chemical Co., St. Louis, MO); injectable labetalol hydrochloride(TrandateR) (5 mg/mL); the internal standard for labetalol assay, 5424444-methyl phenyl) -2-Initylaminol-1 -hydroxyethyl) salicylamide hydrochloridehemihydrate (SCH) (>99% pure) and dilevalol hydrochloride (RR-isomer oflabetalol) (>99% pure) (gifts from Schering Corporation, Bloomfield, NJ); 3-APB (>98% pure) (lot # 243006 584) (Fluka AG, Federal Republic ofGermany); the internal standard for 3-APB assay, 1 -methyl-2-phenoxyethylamine(MPE), (90% pure) (lot # 00128HL) (Aldrich Chemical Co., Milwaukee, WI);injectable norepinephrine bitartrate (LevophedR); injectable phentolaminemesylate (RogitineR); injectable propranolol hydrochloride (InderalR); injectablethiopental sodium (1g/vial) (PentothalR); injectable ampicillin (250 mg/vial)(PenbritinR); injectable gentamicin sulphate (40 mg/vial) (GaramycinR);injectable Atropine sulphate (0.6 mg/mL); halothane (FluothaneR); injectableheparin (HepaleanR); sodium chloride for injection USP (Abbott Laboratories,Montreal, Quebec). All the injectable drug formulations were obtained from thePharmacy Department, Grace Hospital, Vancouver, B.C.Heptafluorobutyric anhydride and triethylamine (Sequanal Grade) (PierceChemical Co., Rockford, IL); ACS reagent grade potassium dihydrogenorthophosphate (monobasic), potassium carbonate, sodium acetate, sodium1314bicarbonate, and tris(hydroxymethyl)aminomethane (TRIS free base) (BDHChemicals, Toronto, Ontario); phosphoric acid, glacial acetic acid andammonium hydroxide (all USP grades) (Fisher Scientific Co., Fair Lawn, NJ); 13-glucuronidase, (GlucuraseR, from bovine liver, Sigma G-4882), aryl-sulfatasefrom Aerobacter aerogenes (Sigma S-1629), glucose standard solution (1mg/mL), zinc sulphate (0.3 N), barium hydroxide (0.3 N), o-dianisidinedihydrochloride, PGO capsules (500 I.U. glucose oxidase, 100 Purpurogallinunits peroxidase and buffer salts), lactate dehydrogenase, glycine buffer, andnicotinamide adenine dinucleotide (NAD) (Sigma Chemical Co., St. Louis Co.,MO), and ACS reagent grade sodium hydroxide pellets (Fisher Scientific Co.,Fair Lawn, NJ).Diethyl ether, ethyl acetate, and toluene (all glass distilled, pesticidegrade), and acetonitrile (Caledon Laboratories, Georgetown, Ontario); methanol(OmnisolvR), BDH Chemicals (Toronto, Ontario). Distilled water of high purity(referred to in the text as "distilled water") was produced on-site by reverseosmosis, followed by filtration through ion-exchange cartridges and 0.45 p.membrane filter (Milli-QR) (Millipore, Mississauga, Ontario).Nitrogen USP (Union Carbide Canada Ltd., Toronto, Ontario); pre-purified helium (for degassing HPLC mobile phase) and ultra-pure grade helium(for GC-MSD) (Matheson Gas Products Canada Ltd., Edmonton, Alberta).Needles and plastic disposable Luer-LokR syringes for drugadministration and sample collection (Beckton-Dickinson Canada, Mississauga,Ontario); membrane filters (0.45p.) (Millipore, Mississauga, Ontario); disposableplastic pipet tips (National Scientific, San Rafael, CA); borosilicate glass pasteurpipets (John Scientific, Toronto, Ontario); heparinized blood gas syringes(Marquest Medical Products Inc., Englewood, CO); heparinized VacutainerRtubes (Vacutainer Systems, Rutherford, NJ); 15 mL PyrexR disposable culturetubes (Corning Glass Works, Corning, NY); polytetrafluoroethylene (PTFE)lined screw caps (Canlab, Vancouver, B.C.); polystyrene tubes (EvergreenScientific International Inc., Los Angeles, CA) and silicone rubber tubing forcatheter preparation (Dow Corning, Midland, MI).2.1.1. Preparation of Standard Solutions and BuffersStandard stock solutions (1 mg/mL) of labetalol hydrochloride, SCH, 3-APB, and MPE were prepared by dissolving accurately weighed quantities of therespective analyte in methanol. The stock solutions were stored at 4°C. Thelabetalol and SCH stock solutions were used within three months of preparationwhile 3-APB and MPE stock solutions were used within one month ofpreparation. No evidence of degradation was found within these time periods.Dilutions of the stock solutions were made with distilled water to yield 1 pg/mL,100 ng/mL and 10 ng/mL concentrations. Standard solutions of labetalol andSCH in 0.05 M phosphoric acid were used in labetalol extraction recoverystudies whereas standard solutions of 3-APB and MPE in toluene were used in 3-APB extraction recovery studies.Phosphate buffer (0.015 M, pH 3.1) was prepared by dissolvingpotassium dihydrogen orthophosphate (monobasic) in distilled water. The pH ofthe solution was adjusted to 3.1 (range: 2.95-3.20) with phosphoric acid.A solution of triethylamine in toluene (0.0125 M) was prepared bydiluting triethylamine with toluene. Four or five pellets of sodium hydroxidewere added to the solution.Carbonate buffer (1M, pH 9.5) was prepared by dissolving potassiumcarbonate and sodium bicarbonate in distilled water. The pH was adjusted to 9.51516(range: 9.40-9.65) using either concentrated hydrochloric acid or 5 N sodiumhydroxide. The pH of the buffer was determined daily prior to use.Ammonium hydroxide solution (4%) was prepared by diluting ammoniumhydroxide USP with distilled water.Sodium acetate solution (0.2 M) was prepared by dissolving sodiumacetate in distilled water and adjusting the final pH to 5.0 using glacial aceticacid.Tris(hydroxymethyl)aminomethane (TRIS) (0.05 M) was prepared bydissolving TRIS free base in distilled water at room temperature (---.22 °C) andadjusting to a final pH of 7.5 using 1 N hydrochloric acid.0-dianisidine dihydrochloride (50 mg/vial) was reconstituted in 20 mLdistilled water. A 1.6 mL aliquot of this solution was mixed with 100 mLdistilled water and a PGO capsule (see section 2.1.) was added and dissolved.The final solution was stable for up to one month at 4°C. Nicotinamide adeninedinucleotide (NAD) (50 mg) was dissolved in 20 mL distilled water. To thissolution, 10 mL of glycine buffer and 0.5 mL lactate dehydrogenase were added.The final solution was stored at 4°C for not more than two days.2.2. Equipment and Instrumentation2.2.1. High-Performance Liquid ChromatographyAn integrated HP 1090 liquid chromatograph system (Hewlett Packard Ltd.,Palo Alto, CA) comprising a microbore solvent delivery system, a sample loopinjector of 250 AL capacity, autoinjector, autosampler, a Model 310 HP (9000series) workstation for acquiring, integrating and storing data files, a Hypersil-ODS reverse phase microbore column (200 x 2.1 mm, 5/2 particle size), a17Hypersil-ODS reverse phase microbore guard column (20 x 2.1 mm, 5/A), an on-line solvent filter assembly, and flexible microbore stainless steel tubings (0.12mm i.d.) (Hewlett Packard Ltd., Palo Alto, CA). A Model 1046Aprogrammable fluorescence detector with replaceable excitation and emissionslits and cut-off filters and interfaced to the HP 1090 liquid chromatographthrough an analog-to digital converter (Hewlett Packard Ltd., Palo Alto, CA).2.2.2. Gas Chromatography with Mass Selective DetectionA Model 5890 (Series II) gas chromatograph equipped with a split-splitless capillary inlet system, a Model 7673 autoinjector, Model G1030Aworkstation (DOS Series) and connected to a Model 5971A quadrupole massselective detector (all from Hewlett Packard Ltd., Avondale, PA); cross-linkedfused silica capillary column (25m x 0.31 mm i.d., film thickness 0.25, 5%phenylmethylsilicone) (Ultra 2) and 4 mm borosilicate glass inlet liner (HewlettPackard Ltd., Avondale, PA); silicone rubber septa (ThermogreenR LB-2)(Supelco, Bellafonte, CA).2.2.3. Physiological MonitoringA Beckman R-711 Dynograph Recorder (Sensormedics, Anaheim, CA);strain-gauge transducers (Statham model P23Db, Gould Inc., Oxnard, CA);Gould DTX disposable pressure transducers (Spectramed Inc., Oxnard, CA);cardiotachometers (Model 9857, Sensormedics, Anaheim, CA); transit-timeblood flow transducers (Transonic Systems Inc., Ithaca, NY); Apple Ilecomputer and computer data acquisition system consisting of Interactive Systems(Daisy Electronics, Newton Square, PA), analog to digital converter and clockcard (Mountain Software, Scott's Valley, CA). A IL 1306 pH/blood gasanalyzer (Allied Instrumentation Laboratory, Milan, Italy) and HemoximeterR(Radiometer, Copenhagen, Denmark).2.2.4. General ExperimentalAn automatic Speed VacR concentrator (Model AS 290, Savant,Farmingdale, NY); a vortex-type mixer (Vortex-GenieR) and incubation oven(IsotempR model 350) (Fisher Scientific Industries, Springfield, MA); IECmodel 2K centrifuge (Damon/IEC division, Needham Hts., MA); rotating typemixer (LabquakeR model 415-110, Lab Industries, Berkeley, CA); infusionpump (Harvard model 944) (Harvard Apparatus, Millis, MA); Pye Unicam SP8-400 spectrophotometer (Pye Unicam Ltd., Cambridge, UK).2.3. Development of a HPLC Assay with Fluorescence Detection for theQuantitation of Labetalol in Biological Fluids of Sheep2.3.1. Optimization of Mobile PhaseThe following parameters were studied in an attempt to determine theoptimum composition and flow rate of the mobile phase to be used to obtainsatisfactory chromatography of labetalol and SCH on a C-18 microbore column:1) pH of the phosphate buffer (from 2.5 to 7.5).2) molarity of the phosphate buffer (1 to 20 mM).3) selection of organic modifier (methanol and acetonitrile)4) proportion of organic modifier (0-70%).5) mobile phase flow rate (0.3 to 0.8 mL/min).1819The sample for injection was prepared by mixing 1 mL aliquots of aqueoussolutions of labetalol and SCH (lmg/mL each) and 10 ttL of the mixture wasinjected into the HPLC under varying mobile phase conditions. UV detection at254 nm was used in these initial experiments. The mobile phase variables wereevaluated against the following: separation of drug and internal standard, theirpeak shapes and symmetry quantitated by the assymmetry factor. Separationwas evaluated by the resolution factor (R s), which is calculated as [R, = 2 *(difference in retention times) ÷ (sum of peak widths at base)], peak shape by thepeak width at base and peak symmetry by the assymmetry factor, which isobtained by dividing the peak at its apex into two halves and taking the ratio ofthe rear half to front half areas.2.3.2. Optimization of DetectionThe excitation and emission wavelengths that provide the best signal-to-noiseratio were determined as follows. A 10 R1_, aliquot of a 1 i.tg/mL aqueoussolution of labetalol was injected into the HPLC with the fluorescence detectoron line. The labetalol peak (from a knowledge of retention time obtained usingUV detection) was "trapped" in the detector cell by turning the pump off whenthe peak was beginning to elute. With the excitation wavelength set at zero, theemission wavelength was scanned from 190 to 600 nm, in increments of 10 nm.Emission peaks were chosen from a fluorescence intensity vs emissionwavelength plot. The excitation wavelength was then scanned against each ofthe emission peaks. The process was repeated with the scanning done overnarrow ranges at increments of 1 nm to obtain the optimum wavelength pair.20Sensitivity of detection was further optimized by using excitation andemission slits of varying widths (1, 2 and 4 mm) and emission filters of differentcut-off values (210 to 400 nm).2.3.3. Optimization of ExtractionA sample of an aqueous solution of labetalol (1 lig/mL) was subjected toextraction and the following parameters were studied: (i) pH of the buffer, (ii)extraction efficiency of solvents (dichloromethane, ethyl acetate and toluene),(iii) volume of the organic solvent, (iv) volume of aqueous phase, and (v)duration of mixing. The organic phase was separated, dried and reconstituted in500 µL of 0.05 M phosphoric acid and 50^was injected into the HPLC. Inaddition, a two-step extraction, in which the separated organic layer was re-extracted with 0.05 M phosphoric acid, was also attempted. In the latter case,an aliquot of aqueous layer at the end of the second step of extraction wasdirectly injected into the HPLC. Following preliminary optimization, theextractions were repeated with 100, 250, 500 and 1000 !IL of biological matrices(maternal and fetal plasma, amniotic and tracheal fluid) spiked with labetalol.2.3.4. Procedures for the Analysis of Labetalol in Biological Fluids2.3.4.1. HPLC Operating ConditionsChromatography was achieved on a 200 x 2.1 mm analytical column,which was connected in series with a guard column (20 x 2.1 mm) and an on-line solvent filter. The connections between the solvent delivery system, thecolumns and the detector were made with 0.12 mm i.d. flexible stainless steel21tubing. The mobile phase used was pH 3.1 phosphate buffer (0.015 M) andacetonitrile in a ratio of 56:44. The phosphate buffer was first filtered through amembrane filter of 0.45 A pore size aided by suction. The buffer andacetonitrile, which were pumped separately in the binary HP 1090 liquidchromatograph system, were degassed with helium. The flow rate used was 0.5mL/min. The fluorescence intensity of labetalol was monitored at an excitationalwavelength of 196 nm and an emission wavelength of 412 nm with a 370 nmemission cut-off filter. The "Photo Multiplier Tube Gain" (i.e., signalattenuation) used was 17 or 16 in a scale of 1-18. The optical system of thedetector included a 2 mm wide excitational slit and two 4 mm wide emissionslits.2.3.4.2. Extraction of Labetalol from Biological FluidsThe procedure used for the extraction of labetalol from plasma, amnioticand tracheal fluid samples is as follows. To 0.25 mL of the biological fluid in aglass culture tube, 100 !IL of internal standard solution (1 tg/mL) and 0.5 mL ofpH 9.5 carbonate buffer (1 M) were added and the total volume adjusted to 0.75mL with distilled water. The samples were then extracted with 6 mL of ethylacetate by mixing for 20 min on a rotary shaker. The tubes were thenrefrigerated for 15 min to break any emulsion that might have formed duringmixing. This was followed by centrifugation at 1000 g for 6 min. The organiclayer was transferred to a clean, dry screw-capped glass tube and mixed with 0.6mL of 0.05 M phosphoric acid for 20 min and then centrifuged. The organiclayer was discarded and the aqueous layer transferred to 300 AL microvials and60 ILL was injected into the HPLC.2.3.4.3. Preparation of Calibration CurveStandard solutions of labetalol and the internal standard were preparedfrom methanolic stock solutions (1 mg/mL) by dilution with HPLC grade waterto produce concentrations ranging from 10 to 1000 ng/mL. Blank biologicalfluid samples (0.25 mL) were spiked with varying amounts of labetalol andidentical amounts of the internal standard (100 ng). The spiked samples wereextracted as described previously. Duplicate samples were prepared andanalyzed for each of the labetalol concentrations in the standard curve. Thecalibration curves were constructed by plotting the mean labetalol/internalstandard peak area ratio against the amount of labetalol added to the sample.2.3.4.4. Quantitation of Labetalol in Biological Fluid SamplesDuplicate aliquots of the biological fluid samples were extractedsimultaneously with the calibration curve samples, prepared from the respectivebiological fluid. Labetalol concentration in the biological fluid sample wasdetermined from the peak area ratio corresponding to the sample and the linearregression equation of the standard curve. Samples with peak area ratios lowerthan that of the lowest concentration or higher than that of the highestconcentration of the calibration curve were not quantitated.222.3.5. Labetalol Assay Validation2.3.5.1. Precision of QuantitationIntra-sample variation was studied using replicate samples (n=4) at eachof the concentrations of the calibration curve in each of the biological fluids.The percent coefficients of variation were determined. Variability due to theinjector was studied by multiple injections of standard solution of labetalol (1 p.g/mL).2.3.5.2. Extraction Recovery StudiesThe efficiency of extraction of labetalol from each of the biological fluidswas determined over the entire concentration range of the calibration curve bycomparing the peak area ratio of the extracted samples with that of directinjection of a corresponding amount of labetalol.2.3.5.3. Determination of Minimum Quantitation LimitThe minimum limit of quantitation was defined as the amount that yields asignal-to-noise ratio of at least 3 and an intra-sample coefficient of variation ofless than 10% .2.3.6. Analysis of Glucuronide and Sulphate Conjugates of LabetalolUrine and bile samples obtained from adult non-pregnant sheep followingbolus administration of labetalol and amniotic fluid samples obtained followingdirect fetal administration of labetalol were also analyzed for the presence ofconjugated metabolites of labetalol using the general procedure described byBrashear et al., 1988. Aliquots of urine (50 pt each), bile and amniotic fluid(250 [IL each) samples were treated with 0.5 mL pH 5.0 sodium acetate buffer2324(0.2 M) and 0.5 mL GlucuraseR for the determination of glucuronide and 0.5mL pH 7.5 Tris buffer (0.05 M) and 30 "IL of aryl-sulfatase preparation forsulphate determination. The enzyme treated samples were incubated overnightat 37°C in a water bath with gentle shaking. Following incubation, the sampleswere cooled to room temperature and the liberated labetalol was quantitated aspreviously described. Glucuronide or sulphate conjugate concentrations wereexpressed as the difference between the post-incubation and pre-incubationlabetalol concentrations.2.4. Development of a GC-MSD Assay for the Identification andQuantitation of 3-APB, an Oxidative Metabolite of Labetalol, in theBiological Fluids of Sheep2.4.1. Optimization of GC ConditionsSamples of 1 mL each of methanolic solutions of 3-APB and MPE (1 pig/mL) were mixed and dried under a stream of nitrogen at 30°C, reconstituted in1 mL toluene and derivatized with HFBA at 55°C for 1 h. The excessderivatizing reagent was neutralized by vortex mixing with 2 mL 0.067 Mphosphate buffer (pH 6). The organic layer was removed to autosampler vialsand 1 !IL was injected onto the GC. The default derivatization conditions usedin the preliminary experiments were from an assay method for ritodrine,developed in this laboratory (Wright et al., 1991 a). The following parameterswere studied with respect to separation of the drug and internal standard, peakshape and symmetry.1) Injector temperature: 160, 180, 200, 220 and 250°C.2) Initial column temperature: 90, 100, 125 and 150°C.3) Column temperature ramping: 5, 10 and 15°C/min.2.4.2. Optimization of Derivatization and ExtractionThe following factors were studied:1) Duration of HFBA derivatization (15 min to 2 h).2) Neutralization of HFBA (water, pH 6.0 phosphate buffer (0.067 M),water + 4% ammonium hydroxide).3) Adjustment of sample pH (7.0-14.0) using carbonate buffers andsodium hydroxide.4) Extraction efficiency of solvents (ethyl acetate, ether, hexane andtoluene).5) Volume of solvent and duration of mixing.6) Drying conditions (nitrogen vs vacuum, temperature, incomplete vscomplete drying).7) Reconstitution volume (0.1 to 1.0 mL).2.4.3. Optimization of Sensitivity and SelectivityThe following steps were used in the optimization of selectivity andsensitivity:1) Selection of two ions from the mass spectrum of 3-APB derivativeobtained in the scan mode that are diagnostic and with at least one ofthem having a relative abundance of 50% or more.2) Calculation of the dwell time required to obtain a minimum number of10 scans to define each peak.3) Selection of the appropriate tuning method (reagent and target ions).254) Determination of the exact fractional mass (correct to two decimalplaces) for selected ion monitoring that produces the best signal-to-noise ratio.2.4.4. Procedures for the Analysis of 3-APB in Biological Fluids2.4.4.1. GC-MSD Operating ConditionsA cross-linked fused-silica capillary column (25m x 0.31 mm i.d. , filmthickness 0.25 , 5% phenylmethylsilicone) and a wide bore (4 mm i.d.)borosilicate glass inlet liner with silanized glass wool plug were used forchromatography. The splitless mode of injection was employed in all cases.The GC injection port temperature was kept at 200°C. The oven temperaturewas initially set at 100°C and following injection was raised to 200°C at the rateof 15°C/min and then to 280°C at the rate of 30°C/min. The final temperature(280°C) was maintained for 1 min to give a run time of 11.33 min. Helium wasused as the carrier gas at a flow rate of 30 cm/sec. The electron energy used forEI was 70 eV. Tuning of the mass selective detector was accomplished usingPFTBA as the tuning reagent. Two groups of ions were monitored in the SIMmode: m/z 347.4 and 134.0 in group I (internal standard) and 345.4 and 132.0(3-APB) and the switch (from group I to II) was made at 8.15 min. A dwelltime of 200 ms was used to obtain a detection frequency of 2.25 cycles/sec.2.4.4.2. Extraction and Derivatization of 3-APB from Biological FluidsThe biological fluids studied were urine, bile, and plasma obtained fromadult non-pregnant sheep and fetal plasma and amniotic fluid obtained from2627pregnant sheep. One mL samples of the biological fluid were added to clean 10mL borosilicate glass tubes with PTFE lined screw caps (Corning Laboratory,Corning, NY). Subsequently, 100 juL of aqueous internal standard solution (1 ttg/mL) and 300 pL of 5N NaOH were added and this mixture was extracted with6 mL of n-hexane by tumble mixing for 5 min. The tubes were then chilled (at -20°C for 15 min) to facilitate breakage of any emulsion formed during mixing.Following centrifugation for 3 min at 1000 g, the organic layer was separatedwith borosilicate Pasteur pipets and transferred to clean tubes. The drying of thesolvent was accomplished using the automatic Speed VacR concentrator atambient temperature, using a target vaccuum of 2000 mtorr. The samples werereconstituted with 300 (IL of 0.0125 M solution of triethylamine in toluene andtreated with 20 jut of HFBA at 55°C for 1 h for derivatization. The sampleswere then cooled to room temperature and the derivatizing reagent wasneutralized by treating first with 0.5 mL distilled water and then with 0.5 mL4% ammonium hydroxide solution with a 30 sec vortexing after each addition.The aqueous layer was discarded while the organic layer was transferred toautosampler vial inserts and 1 [iL was injected into the GC.2.4.4.3. Preparation of Calibration CurveBlank biological fluid (1 mL) samples were spiked with varying amounts of3-APB (range: 0.5-1000 ng), 100 ng of internal standard and 300 !IL of 5NNaOH. After adjusting the total volume of the aqueous phase to 1.5 mL withdistilled water, the samples were extracted and derivatized as described above.The peak area ratio (3-APB/internal standard) was plotted against the amountadded to yield the calibration curve. Calibration curves with six points,prepared in duplicate, were used for routine sample analyis. A calibration curvein the appropriate biological fluid was prepared each time the samples wereanalyzed.2.4.4.4. Quantitation of 3-APB in Biological Fluid SamplesConcentrations of 3-APB in biological fluids was determined as describedfor labetalol (section 2.3.4.4.).2.4.5. Validation of 3-APB Assay2.4.5.1. Precision of QuantitationIntra-sample variation was studied using replicate samples (n=3) at eachof the concentrations of the calibration curve in each of the biological fluids.The percent coefficient of variations were determined. Variability due to theinjector was studied by multiple injections (1 ilL) of standard solution of 3-APB(1 µg/mL).2.4.5.2. Extraction Recovery StudiesSpecified amounts of 3-APB (in aqueous solution) were added to blankbiological fluid (1 mL) samples and the spiked samples were extracted withoutthe internal standard. The organic layer was separated and dried, followingwhich, 100 ng of internal standard (in toluene) was added and the samples weredried again and derivatized as described previously. The amount of 3-APBrecovered by the extraction procedure was determined using a calibration curve28constructed by derivatizing corresponding amounts of standard 3-APB (intoluene) with 100 ng of internal standard (in toluene) (i.e. , without extraction).2.4.5.3. Determination of Minimum Quantitation LimitThe minimum quantitation limit for 3-APB was determined as explainedpreviously (section 2.3.5.3.).2.4.6. Analysis of Glucuronide and Sulphate Conjugates of 3-APBThe conjugates of 3-APB were analyzed in identical manner as describedfor labetalol (section 2.3.6.).2.5. Standard Procedures for Sheep Experiments2.5.1. Recording of Hemodynamic ParametersThe various parameters that were continuously monitored (varies with thetype of experiment; see section 2.7.) include fetal and maternal arterialpressures, fetal hind limb venous pressure, tracheal and amniotic pressures,which were measured with strain-gauge manometers (Statham Model P23Db,Gould Inc., Oxnard, CA) or disposable DTX transducers (Spectramed, Oxnard,CA). Fetal and maternal heart rates were measured from the arterial pulse bymeans of cardiotachometers (Model 9857, Sensormedics, Anaheim, CA).Femoral and hind limb blood flows were measured with transit-time flowtransducers (Transonic Corporations Inc., Ithaca, NY). All of these variableswere continuously recorded using a Beckman R-711 polygraph recorder. The2930analog signals were converted simultaneously to digital form through an analogto digital conversion board (Daisi Electronics, Newton Square, PA) and an on-line Apple IIe computer (Apple Computers Inc., Cupertino, CA) equipped with aclock board (Mountain Computers Inc., Scotts Valley, CA) (Kwan, 1989). Thesampling rate was 2.5 Hz. At the end of each minute, the measurements wereaveraged, fetal arterial pressure corrected for amniotic pressure and the variablevalues displayed on the monitor. Every 30 minutes, the minute averagemeasurements were automatically transferred to floppy diskettes for subsequentanalysis.2.5.2. Blood Gas AnalysisBlood pH, p02 , and pCO2 were measured by an IL 1306 pH/blood gasanalyzer following injection of -200 [IL whole blood. The instrument alsocalculates base excess and bicarbonate. Blood oxygen saturation and hemoglobincontent were measured in duplicate using a HemoximeterR. Oxygen content wascalculated as hemoglobin concentration (g/100 mL) * oxygen saturation (%) *0.01392.5.3. Glucose MeasurementGlucose concentrations were measured in whole blood and amniotic fluid(where applicable) samples. Freshly drawn samples were transferred toheparinized tubes and 0.2 mL was added (within 30 min of collection) topolystyrene tubes containing 0.9 mL distilled water. To this mixture was added0.55 mL of zinc sulphate (0.3 N), vortex mixed and the mixture was allowed tostand for 10 minutes. Barium hydroxide (0.3 N), 0.55 mL, was then added,31followed by vortex mixing and then the solution was allowed to stand for afurther 10 minutes prior to centrifugation at 4000 rpm for 15 min. Thesupernatant was transferred through cotton-tipped pasteur pipets to cleanpolystyrene tubes, covered with ParafilmR and refrigerated until analysis. Allsamples were analyzed in duplicate within two weeks using glucose assay kits.The assay method involves enzymatic oxidation of glucose to form gluconic acidand hydrogen peroxide. The hydrogen peroxide then oxidizes o-dianisidine (inthe presence of peroxidase) to a product which can be quantitatedcolorimetrically.2.5.4. Lactate MeasurementFor the analysis of lactate, 0.3 mL of the sample (whole blood oramniotic fluid) was added to polystyrene tubes containing 0.6 mL perchloric acid(8%) followed by centrifugation at 4000 rpm for 15 min. The supernatant wasremoved to clean polystyrene tubes, covered with ParafilmR and refrigerateduntil analysis. The samples were analyzed in duplicate within 2 weeks usinglactate assay kits, which employ enzymatic conversion of lactate in the presenceof nicotinamide adenine dinucleotide (NAD) to pyruvate and the reduced form ofNAD (NADH). The NADH concentration, which is equivalent to theconcentration of lactate in the sample, was then determinedspectrophotometrically.2.6. Animal Preparation2.6.1. General Maintenance32Both pregnant and non-pregnant ewes of Suffolk, Finn and Dorset mixedbreed were used in these studies. The animals were brought into the animal unitat the Children's Variety Research Center, at least 1 week prior to surgery, andkept in groups of 2 or more in large pens in full view of one another. Theanimals received a standard diet and free access to water. Ethical approval forthe studies was obtained from the Animal Care Committee of the University ofBritish Columbia and the procedures used were in accordance to the guidelinesof the Canadian Council on Animal Care.2.6.2. Pregnant Sheep StudiesEleven time dated pregnant sheep were operated on between 115 and125 days of gestation (term 145 days). Food was withheld for about 18 hprior to surgery. Aseptic techniques were employed throughout the surgicalprocedure. Following intravenous atropine (3 mg) administration to controlsalivation, anesthesia was induced with intravenous sodium pentothal (1g). Theanimals were intubated with an endotracheal tube and anesthesia was maintainedwith a mixture of halothane (1-2%), nitrous oxide (60%) and oxygen. Anintravenous bolus injection of 500 mg ampicillin and 5% dextrose solution, at arate of 1.0-1.5 mL/min, via an intravenous drip, were administered to the ewe.The ewe's abdomen was shaved, and the surgical area was sterilized with 10%povidone-iodine topical solution, while other areas were covered with sterilesheets and drapes. A midline abdominal incision was made in the ewe and theuterus identified. Access to the head of the fetus was gained through an incisionof the uterine wall in an area devoid of placental cotyledons and major bloodvessels. With a small incision, the trachea was exposed. The tracheal catheter(2.2 mm o.d.), was inserted through a small incision 1-2 cm below the larynx33between two rings of cartilage and advanced 4-5 cm into the trachea. Thetracheal catheter did not obstruct lung fluid efflux from the airway into thepharynx. The catheter was anchored onto the skin overlying the incision withthe 3-0 silk attached to the catheter. A drop of KrazyglueR (Feature ProductsInc., Mississauga, Ontario) was applied on the catheter's point of entry to thetrachea. The tracheal incision was then closed. A catheter was placed in theamniotic fluid and anchored to the neck. After this, a second small uterineincision was made to expose the fetal hindlimbs. The hindquarters of the fetuswere withdrawn from the uterus. An incision of about 3 cm in length was madeabove the femoral arterial pulse in the groin. The femoral artery was exposedand 3 pieces of 3-0 silk sutures were passed underneath the vessel. After thevessel was tied off with the distal silk and temporarily constricted with theproximal one, a partial cut was made on the vessel between the middle and thedistal sutures. Approximately 5 to 6 cm of the catheter was threaded through thevessel into the descending aorta. Then the catheter was secured to the vesselwith all three silk sutures and a drop of KrazyglueR was applied on the site ofentry. The catheter was anchored to the adjacent muscle on either side of theincision with the sutures. Both the right and left femoral arteries werecatheterized in this manner. In a similar manner, silicone rubber catheter wasalso placed in the right lateral tarsal vein and a small branch of the uterine veindraining the uterine horn containing the operated fetus. In the case of tarsal veincatheter, approximately 11 to 12 cm of the silastic catheter was inserted into thevessel to reach the inferior vena cava. To catheterize the common umbilicalvein, a small incision was made in the umbilicus overlying one of the twoumbilical veins. Two sutures were placed, in parallel, through the vessel wall atright angles to the long axis. Using an 18-gauge needle, a hole was made in thevessel wall between the sutures and a silicone catheter inserted for — 2 cm, so34that the tip lay in the intra-abdominal common umbilical vein. The sutures weretied in a "figure 8" fashion around the catheters and a drop of KrazyglueR wasapplied. For fetal bolus studies, catheters were placed in the pudendo-epigastricartery (HLA) and vein (HLV) of right hind limb and a 4R or 4S series transit-time blood flow transducer (Transonic Corporations Inc., Ithaca, NY) wasplaced around the right external iliac artery distal to the origin of the circumflexiliac artery (Fig 2). The fetus was then gently returned to the uterus and theuterine incision closed with a continuous 2-0 gut chromic suture and thenoversewn. Amniotic fluid lost during surgery was replaced with irrigation saline(Travenol Canada Inc., Mississauga, Ontario). The catheters were filled withheparinized normal saline (1.2 U heparin/mL), tunnelled subcutaneously andexited through a small incision on the maternal right flank. The ewe's midlineabdominal incision was closed in layers and the right flank incision was sewn upas well. Medical adhesive spray was applied onto the incisions. Finally, thematernal femoral artery and vein were catheterized. All the catheters werecapped and stored in a denim pouch which was secured in place with twoadhesive bandages on the right flank. The ewe's abdomen was then wrappedaround with elastic crepe bandages. All vascular catheters were flushed dailywith 2 mL of heparinized normal saline. In the case of umbilical venouscatheter, an additional 0.5 mL heparin was used to prevent clot formation.Ampicillin (500 mg) and gentamicin (40 mg) were administered prophylacticallyto the ewe on the day of surgery and for the first four days following surgerywhile ampicillin (500 mg) and gentamicin (10 mg) were administered to the fetusat the time of surgery. Ampicillin (500 mg) and gentamicin (20 mg) were alsoadministered into the amniotic cavity on a daily basis until delivery. The eweswere moved to holding pens in the company of other sheep and were allowed toFemoralcatheterryDeep Femora11Hindlimb Artery (& Vein)^ArtePudendoepigastricInternal Mix tArteriesUmbilical Arterkis Femoral ArteryCatheter ArteryLateralTarsal Vein .Tarsal VeinCaiheter35FIG 2: Schematic diagram of ovine fetal hind limb to illustrate the positionof the hind limb catheters and flowmeter.recover for at least 5 days following surgery. The catheters were flushed dailywith 2 mL hepaiinized normal saline (1.2 U heparin/mL).2.6.3. Nonpregnant Sheep StudiesNonpregnant Dorset or Suffolk breed ewes aged 5-11 years were used inthese studies. The ewes were sedated with i.v. sodium pentothal (1-1.5 g) andanesthetised with a mixture of halothane (1-2%), nitrous oxide (60%) andoxygen. Silicone rubber catheters were implanted in the femoral artery and veinin one limb while a 6R-series transit-time blood flow transducer (TransonicSystems Inc., Ithaca, NY) was placed on the femoral artery of the other hindlimb. A catheter was also placed in the jugular vein for drug administration.An abdominal incision to the right of the umbilicus was made to gain access tothe gall bladder, where a catheter was placed to allow bile collection. Thecatheters were exteriorized, tunnelled subcutaneously through an incision on theflank and secured in a denim pouch. The catheters were filled with heparinisedsaline and capped when not in use. Ampicillin (500 mg) and gentamicin (40 mg)were administered prophylactically on the day of surgery and the following fourdays. The ewes were allowed to recover for at least five days before they wereused in the experiments. Just before the beginning of each experiment, a Foleycatheter was inserted for total urine drainage.362.7. Experimental Protocols2.7.1. Maternal Bolus Studies37A total of fourteen experiments were performed on eleven ewes. Nineexperiments involved administration of a 100 mg dose of labetalol (20 mL)(TrandateR), administered as an intravenous bolus through the maternal femoralvein. The catheter was flushed immediately with heparinized saline (10 mL).The other five experiments were control studies involving a bolus of normalsaline (20 mL) instead of labetalol. In both types of experiments, samples forlabetalol analysis were obtained from maternal artery, amniotic and fetal trachealfluid (3 mL each), fetal artery, umbilical and uterine veins (1.5 mL each) at -30,-15, 3, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 360 minand then every 2 h till 12 h, as well as at 24 h and 48 h following drugadministration. Simultaneously, maternal and fetal arterial samples (0.5 mL)were taken for blood gas analysis. The fetal blood taken for each sample wasreplaced with an equal volume of drug-free maternal blood, collected at thebeginning of the experiment. The blood samples were immediately transferredto heparinized tubes and centrifuged at 3000 g for 15 min to harvest the plasma.The plasma, as well as the amniotic and tracheal fluid samples, were transferredto clean PTFE lined screw capped glass tubes and stored at -20 °C until analysis.In six of the experiments (four labetalol and two control experiments), fetal andmaternal blood and amniotic fluid samples were also analyzed for glucose andlactic acid. In those cases, an aliquot was removed from the samples for theanalysis of glucose and lactate before they were centrifuged.For at least 24 h before and 48 h after labetalol or saline administration,fetal and maternal arterial pressures, tracheal and amniotic pressures, fetal andmaternal heart rates were continuously monitored on a polygraph recorder aswell as processed by an on-line computerized data acquisition system asdescribed previously.2.7.2. Bolus Studies in Nonpregnant Sheep2.7.2.1. Labetalol BolusFive animals were used in this study. A 100 mg dose of labetalol wasadministered as an intravenous bolus through the jugular vein. Blood samples (3mL) for the quantitation of labetalol, glucose and lactate were obtained fromfemoral artery and vein at -30, -15, 3, 10, 15, 20, 30, 45 and 60 min, every 30min until 4 h and then at 5, 6, 8, 10, 12 and 24 h following drug administration.The blood samples were immediately transferred to heparinized tubes, aliquotsremoved for glucose and lactate determinations and centrifuged at 3000 x g for15 min to harvest the plasma. Samples (0.5 mL) for blood gas analysis (pH,base excess, p02 , pCO2 , hemoglobin content and percent oxygen saturation)were also obtained simultaneously from femoral artery and vein. Urine wascollected continuously and aliquots (10 mL) corresponding to fixed intervals (0-30 min, 30-60 min and then every hour until 6 h, and then at 6-8, 8-10, 10-12and 12-24 h following drug administration) were retained. Bile samples wereobtained from only two of the animals (E#617 and 248). Since continuousdraining of bile will deplete the physiologically essential components (e.g. bilesalts), samples were obtained over discrete half-hour intervals by gravity assisteddraining at 30 min, 2, 4 and 10 h following labetalol administration. Theplasma, as well as the urine and bile samples were stored at -20°C until analysis.The urine and bile samples and some of the arterial plasma samples were alsoused for the identification and quantitation of 3-APB, an oxidative metabolite oflabetalol.3839The femoral arterial and venous pressures, heart rate and femoral bloodflow were continuously monitored on a polygraph recorder as well as processedby an on-line computerized data acquisition system as described previously.2.7.2.2. Dilevalol (RR -isomer of Labetalol) BolusThis study was conducted only in two animals (E#105 and E#248). A 25mg dose of dilevalol (in 10 mL normal saline) was administered through thejugular vein. The sampling and recording protocol used was identical to thatused for labetalol (section 2.7.2.1.). Femoral blood flow data was obtainedfrom only one animal (E# 105).2.7.3. Infusion Studies in Nonpregnant SheepFive animals were used in these studies. Injectable labetalol was infusedthrough the jugular vein at a constant rate of 0.5 mg/min immediately followinga 100 mg bolus and the infusion was continued for 6 h. Blood samples (3 mL)for the quantitation of labetalol, glucose and lactate were obtained from femoralartery and vein at -30, -15, 15, 30, 45, 60, 90, and 120 min, and every houruntil 6 h during infusion and at 15, 30, 45, 60, 90, and 120 min, 3, 4, 6 and 18h post-infusion. The blood samples were immediately transferred to heparinizedtubes, aliquots removed for glucose and lactate determinations and centrifuged at3000 g for 15 min to harvest the plasma. Samples (0.5 mL) for blood gasanalysis (pH, base excess, p0 2 , pCO2 , hemoglobin content and oxygensaturation) were also obtained simultaneously from femoral artery and vein.40The femoral arterial and venous pressures, heart rate and femoral bloodflow were continuously monitored on a polygraph recorder as well as processedby an on-line computerized data acquisition system as described previously.2.7.3.1. Norepinephrine (NE) ChallengeThe degree of x-adrenergic blockade caused by labetalol infusion wasassessed by intra-arterial injections of NE. Five different doses of NE (0.025,0.05, 0.1, 0.2 and 0.4 Kg/kg; q. s. to 3 mL with normal saline) and a controldose (3 mL saline) were administered through the femoral artery in a randomisedorder. The injections were followed by a 5 mL saline flush. The doses wereseparated by 6 min. The graded NE challenge was repeated three times - at 30min before the start of labetalol infusion, at 2 and 5 h after the start of infusion.The response was calculated as the magnitude of change in femoral blood flowfrom baseline values, i.e., Emax-Ezero , where Emax represents the maximumchange in flow following the injection and Euro , the mean flow corresponding tothe minute preceeding the injection.2.7.4. Intra-arterial Administration of LabetalolThe direct vascular effects of labetalol were studied with experimentsinvolving intra-arterial injections of labetalol, which were conducted in naiveanimals (i.e., no drug was administered for at least 72 h before the experiment).2.7.4.1. Studies in Adult Nonpregnant Sheep41In five adult nonpregnant sheep, doses of labetalol ranging from 0.001 to1 mg in saline were injected intra-arterially, i.e., via the femoral arterialcatheter, starting with the lowest dose and separated by a 10 min interval.Control injections (5 mL normal saline) were administered at the beginning andend of the experiment. The changes in femoral blood flow over 10 sec intervalswere recorded. A dose-response plot was constructed in each case. In twoanimals, the intra-arterial injections (0.1 and 1.0 mg labetalol) were repeatedfollowing administration of phentolamine (30 mg i.v. bolus followed bycontinuous infusion at the rate of 1 mg/min for 90 min) to produce oc-blockadeand in one animal, following administration of propranolol (20 mg infused over10 min), to produce 0-blockade. Alpha blockade was indicated by the lack ofany vasoconstictory response to intra-arterial injection of 0.8 i.tg/kg of NE,which produces complete vasoconstriction (flow momentarily reduced to 0mL/min) in the control sheep. Lack of pressor response to 1 pg/kg ofisoprenaline (increase in heart rate to 200 bpm) was taken as an indication of f3-blockade. The change in flow was calculated as E.-E zra , as explainedpreviously under NE challenge (section 2.7.3.1.).For at least one hour before, during and for at least one hour after theexperiments, the femoral arterial and venous pressures, heart rate and femoralblood flow were continuously monitored on a polygraph recorder as well asprocessed by an on-line computerized data acquisition system as describedpreviously.2.7.4.2. Studies in Fetal SheepThe fetal hind limb preparation was used for these studies (Fig 2), whichwere conducted in a total of six animals. The following doses of labetalol were42injected through the external pudendal epigastric artery (HLA): 0 (control), 0.1,1.0, 10, 25, 50, 100, 250 and 500 lug. The volume of the injection in all caseswas 3 mL, which was followed by a 5 mL saline flush. The doses wereseparated by a 6 min interval. The changes in hind limb blood flow, arterialpressure and hind limb venous pressure over 10 sec intervals were recorded.2.7.5. Fetal Bolus StudiesA total of nine experiments were performed on seven chronicallyinstrumented fetal lambs. In five of the experiments, a 4 mg dose of labetalolwas administered as an intravenous bolus through the fetal tarsal vein while inthe other four, 2 mL of saline was injected, to serve as controls. Samples forthe determination of labetalol, glucose and lactic acid were obtained from fetalhind limb artery and vein (HLA and HLV respectively) (1.5 mL each), amnioticfluid, tracheal fluid (only for labetalol analysis) and maternal femoral artery (3mL each) at -30, -15, 3, 10, 15, 20, 30, 45 and 60 min, every 30 min until 4 hand then at 5, 6, 8, 10, 12 and 24 h following drug administration. Samples(0.5 mL) for blood gas analysis (pH, base excess, p0 2 , pCO2 , hemoglobincontent and percent oxygen saturation) were also obtained simultaneously fromthe fetal and maternal arteries. The fetal blood withdrawn at each sample wasreplaced via the tarsal vein with an equal volume of drug-free maternal blood,collected at the beginning of each experiment. The blood samples wereimmediately transferred to heparinized tubes on ice and aliquots removed for thedetermination of glucose and lactate. The remainder of the blood samples werecentrifuged at 3000 g for 15 min to harvest the plasma. The plasma, as well asthe amniotic and tracheal fluid samples, were transferred to clean PTFE linedscrew capped glass tubes and stored at -20°C until analysis.43Fetal and maternal arterial, fetal hind limb venous, amniotic and trachealpressures, fetal and maternal heart rates and fetal external iliac artery blood flowwere monitored continuously for at least 24 h before and after the experiment.2.8. Data Analysis2.8.1. Pharmacokinetic Analysis2.8.1.1. Selection of Weighting FactorPlasma concentration data were weighted before they were subjected tocurve-fitting. An appropriate weighting factor was chosen by the method ofAlbert et al., 1974. The steps involved in this method are as follows. First,concentration-time data from the individual subjects were pooled and estimatesof mean concentrations at each time point and the associated variances werecalculated. The variance of the concentrations is related to the meanconcentration values by the following equation:ln G2 = ln a + n ln Cwhere In refers to natural logarithm, 62 is the variance, C is the meanconcentration and a and n are constants. The appropriate weighting factor is -n,i.e., any concentration C t is weighted as (Cd -n.2.8.1.2. Pharmacokinetic FittingThe weighted plasma drug concentration-time data were fitted by thecomputer program AUTOANR (Sedman and Wagner, 1976) or JANAR(Statistical Consultants Inc., Lexington, KY) to choose the most appropriateequation to describe the decline in plasma concentrations and to obtain initialestimates of exponents and constants (e.g. P, A, B, 7E, a, and 0). The initialestimates were used in iterative fitting to obtain final estimates usingPCNONLINR (version 3.0) (Statistical Consultants Inc. Lexington, KY).2.8.1.3. General Calculation of Pharmacokinetic ParametersWith the exception of p, the terminal elimination rate constant, which wasobtained through model specific fitting, the rest of the pharmacokineticparameters were calculated in a model independent manner. The formulae usedin the estimation of pharmacokinetic parameters were obtained from Gibaldi andPerrier, 1982, unless otherwise specified.The area under the plasma concentration-time curve (AUC) and areaunder the first moment curve (AUMC) were calculated as follows:AUCo c° = AUCot-last + AUC t-Iastc°AUMC0 0° AUMC0t-last AUMC t_iast c"where t-last represents the time of the last sample. The terms AUC ot-last andAUMCot-last were determined by trapezoidal approximations. An estimate ofAUC t_last °° was made according to the equation44AUC t-last °°^Ct-lastiOand an estimate of AUMC t-last c° was made by the equation (Gouyette, 1983)AUMC t_iast °° = t-last * Cr-last/0 + Ct-last/132Terminal elimination half-life (t 1/213) was calculated astw = 0.693/0The mean residence time (MRT) was estimated by the equationMRT = AUMCoc"/AUCe°Total body clearance (CL) following an intravenous bolus asCL = Dose/AUCe°while steady-state clearance (CL„), i.e. , during constant rate infusion to steady-state ascLss = Ico/csswhere lco is the infusion rate and C ss is the steady-state concentration oflabetalol. The apparent volumes of distribution were calculated asVDa„a = Dose/(AUCe/(3)VDss = Dose * AUMC/(AUC) 2452.8.1.4. Calculation of Transplacental and Nonplacental ClearancesMaternal and fetal transplacental clearances were calculated by amodification of the equation derived by Anderson et al. 1980a, for steady-stateinfusion (Eqn. 1):[CLfp] ss = [CLf]SS * (FssiMs)^Eqn. 1where [CL fp] ss is fetal transplacental clearance, [C1.4] ss is fetal total bodyclearance, F and M are the concentrations in the fetus and mother, respectively,following maternal infusion to steady-state. Integrating the above equation fromtime zero to infinity yieldsCL4, = CLf * (-FAuc/MAuc)^Eqn. 2where CLfi, and CLf represent the time averaged fetal transplacental and totalbody clearances respectively, while FAUC and MAUL represent the fetal andmaternal AUC from time zero to infinity, respectively. An estimate of CL f wasobtained following direct fetal intravenous labetalol administration (see section2.7.5.) while F^/MAUC- -AUC was obtained in the studies following maternal drugadministration (see section 2.7.1.). The maternal transplacental clearance(CL.p) could be calculated in a similar manner from the maternal total bodyclearance (CLm) (Eqn. 3).CL„,p = CLm * (M^1-AUC - AUC,^Eqn. 3The value of CLm was obtained from maternal i.v. bolus administration oflabetalol (see section 2.7.1.), while MAUC/FAUC was determined followingdirect fetal administration (see section 2.7.5.). The maternal nonplacental andfetal nonplacental clearances^and CLf,.,, respectively) were calculated asthe difference between the total body clearance and transplacental clearance(Eqns. 4 & 5).CLfi, = CLf - CLf,^Eqn. 44647CLnin = CLm - CLmp^Eqn. 52.8.2. Analysis of Hemodynamic and Metabolic DataThe arterial pressure, blood flow and heart rate values were averagedover 30 min periods. The mean values obtained from all the animals along withthe SEM were plotted against time. Glucose and lactate concentrations wereexpressed as mM. The fluxes of glucose, lactate and labetalol across the hindlimb were calculated as [(arterial concentration - venous concentration) * hindlimb blood flow]. Fetal hind limb vascular resistance was calculated as [(arterialpressure - hind limb venous pressure) ÷ (hind limb blood flow)].2.8.2. Statistical AnalysisThe blood gas parameters, concentrations of glucose, lactate, hind limbfluxes of glucose and lactate following labetalol administration were analyzed forstatistical significance against changes in control experiments (in the case ofmaternal bolus and feta bolus experiments) by Multivariate Analysis of Variance(MANOVA) or against pre-experiment values (in the case of experiments in non-pregnant animals) by Fisher's Least Square Difference using SPSS/PC+program (SPSS Inc., Chicago, IL). Changes in arterial pressure, heart rate andflow values following labetalol administration were tested for significantdifference from control values by Fisher's Least Square Difference.Values are expressed as mean ± standard error of the mean, unlessotherwise specified. The level of significance used in all cases was equal to0.05.3. RESULTS3.1. Development of a Microbore HPLC Assay with Fluorescence Detectionfor the Quantitation of Labetalol in Biological Fluids.3.1.1. Optimization of Mobile Phase.The pH of the phosphate buffer used in the mobile phase had significanteffect on the retention time, peak shape and resolution. pH values greater than 4.0provided broad peaks (peak width >2 min). The optimum pH range was found tobe 2.8-3.2. Increasing the molarity of the phosphate buffer (from 1 mM to 20 mMin steps of 5 mM) decreased the peak width and the retention time (Fig 3). Theideal concentration was between 15-20 mM.Acetonitrile and methanol were studied as organic modifiers in combinationwith pH 3.1 phosphate buffer (0.015 M). Less tailing of labetalol and SCH peakswas observed with acetonitrile than with methanol at similar concentrations. Bestresults in teens of retention time (4.1 min for labetalol and 6.0 min for SCH), peaksymmetry (1.05-1.15) and resolution between labetalol and SCH (>1.5) wereobtained with a flow rate of 0.5 mL/min with 44% acetonitrile.3.1.2. Optimization of Detection of LabetalolThe optimum excitation and emission wavelengths, determined fromscanning the trapped labetalol peak, were found to be 196 and 412 nm,respectively. The signal-to-noise ratio was significantly improved by using a 2mm excitation slit and two 4 mm emission slits. Use of emission cut-off filters4849FIG 3: Effect of molarity of phosphate buffer in the mobile phase on thepeak width and retention time of labetalol on a C-18 column.resulted in reduction in the background noise and the 370 nm filter provided theoptimum sensitivity.3.1.3. Optimization of Extraction of LabetalolOf the four solvents studied (dichloromethane, ether, ethyl acetate andtoluene), only ether and ethyl acetate provided an extraction efficiency of 50%(Fig 4). Further optimizations were done using ethyl acetate. The extractionefficiency improved with an increase in sample pH from 6.0, but no appreciableincrease was seen beyond 9.0. The optimum volume of ethyl acetate was 6 mL,which provides a phase volume ratio (organic to aqueous) of 5 and the duration ofmixing was optimized at 20 min. Under these conditions, the absolute recovery oflabetalol was about 81%.Two-step extractions were also attempted. Extraction with 2 x 3mL ethylacetate followed by drying of the pooled solvent and reconstitution provided about85% recovery. Extraction with 6 mL ethyl acetate followed by re-extraction of theethyl acetate layer with dilute phosphoric acid (0.01, 0.05 or 0.1 M) provided acleaner chromatogram as compared to single extraction (Fig 5), but the absoluterecovery decreased to about 75%.3.1.4. Extraction of Labetalol from Biological FluidsThe procedure used in the extraction of labetalol from biological fluids isshown in Fig 6. Representative chromatograms obtained from spiked biologicalfluids following a two-step extraction using 6 mL ethyl acetate and 0.6 mL 0.01 Mphosphoric acid are shown in Fig 7.50100 -81.1% 84.2% A CFIG 4: Absolute extraction recovery of labetalol with different solvents.A: Dichloromethane, B: Ethyl acetate, C: Diethyl ether and D:Toluene.51LL0A52711-i98-255-121^2^3^i^5^6^ 1^2^3^i^5 498-255-12FIG 5: HPLC Chromatograms obtained from blank sheep plasmafollowing a two-step (A) and one-step (B) extraction. Aninterfering peak was seen at around 3.5 min following one-stepextraction (B).Discard organic layer20 min extraction5 min spinPlasma (or other fluid) 250 uL + Int. Std.(100 ng)+ 500 uL pH 9.5 (1M) buffer + 6 mL ethyl acetate 20 min extraction20 min chilling5 min spinDiscard aqueous layerTo the organic layer, add 600 uLof 0.01M phosphoric acidInject 60 uLFIG 6: Optimized procedure used for the extraction of labetalol from biological fluids.1=t)A1 000 -8007B7007.600 .:5007>600 ->400E400 -E 3002007200 -100 -^A0 .Csea.]500.40070Time (min.)8.0> >E 300': E2007100 -0.0 8 .^1FIG 7: Superimposed HPLC chromatograms of blank and spiked (100 nglabetalol) biological fluids (1 - Labetalol and 2 - Internal Standard):A: Pregnant sheep plasma, B: Amniotic fluid, C: Fetal plasma and D:Fetal tracheal fluid.0.0Time (min.)^ Time (min.)8 . o^0 0^8 . 0Time (min.)D700 -:Sea:500:400 -:^300 -:^I200710073.1.5. Validation of Labetalol Assay3.1.5.1. Precision of QuantitationA typical calibration curve following extraction from sheep plasma isshown in Fig 8. The coefficients of determination were between 0.996 and 1.000following extraction from various biological fluids. The coefficients of variationover the calibration range are shown in Table 1. The mean intra-samplecoefficient of variation over the concentration range in plasma was found to be2.95 ± 2.76%, while in the case of amniotic and fetal tracheal fluids, the valueswere 3.85 ± 3.2% and 4.12 ± 2.7% respectively. The coefficient of intra-injectionvariability (a measure of variability due to the injector) was consistently less than0.5%3.1.5.2. Extraction Recovery StudiesThe mean extraction recovery of labetalol from plasma over the entirerange studied (i.e. 0.5-120 ng) was 76.01 ± 2.8%. Extraction recovery, as afunction of amount added, is shown in Fig 9. Mean extraction recoveries fromamniotic and tracheal fluids over the calibration range (0.5-20 ng) were found tobe 70.56 ± 3.76% and 75.12 ± 8.8%, respectively.3.1.5.3. Minimum Quantitation Limit of Labetalol AssayThe minimum quantifiable limit in this assay was .-- 30 pg of labetalolinjected (absolute sensitivity). In terms of amount added (apparent sensitivity),this was equivalent to about 0.4 ng and in concentration terms was equivalent to552.01.51.00.50.0Y = 0.0212r 2 = 0.9996+ 0.01558 X-,:/10^25 50 75 100 125AMOUNT OF LABETALOL ADDED (ng)FIG 8: A typical calibration curve employed in the quantitation of labetalol (5 -120 ng) in plasma (mean ± SD) (n=3).TABLE 1: INTRA-SAMPLE VARIABILITY IN LABETALOL ASSAYAMOUNT OFLABETALOL ADDED (g)MEAN PEAK AREARATIOCV (%)@0.5 0.0931 9.112.5 0.1122 5.565.0 0.2724 0.8315.0 0.2347 1.6530.0 0.4939 1.0950.0 0.8032 0.67100.0 1.5845 3.29120.0 1.8715 2.15n=4571 0 -0....--Li^75 ->OLIJ50z0 25 -Y --= -0.12 + 0.7548 Xr = 0.9998125 -25^50^75^100AMOUNT ADDED (ng)FIG 9: Extraction recovery of labetalol from sheep plasma as a function ofamount added (mean ± SD) (n=3).12559about 1.6 ng/mL (using 250 ptL volume of sample). Fig 10 shows a chromatogramobtained from plasma spiked with 0.5 ng labetalol.3.2. Development of a GC-MSD Assay for the Identification andQuantitation of 3-APB, an Oxidative Metabolite of Labetalol in theBiological Fluids of Sheep3.2.1. Optimization of GC ConditionsThe splitless mode of injection was used in all cases to maximizesensitivity. The optimum injector temperature was 200°C, with lowertemperatures (160 and 180°C) resulting in tailing peaks and higher temperatures(220-250°C) resulting in reduced peak areas. Significant "cold-trapping" effectwas obtained with initial oven temperature of 100°C or lower, but initialtemperature at 90°C increased the retention time by about 3 min Hence 100°Cwas chosen as the initial temperature. The oven temperature programming of 15°C/min up to 200°C, followed by 30°C/min to 280°C yielded satisfactoryresolution, peak shape and total run time (-11 min).3.2.2. Optimization of Derivatization and Extraction of 3-APBTreatment with HFBA at 55°C for lh (Wright et al., 1991) provided nearcomplete derivatization. No appreciable increase in peak area was seen beyondlh. Neutralization with pH 6.0 phosphate buffer (0.065 M) yielded a complexchromatogram with a high baseline noise. Use of 0.5 mL distilled water followedby 0.5 mL ammonium hydroxide (4%) (Riggs et al., 1983) provided significant400 -300 -200 -1CD . 0Time (min.)FIG 10: HPLC chromatogram obtained from blank sheep plasma spiked with 0.5 nglabetalol (the lowest calibration point) and internal standard. (1 - Labetalol and 2- Internal standard). Approximately 40 pg of labetalol was actually injected.61improvement in the form of a cleaner chromatogram even with 2 11,1. injectionvolume (optimized injection volume = 1p.L).Extraction of 3-APB at different pH conditions of the aqueous phase wasstudied using 1M carbonate buffer (pH 9.5), 1N, 2N, and 5N sodium hydroxidesolutions (200-500 pL volume). Extraction efficiency improved by about 25%with 2N and 5N sodium hydroxide solutions as compared to pH 9.5 buffer or 1Nsodium hydroxide (Fig 11). Of the four solvents studied (ethyl acetate, ether,hexane and toluene), ether and n-hexane provided extraction recoveries > 80%.Hexane was used in subsequent optimization studies. Optimal extraction recoverywas obtained with 6 mL hexane and mixing time of 5 min. Longer durations ofmixing resulted in extensive emulsification, which prevented complete removal ofthe organic layer. No significant differences in recovery were found betweendrying the hexane layer under a stream of nitrogen at 30°C and drying undervacuum using SpeedVacR concentrator at ambient temperature. Also, completedrying of the solvent at ambient temperature did not result in any loss of theanalyte due to volatilization. The reconstitution volume of 300 p,L, was chosen toincrease the apparent sensitivity; volumes less than 300 iiL do not provide anysignificant increase in the signal-to-noise ratio.3.2.3. Derivatization and Extraction of 3-APB from Biological FluidsThe optimized procedure for the derivatization and extraction of 3-APBfrom urine, bile or plasma is shown in Fig 12. The total ion chromatogram of theHFBA derivatives of 3-APB and the internal standard following extraction fromcontrol urine spiked with 100 ng each of 3-APB and MPE and their mass spectraare shown in Fig 13. The suggested m/z assignments for some of the fragmentsare shown in Fig 14.62pH 9.5^NaOH^NaOH^NaOH(1M) (1N) (2N) (5N)FIG 11: Effect of various reagents used for sample pH adjustment (prior to solventextraction) on the absolute recovery of 3-APB in urine.[DERIVATIZE WITH HFBA AT 55 deg C for 1 hrNEUTALIZE HFBA WITH WATER & AMM. HYDROXIDE (4%)INJECT 1 uL OF ORGANIC LAYER INTO GCTUMBLE MIX (6 min) & REMOVE ORGANIC LAYERDRY HEXANE & RECONSTITUTE IN 0.3 mL TEA in TOLUENEURINE, BILE OR PLASMA (1 mL) +100 ng 1-METHOXY-2-PHENYLETHYLAMINE +0.3 mL 5N NaOH + 6 mL n-HEXANEFIG 12: Optimized procedure used for the extraction of 3-APB from biological fluids.Abundance40000Scan 293 (8.015 min): 0100121.D2 43500030000250002000015000100005000 169107^13494240226192^ 347Time - > 50^100 150 200 250 30064Fig 13: EI GC-MS following HFBA derivatization of standard 3-APB and MPE (internalstandard). A: Total Ion Chromatogram; B: Mass Spectrum of 3-APB Derivative andC: Mass Spectrum of the MPE Derivative.Abundance250000-200000-150000-•100000 -50000-1/2^->6CaFrOO-H--------------------CH,TIC:'STD• 1.,,0100121.D3-APE:C,FrOC-HCH.-,_____000^6.50 7.00 7.50 8.00 T^8.50 9.00 9.50 10.00Abundance91 1'7Scan 308^(8.219 min): 0100121.D180001600013214000120002411000080006000-40006934516925420000 -(44^I;Jll iI J. .^,^,^.t I^1 4 1 1Time -> 50^100^150 200 ^' -^250^300ABCFig 14: 3-APB Derivative: Suggested m/z Assignments for the mass spectrumMW=34565C3 F7 0IIC-FiN169OTHER IONSm/z44^CH3-CH-NH269 CF3117^CH=CH-CH2-C6H5 and/orCH3-C =CH-C6H5132^CH3-CH=CH-CH2-C6H53.2.4. Validation of 3-APB Assay3.2.4.1. Precision of QuantitationFig 15 shows a typical calibration curve (peak area ratio expressed as mean± SD) obtained following extraction and derivatization of spiked urine samples.Quantitation was found to be linear in the examined range of 0.5-1000 ng 3-APB(0.98 r2 1.00). The intra-sample coefficients of variation (n=5) were found tobe less than 12% in all cases over the entire calibration range. Inter-day variabilitywas overcome by running a fresh standard curve every time the samples wereanalyzed.3.2.4.2. Extraction Recovery StudiesThe efficiency of the extraction procedure was studied at five differentconcentrations (10, 100, 250, 500 and 1000 ng/mL of urine/bile) (n=3) and themean extraction recovery was found to be 102.9 ± 4.9% from urine and 98.3 ±1.45% from bile.3.2.4.3. Minimum Quantitation Limit of 3-APB AssayThe minimum limit of quantitation was found to be equivalent to about 2pg of 3-APB (amount injected) or 0.5 ng/mL (in concentration terms).663.3. Maternal Bolus Studies10.0 -1.0 -0.1 -1.0E-2 -1.0E-3 -A = -2.300, B = 0.957 & r 2 = 0.9991.0E 4 ^0.1 1.0^10.0^100.0AMOUNT 3-APB ADDED (ng)1000.0Fig 15: Typical calibration curve following extraction of 3-APB (0.5 - 1000 ng) fromurine (mean ± SD; n=3).3.3.1. Experimental DetailsTable 2 lists the particulars about the animals used in the study. Thegestational age of the ewes in the labetalol group was 132.6 ± 1.6 days while inthe control group it was 132.8 ± 1.4 days. The mean birth weight of the operatedfetus was 3255 ± 207 g in the labetalol group and 2914 ± 154 g in the controlgroup. The fetal and maternal cardiovascular and acid-base status were within thenormal range at the beginning of each experiment and the mean values are shownin Table 3.3.3.2. PharmacokineticsFig 16A shows a typical disposition profile of labetalol in the maternal andfetal femoral artery, amniotic and tracheal fluid compartments of the pregnantsheep (E# 109) following a 100 mg bolus. In the mother, the maximumconcentration (at 3 min) was in the range of 1.1-2.3 gg/mL. The weighting factorfor the maternal labetalol concentrations was determined from the plot of logvariance vs mean concentration (Albert et al., 1974). The slope of the plot wasfound to be 1.65 and accordingly a weighting factor of [1/C 2] was chosen. Thedisposition in the maternal plasma is best described by a tri-exponential equationof the type Pe-nt + Acca + Be-Bt (except in one animal, where the data is best-fitted by a bi-exponential equation of the form Acat + Be -I3t) with a very rapiddistribution phase. The maternal pharmacokinetic parameters calculated for theindividual subjects are shown in Table 4. The mean total body clearance was135.86 ± 16.93 L/h (30.8 ± 3.83 mL/min/kg) while the mean terminal eliminationhalf-life was 2.79 ± 0.66 h. The apparent volume of distribution calculated asVDarea was 477.51 ± 52.98 L (6.48 ± 0.72 L/kg) while the nonparametric68TABLE 2: MATERNAL LABETALOL BOLUS: EXPERIMENTAL DETAILS: EWE#BODY^GEST.WT. AGE(Kg)^(days)# ofFetus TERM(days)BIRTH WT.OF FETUS(grams)SAMPLES OBTAINED^LACTATE GLUCOSEI. LABETALOL EXPERIMENTS:145 76.4^139 1 144 2364* MA, FA & TR NO NO109 82.7^127 1 144 4082* MA, FA, UTV, UV & AMN NO NO091 73.2^142 1 145 3728* MV, FTV, UV, UTV, AMN & TR NO NO127 85.9^133 2 141 2933*, 3478 MA, FA, UV, AMN & TR NO NO248 80.0^131 2 141 2550*, 2730 MA, FA, UV, UTV & AMN NO NONTG 40.5^130 1 142 3300* MA, FA & AMN YES YES105 69.1^131 2 138 3165*, 2535 MA, FA, AMN & TR YES NO137 81.0^129 2 135 3063*, 2874 MA, FA, AMN, & TR YES YES201 73.8^131 2 139 4110*, 3320 MA, FA, UV, UTV & AMN YES YESH. CONTROL EXPERIMENTS:127 85.9^133 2 141 2933*, 3478 MA, FA, UV, AMN & TR NO NO141 75.0^138 2 140 @*, 3092 MA, FA, UV, UTV, AMN & TR NO NO248 80.0^131 2 141 2550*, 2730 MA, FA, UV, UTV & AMN NO NONTG 40.5^130 1 142 3300* MA, FA & AMN YES YES617 82.3^132 2 138 2873*, 2547 MA, FA, UV, AMN & TR YES YES* Operated fetus@ Weight not available; fetus trampled upon by ewe after unsupervised delivery.70TABLE 3: PRE-EXPERIMENTAL MATERNAL AND FETAL BLOOD GASPARAMETERS (mean ± SEM).PARAMETER MATERNAL ARTERIAL FETAL ARTERIALA. MATERNAL BOLUS EXPERIMENTS (n=8)7.356 ± 0.01722.3 ± 1.450.2 ± 0.7pH^ 7.481 ± 0.016p02 (mm Hg)^ 137.5 ± 6.9pCO2 (mm Hg) 36.1 ± 1.0Base Excess (mEq/L) 4.3 ± 0.5 2.3 ± 0.9Hemoglobin (g/100 mL) 9.1 ± 0.4 11.1 ± 0.402 Saturation (%) 100.7 ± 4.2 57.8 ± 2.202 Content (mL 0 2/100 mL) 12.7 ± 1.1 9.0 ± 0.9B. FETAL BOLUS STUDIES (n=5)pH 7.505 ± 0.011 7.351 ± 0.017p02 (nun Hg) 131.1 ± 5.8 25.4 ± 1.9pCO2 (mm Hg) 33.3 ± 1.1 47.3 ± 1.4Base Excess (mEq/L) 3.8 ± 0.5 1.6 ± 0.9Hemoglobin (g/100 mL) 9.5 ± 0.6 10.8 ± 0.802 Saturation (%) 99.3 ± 3.1 65.7 ± 2.802 Content (mL 02/100 mL) 12.8 ± 1.7 10.2 ± 1.10OD13o-o_.o^100^\^ -^NAN.0N...6,6.-A ^.,-.A-- N.^AA -A 0 ---..,,A..^0 0\2 1:1.. ti^^61434.-4" • • 0i^-0--10/ \O0O100071^— o MA• — • FAo —o AMA - A TR•A^V12TIME (h)FIG 16: Representative plots of labetalol concentrations in two experimentsfollowing a 100 mg maternal intravenous bolus administration.(A - E# 109 and B - E# 201). (MA: Maternal arterial plasma, FA: Fetal arterialplasma, UT: Uterine venous plasma, UV: Umbilical venous plasma, AM:Amniotic fluid and TR: Tracheal fluid).TABLE 4: LABETALOL PHARMACOKINETICS IN PREGNANT SHEEP FOLLOWINGMATERNAL BOLUS ADMINISTRATION.A. EWEEWE #^AUC A^C tv TBCL^Vdarea Vd 1VIRT(mg*h/L) (mg*h-/L) (1/%1 (L/hl (L) (L) (h)E145^0.581 0.964 0.322 2.15 172.12^534.50 285.58 1.66E109^0.624 0.939 0.320 2.17 160.26^500.81 241.16 1.50ENTG^0.518 0.591 0.347 2.00 193.05^556.34 220.26 1.14E105^0.997 1.944 0.359 1.93 100.3^279.39 195.57 1.95E201^0.977 2.073 0.196 3.54 102.35^522.19 217.18 2.12E248^0.658 0.771 0.52 1.33 151.94^292.19 178.07 1.17E127^1.409 4.336 0.108 6.42 70.97^657.15 218.41 3.08MEAN^0.823 1.660 0.310 2.79 135.86^477.51 222.32 1.80S.E.M.^0.121 0.496 0.049 0.655 16.930^52.975 12.979 0.25B. FETUSEWE #^8 ty Cmax AUC AUMC AUC(1/h) (hi (ng/mL) (mg*h/L)^(mg*h-4/L) (F/M) %E145^0.265 2.62 15.4 0.058^0.123 9.91E109^0.137 5.06 66.4 0.131 0.551 19.23ENTG^0.232 2.99 23.1 0.087 0.379 16.84E105^0.238 2.91 42.8 0.157 0.930 15.70E201^0.160 4.33 36.3 0.136 0.814 13.90E248^0.130 5.33 24.0 0.117 0.837 17.74E137^0.140 4.95 40.7 0.107 0.714 ----@E127^0.212 1.42 21.2 0.102 0.404 7.25MEAN^0.189 3.71 33.7 0.112 0.594 14.37S.E.M.^0.019 0.50 5.8 0.011 0.098 1.5472@ Maternal sampling incomplete.73estimate (VD ss) was 222.32 ± 12.98 L (3.02 ± 0.18 L/kg). Labetalol showed amono or bi-exponential decline in the fetal plasma, with the terminal eliminationrate constant significantly smaller than that seen in the maternal plasma (0.189 ±0.019 Ir 1 vs 0.31 ± 0.04911-1 ) and a mean half-life of 3.71 ± 0.5 h, which wassignificantly higher than that in the mother (2.79 ± 0.66 h) (Student's t-test). Thepeak concentration in the fetal plasma (33.7 ± 5.8 ng/mL), which is roughly 10%of that in the maternal plasma, was observed at 3 min in all but one animal, wheretmax was 10 min. The characteristic features of disposition of labetalol in thefetus are shown in Table 4. The extent of fetal exposure to labetalol following thematernal bolus, as expressed by the mean fetal to maternal plasma AUC ratio was14.37 ± 1.54%. Labetalol accumulated in the fetal tracheal fluid withconcentrations consistently higher than that in fetal plasma after 30 min. Thetracheal fluid to fetal plasma labetalol concentration ratio beyond 2 h was — 2 to 4.The drug appeared in the amniotic fluid at a much slower rate as compared to thefetal tracheal fluid, but beyond 4 h, similar concentrations are seen in both thefluid compartments. The drug persisted in these fluid compartments for 24-48 h,while the concentrations in fetal arterial plasma fell below the limits of detectionbetween 6-12 h. Fig 16B shows the uteroplacental and fetoplacental arterio-venous labetalol concentration profile (E# 201). The concentrations in the uterinevein were mostly identical to those in the maternal artery and there was nosignificant difference between the labetalol AUCs (0.873 ± 0.09 mgh/L in theuterine vein and 0.823 ± 0.12 mgh/L in the maternal artery; paired t-test). In theumbilical vein, the peak labetalol concentration (57.3 ± 14.3 ng/mL) tended to behigher than the peak concentration in the femoral artery (33.7 ± 5.8 ng/mL), butthe difference in the peak concentrations as well as the AUCs (0.133 ± 0.02 mgh/Lin the umbilical vein and 0.113 ± 0.033 mgh/L in the fetal artery) were notstatistically significant (paired t-test).3.3.3. Hemodynamic EffectsNo consistent changes in the maternal cardiovascular parameters (meanarterial pressure and heart rate) were seen following labetalol administration. Themean values obtained from all the animals are shown in Fig 17. In two of theanimals (ENTG and E105), an initial phase of hypotension (maximum change ofabout 10 mm Hg in the mean arterial pressure) lasting 1-3 h, with a correspondingincrease in the heart rate (maximum change of about 20 bpm) was observed whilethe other animals did not show any change. There were no apparent changes inthe fetal mean arterial pressure and heart rate values in any of the animalsfollowing maternal labetalol administration (Fig 18).3.3.3. Metabolic Effects3.3.3.1. Changes in Blood Gas ParametersSignificant changes were seen in the fetal and maternal arterial blood gasparameters following maternal labetalol (Fig 19). In the fetus, pH decreasedgradually over 2 h and remained significantly below the control values until 12 h.An almost corresponding decrease in the base excess was also seen, with the meanvalue at 4 h (-4.7 ± 1.34 mEq/L) representing a maximum decrease of 7 mEq/Lfrom the control value of 2.3 ± 0.63 mEq/L. Fetal p02 decreased significantlywith the mean value at 3 min, which represents the maximum change, being 18.6± 1.53 mm Hg (control value = 22.0 ± 1.39 mm Hg). Fetal pCO2 increasedsignificantly with the maximum change occurring at 30 min (52.8 ± 1.77 mm Hg;control = 50.1 ± 1.0 mm Hg). Fetal 02 saturation fell rapidly by 27 ± 6.5%74cai",-0150 -125 -E-4E-4 1007511075FIG 17: Effect of a 100 mg i.v. labetalol bolus on maternal heart rate and arterial pressure(mean ± SEM) (n=6).Lo,' I1/°1^T -TO (i) - (13,^i°^1\'1 1.\(1)./C6-(I)-(1)-criv^T^j)11 111^10-0n.,1\10 1 10 112^5TIME (h)TV)10090 -80 -14(1) 70 -a.601. -j) - (1). 11- 1-^I O.I IY) I,o,f T/ 1 ?\6kci .1\1 o7 0]of p^oi I or?^1 T. /II5^8^11TIME (h)10I/ A76FIG 18: Effect of a 100 mg i.v. labetalol bolus on fetal heart rate and arterial pressure(mean ± SEM) (n=7).180160E-1^140120rolicf"\i-Jo_io , 01Is ,10/1\1 .110(111^101,i0,(1).10/Tq 1l ,0111^TI2^5TIME (h)8^11605040Cf)30o'N 10\ 1 1^I 1^T0.° 11°1\0/1 \10 /Troi °I\,11/7\-[ I (I)I^1 1.Y7 011°1^T-Ti2^5^8^11TIME (h)7.800 - • •A7.500 • coo°t;•.0•7.400 -•7.300 - %Ise= -•-- 11? f.7.200 11■•••^OO 1180-1002523 •A^*Tr^°•144til^I19^15110 -0 too80 •50Cf1(5";0404111 . -6-T•, .•L-^L•Yr.^••30•77FIG 19: Fetal and maternal arterial blood gas parameters (mean ± SEM) before and aftera 100 mg maternal intravenous bolus administration .of labetalol. (MA: Maternalarterial blood and FA: Fetal arterial blood). Asterisks (*) denote. significantdifference from control values.• • FA-^0^2^6^8^11^24^-1 0^2^6^8 .^11^24TIME (h) TIME (h)78following drug administration and stayed significantly decreased for 4 h. Even at24 h, the fetal 0 2 saturation was lower, although not significantly, than control.The calculated fetal 0 2 content decreased by 28.2 ± 8.1% following labetaloladministration and remained significantly decreased until 5 h (data not shown). Incontrast, the maternal pH and base excess showed only moderate decreases. Themaximum decrease in pH was by 0.015 unit (cf 0.1 unit in the fetus) and baseexcess by 4.3 mEq/L (cf 7.0 mEq/L in the fetus) and the values were back tonormal by 4h, unlike that in the fetus. A gradual but significant decrease in pCO 2over 4h was observed (35.8 ± 0.8 to 30.2 ± 1.7 mm Hg) as well as a trend towardsincrease in p0 2 (not statistically significant) over 2-10 h. Maternal 0 2 saturationand content remained unchanged (data not shown).3.3.3.2. Blood Glucose and Lactate LevelsFig 20 shows the changes in lactic acid and glucose concentrations in fetaland maternal arterial blood and in the amniotic fluid. Significant lactic acidosisoccurred in both the fetus and ewe following labetalol administration, but theeffect was much more pronounced in the fetus and lasted longer (12-24 h).Accumulation of lactic acid, persisting beyond 24 h, was also seen in the amnioticfluid. The changes in the maternal lactate concentrations were characterized by arapid increase in the first hour, with the peak concentration, which was attained at60-90 min, remaining essentially unchanged till 5-6 h and then a return to baselineconcentrations by 12 h. In the fetus, the lactate concentration continued to rise forabout 4 h and then reached a plateau. The rate of decline in the fetus was muchslower than that in the mother and baseline values were not reached before 24 h.The amniotic fluid lactate concentrations in the first 2 h show a distinct lag incomparison to the fetal blood lactate concentrations and peak concentrations wereA-A AMo-o MA^•^ • FA48245^8^112-1 079UzOCDU)OTIME (h)FIG 20: Glucose and lactic acid concentrations (mean ± SEM) before and after a 100 mgmaternal intravenous bolus administration of labetalol. (MA: Maternal arterialblood, FA: Fetal arterial blood and AM: Amniotic fluid).80reached by 6 h. Beyond 10 h, the decline in the amniotic fluid lactate was muchslower than in the fetus with the mean 24 h concentration being roughly twice thefetal arterial concentration.Blood glucose levels also rose in the ewe and fetus following labetaloldosing, but in this case, the effect was more pronounced in the ewe and theamniotic fluid glucose concentration showed only a marginal increase. In contrastto the situation with lactate levels, the changes in the fetal glucose concentrationsparalleled those in the ewe. In all the animals studied, the fetal blood gasparameters and lactate concentrations returned to control values between 24-48 h.The control experiments did not show any change in fetal or maternal lactate andglucose concentrations (Fig 21) nor any consistent deviation in any of the bloodgas parameters (Fig 22).3.4. Labetalol Bolus Studies in Non-pregnant Sheep3.4.1. PharmacokineticsThe mean weight of the ewes (n=5) used in the study was 74.9 ± 4.0 kg.The disposition of labetalol in sheep arterial plasma following a 100 mg i.v. bolusadministration is shown in Fig 23. The weighting factor chosen (Albert et al.,1974) for pharmacokinetic fitting of plasma labetalol concentrations was 1/C 2 .The decline of labetalol concentration was best described by a triexponentialequation of the type Pert + Ae -at + Be-13t. The estimates of the pharmacokineticparameters obtained by nonparametric analysis of the arterial plasma concentrationdata are listed in Table 6. The total body clearance, elimination half-life and4.0 - DAM0-0 FA A-A MA81O^5 8A AE 3.0 -AAzOu 2.0UTJOU 1.00.0-13.02.0 -01.0 -0.0 ^- 1rhAA1 1_11 11- — A A1- ^  IA^A**^- 0 - 0 0- 0-o D-022 5^8^11TIME (h)FIG 21 Glucose and lactic acid concentrations (mean ± SEM) before and after a 20 mLcontrol saline administration. (MA: Maternal arterial blood, FA: Fetal arterialblood and AM: Amniotic fluid).▪ 50 •820^ o MATERNAL ARTERIAL^•^ • FETAL ARTERIALI0\ 1(24130._____0,„„7.500^ 0^ 011251^I^120 -1^3^5^7^11TIME (h)cr)c/)•/1\• —__tn0:1605TIME (h)11••T^•c9.-^9,c) 920- 1^1^3^5TIME (h)40C\23011FIG 22: Fetal and maternal arterial blood gas parameters (mean± SEM)following a 20 mL saline administration (control experiment).1 0 0 0100io06TIME (h)10^12FIG 23: Disposition of labetalol in adult sheep plasma following a 100 mg i.v. bolus(mean ± SEM).TABLE 5: COMPARISON OF THE PHARMACOKINTETICS OFLABETALOL IN PREGNANT SHEEP WITH REPORTED VALUES INPREGNANT WOMENPARAMETER^PREG. SHEEP^PREG. WOMENbTOTAL BODY CL^135.9^129.6(L/h)^ (71.0-193.1)^(92.4-188.4)TERMINAL ELIM ty2 2.8 2.5(h) (1.9-6.4) (1.9-3.3)VDss 222c 274(L) (196-286) (250-557)FETAL DRUGEXPOSURE 0.14d 0.49e(ratio) (0.10-0.19) (0.19-0.81)a Mean values; range is given in parentheses.b Pre-eclamptic women; from Rubin et al., 1983.c Non-compartmental determination.d Fetal/maternal plasma AUC ratio.e Cord blood/maternal plasma concentration ratio atdelivery; from Michael, 1979.84TABLE 6:Pharmacokinetics of labetalol in pregnant and non-pregnant sheep. (mean ± SEM; n=5)PARAMETER NONPREGNANT SHEEP PREGNANT SHEEPCL (ml/min/kg) 29.00 ± 2.67 30.80 ± 3.83ty4 (hr) 2.41 ± 0.30 2.79 ± 0.66MRT (hr) 1.85 ± 0.09 1.80 ± 0.25Vdss (liter/kg) 3.22 ± 0.31 3.02 ± 0.18Vdarea (liter/kg) 6.19 ± 1.13 6.49 ± 0.728586volume of distribution were not significantly different from the estimates obtainedfrom pregnant sheep (two-tailed two sample t-test).3.4.2. Metabolism3.4.2.1. Conjugative MetabolismExcretion of labetalol in urine and bile as free drug (unchanged labetalol)and as glucuronide and sulfate was studied to assess the contribution of thesepathways in the overall elimination of labetalol. Cumulative urinary excretion ofunchanged labetalol accounted for 1.61 ± 0.38% of labetalol dose, while theglucuronide and sulfate conjugates accounted for 11.46 ± 2.82 and 1.47 ± 0.74%of the dose respectively (Fig 24). Both glucuronides and sulphate conjugates oflabetalol were also detected in the bile and their concentrations along with that ofunconjugated (free) labetalol are shown in Fig 25.3.4.2.2. Oxidative MetabolismPlasma, urine and bile samples were also analyzed for the presence of 3-APB, an oxidative metabolite of labetalol. While 3-APB could not be detected inthe plasma samples, its presence in urine and bile samples was established by GC-MSD using an authentic standard, as described previously. The total ionchromatogram and the mass spectrum corresponding to the peak at 8.22 min,which is identical to that of authentic standard (Fig 13B), are shown in Fig 26.The urinary excretion profile of 3-APB is shown in Fig 27 (A & B). Thecumulative excretion of 3-APB (Table 7) over 24 h was 43.76 ± 16.4 lig,representing 0.044 ± 0.016% of the dose. The terminal slope of 3-APB urinary15.0010.00 -ITV5.00 -•/1••0.00 •I^1^1-/-/*II^•^I1^IA87MIN FREE^0 GLUCURONIDE^[mil SULFATEB1E4-tTi)800060000 40002000-r"TT12 24TIME (h)FIG 24: Cumulative urinary excretion of labetalol and its conjugates (mean ± SEM).A: Cumulative amount excreted and B: Percentage of labetalol dose recovered inurine as free drug and conjugates.FREE,^INII FREE+ GLUCURONIDE & = FREE + SULPHATE12.00'1:--■W)8.0070i-E:-:-z:C2E-1^4.00zW0z00^0.00 [.75^2^5^9TIME (h)FIG 25: Concentrations of labetalol and its conjugates in adult sheep bile following a 100mg i.v. bolus (E# 617).Abundance TIC:^0101006.D120000 -100000- *80000-60000-40000 VeN620000-0 ,^,M/Z^->^6.00^7.00^8.00^9.00^10.00^11.00Abundance12000-91117Scan 308 (8.222 min): 0101006.D10000-441328000-6000-2414000-692000- 169 345i2541 , 1 1 II^. 11 1^.^1 I. l„.^, , ^,. ^-.  ,^.Time ->^50^100 150 200 250 300FIG 26: Total ion chromatogram (top panel) of urine sample obtained from E#105(nonpregnant) following a 100 mg labetalol bolus. Asterisk denotes the 3-APB peakand the EI mass spectrum corresponding to that peak (bottom panel).896^9^12^180^31 \010TO10.1A "40 90• •---^ •/".•• •f/•30 -r^0 20^•/'110 -/1^0 •0^3 6^9TIME (h)12 18BTIME (h)FIG 27: Excretion of 3-APB in adult non-pregnant sheep urine following a 100 mg labetaloladministration (mean ± SEM; n=5).A: Cumulative excretion over 24 h; B: Excretion rate plot.TIME^CONC. VOLUME AMT EXC CONC. VOLUME AMT EXC(h) (ng/mL) ImL) (ug) (ng/mL) (ml)^(ug)0-0.5 1.11 20 0.02 4.57 58^0.270.5-1.0 564.83 19 10.73 14.48 84^1.221-2 208.75 28 5.79 2.40 110^0.282-3 91.70 27 2.48 2.19 100^0.223-4 45.36 33 1.50 1.93 110^0.214-5 35.56 20 0.71 2.51 112^0.285-6 23.64 37 0.87 1.83 125^0.238-8 14.75 90 1.33 ND 255^0.008-10 9.81 82 0.80 ND 285^0.0010-12 8.35 107 0.68 ND 330^0.0012-24 3.50 580 1.96 ND 2149^0.00TABLE 7: URINARY EXCRETION OF 3-AMINO-1-PHENYL BUTANELABETALOL 100 mg BOLUS: NON-PREGNANT SHEEPLin E/248.0.1 E#543 E#617 E#316TIME(h)CONC.Ing/mL)VOLUME(mL)AMT EXC(ug)CONC.(og/mL)VOLUME(ml)AMT EXC(ug)CONC.(ng/mL)VOLUME AMT(ml)EXC(u9)CONC.(ng/mL)VOLUME(ml)AMT EXC(ug)CONC.(ng/mL)VOLUME(mL)AMT EXCtug)0-0.5 4.39 15 0.07 ND 22 0 70.73 18 1.27 24.05 12 0.29 NS NS0.5-1.0 1138.49 16 18.22 59.02 20 1.18 825.43 15 12.38 68.79 3 0.20 NS NS1.0-1.5 1117.41 15 16.78 50.00 21 1.05 782.94 14 10.98 284.45 18 4.78 NS NS1.5-2.0 958.51 15 14.38 38.94 35 1.38 871.82 12 8.08 273.31 8 2.19 182.47 20 3.852-3 895.00 30 20.85 22.79 85 1.94 499.42 30 14.98 272.77 18 4.91 180.49 20 3.813-4 241.30 30 7.24 12.08 77 0.93 252.32 30 7.57 250.85 15 3.78 127.72 28 3.324-5 103.03 40 4.12 10.80 75 0.81 180.83 30 4.82 167.80 15 2.52 91.53 30 2.755-8 31.33 52 1.63 8.50 120 0.78 118.87 25 2.92 85.18 14 1.19 49.84 40 1.996-8 22.15 95 2.10 3.51 320 1.12 57.98 80 3.48 32.04 48 1.54 20.00 84 1.688-10 13.40 150 2.01 ND 330 0 18.98 72 1.37 12.58 70 0.88 13.26 105 1.3910-12 8.99 140 1.26 ND 325 0 11.35 BB 0.98 4.53 88 0.39 7.80 115 0.9012-24 6.15 800 4.92 ND 225 0 8.01 480 2.78 4.46 380 1.69 1.44 825 0.90CUMULATIVE AMT. EXC. tug):^ 9.17^24.32ND:^Not detected (< 1 ng/mL)NS:^No Sample^ MEAN CUM. AMT. EXCRETEDDILEVALOL 25 mg BOLUS: NON-PREGNANT SHEEPE#105RR E#248RRCUMULATIVE AMT. EXC. (ug): 1.1.11687.1^2.6992excretion rate plot (0.123 ± 0.14 h - I) was not significantly different from that oflabetalol in plasma (0.134 ± 0.13 h - I) (paired t-test), implying that the eliminationrate constant of the metabolite (km) is much higher than that of the parent drug (K)(Gibaldi and Perrier, 1982). Application of the method of residuals provided anestimate of the apparent elimination half-life of the metabolite equal to 13.5 ± 3.8min Evidence for glucuronidation and sulfation of 3-APB was also obtained (Fig28). While only the glucuronide of the metabolite could be detected in urinesamples, both glucuronide and sulfate were seen in the bile samples. Surprisingly,3-APB was also detected in the free form in the bile samples. Urine and bilesamples were further analyzed for the presence of the hydroxylated form of 3-APBand benzylacetone (Fig 29), which could result from oxidation of 3-APB, butneither of these compounds could be detected in any of the samples.3.4.3. Hemodynamic EffectsThe observed changes in the hemodynamic parameters (heart rate, meanarterial pressure, femoral blood flow and vascular resistance) produced bylabetalol administration are shown in Fig 30. A significant decrease in vascularresistance and increase in femoral blood flow were seen following theadministration of labetalol. These vascular changes were associated withsignificant hypotension and the development of tachycardia. Maximum changeswere seen between 1.5 - 2 h following drug administration. The mean arterialpressure remains decreased beyond 2 h, but the change was not statisticallysignificant.3.4.4. Metabolic Effects=FREE & M TOTAL (free+glucuronide)A150-120-990 -z6003000^MI FREE, Ci FREE+GLUCURONIDE &^FREE+SULFATEBir3500tbo400 -z0 300C-4CDCD0▪ 21 1 1 1100 - •0^MIN 0.75^2.0^5.0^9.0TIME (h)FIG 28: Excretion of 3-APB conjugates following a 100 mg labetalol administration.A: Urine (amount) (E#105) and B: Bile (concentration) (E#248).93OHH2N OCHO3-AMINO-1-(4-HYDROXYPHENYL)BUTANEOHCH 3BENZYLACETONECHLABETALOLN-Dealkylation3-AMINO-1-PHENYL BUTANECH 3FIG 29: Oxidative metabolism of labetalol.(Not exhaustive; dotted lines indicate potential pathways; structure of amphetamine isincluded for the sake of comparison; partly from Gal et al., 1988).1251-0,1 IT^1,01 - 01TJ/1 ifl\c( ri I75—1^2^5^11TIME (h):1.■^115105E—^95(248560—1^2^5TIME (h)110112500/.---... 1 1\ *• \I *T * *..--,ri 200^0 III1`0-0.0.--^I I 1 I '0 '0 '0-0150^ \T T T T0.0•0- 0I 1 I I 9.6.6 '0 1.()•01 1 "9^-O, o -`o5^11TIME (h)O10050—1110Cm)U ,._,tu) 100X^T^.<• 4 0.750 E., EE^i ci)-^9^1 1 Til?‘(12 . 10,1 1,^0.5010 w c4^°I\T^T ,6 9 \ I/I Y, J).0/1\o' 1 i 1 T.-61\j)^C4 40C4 ,,,,E 901v) 480cnI I\ 0 9.•g I^1^i_6/1^po^0^- 1 1 1^ ^1 ,_E-ccw*^I 0 - 0.25r242., 70 ^<C---->FIG 30: Hemodynamic changes following labetalol bolus in adult nonpregnant sheep (mean ±SEM) (n=5). (Asterisks denote significant difference from control values).96Femoral arterial and venous blood gas changes induced by labetalol areshown in Fig 31. The marginal but significant decrease in pH and the moreconspicuous decline in base excess suggest the development of metabolic acidosisover the first six hours following labetalol administration. The mean blood baseexcess value at 3 h, which represents the maximum change, shows a decrease frommean control value by 3.8 mEq/L in the arterial and by 4.7 mEq/L in the venousblood. Significant changes were also seen in arterial pCO2 , which showed amaximum decrease of 13.9 ± 2.3% and venous p0 2 , which increased by 52.4 ±6.8%. The calculated arterial and venous oxygen contents are shown in Fig 32.Both the arterial and venous oxygen contents show an increasing trend, but onlythe venous oxygen content changes were significant. Labetalol also causessignificant hyperglycemia and lactic acidosis (Fig 33). The net uptake/output oflabetalol, glucose and lactic acid by the hind limb was calculated from therespective arterio-venous concentrations and the femoral blood flow data using theFick principle, i.e., [(CarteriarCvenous).* flow] and the results are shown in Figs 32and 34. There was a marked and significant uptake of labetalol in the initial 15min after drug injection, which however decreases rapidly and beyond 2 h, therewas no significant uptake or release. There was also net uptake of glucose overthe initial 4 h, after which there was a reversal of flux. In contrast, the venouslactic acid concentrations were consistently higher than or equal to that in theartery (Fig 33), so that there was sustained lactate release from the hind limb over12 h (Fig 34). The total amount of lactate released was calculated as the AUC ofthe lactate output curve and averaged 6.25 ± 1.35 g (0.07 ± 0.015 M). The netuptake of oxygen before and after labetalol administration is shown in Fig 32. Asignificant increase in oxygen uptake across the hind limb at 15 min after theadministration of labetalol was observed.150 -0—o9^10^ r 0o 9 01eTr• •‘•- •••• •-•• --•301\1) 1\ te:t-i***XI\I8^11^24TIME (h)97oARTERIAL ^ •VENOUSFIG 31: Femoral arterial and venous blood gas changes following labetalolbolus in adult nonpregnant sheep (mean ± SEM) (n=5). (Asterisksdenote significant difference from control values).0T0ro-o ARTERIAL •—• VENOUS9820.00• ma 15.0Cx1E--4• 00 0C) -‹(Pi e/ 10.0 --d5.06 O T T TiT^* ' 1 - 00,, Ti ci:6 0- t.^1 1 1 C) (II^1  i^O. O.ie•i11\^T.0^T r •- I^'4? JA, TI .--•.10 .1.\r• ol -.I^ 1.^I^•141511AeiO^O7^11^24TIME (h)12501000B75011.500 1\T^r^TCL) ?‘ T/C1) ('?.0C)>-+250 I07TIME (h)11^24FIG 32: Effect of labetalol on oxygen homeostasis in adult nonpregnant sheep (mean ± SEM)(n=5). (Asterisks denote significant difference from control values).990^ °ARTERIAL^•^ •VENOUS••N t.• *** gyp\ I, 'y  0 •Ttic^lic\ i^4.0^I/0.17* 0*\^2.0^''N.10i0TIME (h)FIG 33: Labetalol bolus in adult nonpregnant sheep: Femoral arterial andvenous blood glucose and lactate concentrations (mean ± SEM) (n=5).(Asterisks denote significant difference from control values).42^3TIME (h)100TIME (h)o^Io050-50Cr)0-100-150-1 0 8^11^24TIME (h)1003.002.001.0000.00CT-1GnFIG 34: Changes in arterio-venous fluxes following labetalol administration inadult nonpregnant sheep (mean ± SEM) (n=5). Positive values indicatenet uptake and negative values indicate net release from the hind limb.(Asterisks denote significant difference from control values).3.5. Dilevalol Bolus Studies in Non-pregnant SheepSince these studies were conducted in only two animals, no statisticalanalysis was performed.3.5.1. PharmacokineticsThe disposition of dilevalol in arterial plasma (Fig 35) was best describedby a triexponential equation. As a default, the concentrations were weighted by1/C2. The estimates of the pharmacokinetic parameters for the two animals arelisted in Table 8.3.5.2. MetabolismThe cumulative urinary excretion of free dilevalol, glucuronide andsulphate conjugates are shown in Fig 36. The cumulative amounts of free drug,glucuronide and sulphate excreted over 24 h following drug administration in E#105 were 0.47, 1.23 and 0.28 mg, respectively, representing 1.9, 4.92 and 1.12%,respectively. The corresponding values in E# 248 were 0.15, 1.09 and 0.20 mg,respectively, representing 0.58, 4.34 and 0.79%, respectively. The concentrationsof free drug and conjugates in bile are shown in Fig 37.The plasma, urine and bile samples were analyzed for 3-APB, before andafter incubation with P-glucuronidase and arylsulfatase preparations. Themetabolite was detected only in the urine and bile samples. The cumulativeamounts of 3-APB excreted in the urine over 24 h following dilevaloladministration (E105RR and E248RR) are shown in Table 7. The cumulativeamount recovered over 24 h in E#105 and E#248 were 26.87 and 2.69 pg,101T IM  1-1 (h)0^ 0 E#105^•^ • E#248FIG 35: Disposition of dilevalol in adult nonpregnant sheep arterial plasmafollowing a 25 mg i.v. bolus.Table 8: Pharmacokinetics of Dilevalol (RR-isomer of Labetalol) in AdultNon-pregnant Sheep Following a 25 mg i.v. Bolus Administration.PARAMETER E# 105 E#248Terminal elimination half-life (h) 5.87 3.70AUC (mg*h/L) 0.162 0.141AUMC (mg*h2/L) 0.276 0.450Total body clearance (mL/min/kg) 42.74 37.10Mean residence time (h) 1.75 3.20Vdss (L/kg) 4.36 7.12Vdarea (L/kg) 21.70 11.8910300OO00• • • • •^ •ATIM^ (h)6^9^12• • FREE^0 ^ 0 SULPHATE^0 ^ A GLUCURONIDEFIG 36: Cumulative urinary excretion of dilevalol as unconjugated (free),glucuronide and sulphate in an adult nonpregnant sheep (E#105).12501000750500O250FREE FREE+GLU^FREE+SUL3.0t=1° 2.0a7' 1.0OU0.02.5■  n^_, F6a^,._ , 4.0 6.0^10.0TIMH (h)FIG 37: Concentrations of unconjugated (free) and conjugated dilevalol in bileof adult nonpregnant sheep following a 25 mg i.v. bolus (E#105).106respectively, representing 0.11 and 0.011%, respectively. The concentrations offree 3-APB and its conjugates in bile samples are shown in Fig 38.3.5.3. Hemodynamic EffectsThe changes in heart rate, mean arterial pressure, femoral vascularresistance and blood flow in E# 105 following a 25 mg dilevalol bolus are shownin Fig 39. The maximum changes seen include a 34.9% decrease in mean arterialpressure, 35.3% increase in heart rate, 66.7% decrease in femoral vascularresistance and a 150.6% increase in femoral blood flow.3.5.4. Metabolic EffectsThe changes in arterial and venous blood gas parameters (E#105) followingdilevalol bolus are shown in Fig 40. The arterial pH and base excess values showan initial trend towards a decrease while no apparent changes were seen in arterialand venous p02 and pCO 2 . The arterial and venous blood glucose and lactateconcentrations (E# 105) are shown in Fig 41. Dilevalol bolus appears to causehyperglycemia and lactic acidosis. The area under the arterial lactateconcentration curve is equal to 59.9 mMh. No marked changes were seen in thearterial and venous oxygen contents (Fig 42). The glucose, lactate and oxygenfluxes across the hind limb are shown in Figs 43 and 42. The glucose fluxshows.a slight increase in hind limb uptake, which was maintained almostthroughout the sampling period. A net release of lactate was seen in the controlperiod. Following dilevalol administration, there was a reversal of flux, indicatingnet uptake, which continues for up to 150 min From 3 to 8 h, the net release oflactate from the hind limb was 2.13 g (23.67 mmol) (the area under the negativeA60.0 - 107I^I FREE^PI; FREE + GLUCURONIDE►•44P41"1•14•►•►4••►•►4•45.01,4 30.0 -1 -)0 ^15.014•14•14•►•►••04••14►•►•►•►•►•►•►4•14•►4►••►4•►4►4►1►•►••1'1•14•►4•14•141.•14•••►4•►4•►•►•►•••►••14••14►•••14••14•►4••►4••►4•14P4••P 4P41414►••••►••►4•►41••104••►►•►4••►4••►•14•••14•►4•14•14•144►4•►►••►••►4••••►•••••14•104••►P4•14►4•►4••14•14•►4••►•►4•114►4•14•14•11•►••14•►1•04•••►►••4411►•►14•••1 4•►4•►4••P4•14•►4►••11'44•1 4•14•►4•►4••04•1 4••••0.0••140 3 6TIME. (h1^18B . FREE^I I FREE + GLU^CZJ FREE +SUL400 -300za,0200z0 100 -2.0I4.0^6.0171 10.0^24.00TIME (h)FIG 38: Excretion of 3-APB and its conjugates following a 25 mg dilevaloladministration in an adult nonpregnant sheep (E#105)A: Urine (amount) and B: Bile (concentration).125-t=1100^r0•0-00 •-•-•\ igi, : ^cn/^‘0 cn• •^p:175^C.1-g---50111250.^/ 0 0.0.o.100^0to' 0.0 \0-0.0.0-0-0•=4^• • /1: 0-•-• •• \Os^WO -•"75f24^I •.•^•W":, 50-10e,_,....• \ /W^n/ • S.• •-• -•-•••\ iv. '•-•- •,•1080-0 HEART RATE^• — • ARTERIAL PRESSURE150-0M " 10 0 -oM5000-1 2^5^8 112.00C.)1.50124 tO1.000 ..o^0 ",0-0-0-0-0O/0 -0 °.° -0.0E 0.50(/)0.00-1 2^5 11TIME (h)FIG 39: Hemodynamic changes in an adult nonpregnant sheep (E#105)following a 25 mg dilevalol bolus.1 1 24• — • VENOUS175125755^8TIME (h)20 — 0 ARTERIAL5^8^11^24TIME (h)FIG 40: Femoral arterial and venous blood gas changes in an adult nonpregnantsheep (E#105) following a 25 mg dilevalol bolus.6.04.02.00.0—1^2^5TIME (h)11 241100^ 0 ARTERIAL^•^ • VENOUS5.04.03.02.0FIG 41: Femoral arterial and venous blood glucose and lactateconcentrations in an adult nonpregnant sheep (E#105) following a 25 mgdilevalol bolus.00 0300200 01005004000 0 0 0 ARTERIAL • — • VENOUS15.0111••ai`^\^Aft \• • w • •64%000  0 0 00Z 0Z0c:) 10.014, 0C.D>-1o 'er5.0-1 2 5^8TIME (h)1 1^24—1^2^5^8^11TIME (h)FIG 42: Effect of dilevalol on oxygen homeostasis in an adult nonpregnant sheep(E# 105).O240^0O10-0-5 50U)0u -5C-10-110--5 50E-1-(C-5 -utie^cbo•I \ • •• `.•92D2^//5 8^11^24^ //5 8^11^24TIME (h)O2•zFIG 43: Changes in arterio-venous fluxes following dilevalol administrationin an adult nonpregnant sheep (E#105). Positive values indicate netuptake and negative values indicate net release from the hind limb.112113flux curve from 3 to 8 h). The net oxygen consumption by the hind limb, asshown by the oxygen flux, roughly doubles following dilevalol administration (Fig41).3.6. Labetalol Infusion in Non-pregnant Sheep3.6.1. Disposition of LabetalolThe total amount of labetalol administered at the end of infusion in each ofthe five experiments was 280 mg. The arterial plasma labetalol concentrationsduring and after the infusion are shown in Fig 44. The slope of the straight linejoining the concentrations from 90 min to 6 h (-0.016 ± 7.98E-3) was notsignificantly different from zero (one-sample, two tailed t-test; p<0.05), indicatingthat labetalol concentrations remained at steady-state over that period. The meanlabetalol concentration during the steady-state ranged from 317.67 to 894.64ng/mL (mean = 454.19 ± 111.05 ng/mL). The clearance of labetalol at steady-state, calculated as the ratio of infusion rate to steady-state plasma concentration,was 17.62 mL/minfkg.3.6.2. Hemodynamic EffectsThe changes in mean arterial pressure, heart rate, femoral vascularresistance and femoral blood flow before, during and after labetalol infusion areshown in Fig 45. There was prolonged hypotension associated with labetalolinfusion and the change was significant from 4 h from the start of the infusionuntil the end of the experiment, at 10 h post-infusion. The hypotension wasaccompanied by transient, but significant, tachycardia, a significant decrease in10001ctSi...-,.616-6-11^T0I0\ ,oqi) 6 6- n-ii I J. ---- .9--- ...00^3 6^9^12^24TIME (h)FIG 44: Arterial plasma labetalol concentrations following combined bolus (100mg) and infusion (0.5 mg/min for 6 h) in adult nonpregnant sheep (mean ±SEM). (Solid line indicates infusion period).TIME (h)TIME (h)FIG 45: Hemodynamic changes during andlabetalol in adult nonpregnant sheepdenotes infusion period and asterisksrespective control values.after continuous infusion of(mean ± SEM) (n=5). Solid linedenote significant difference fromfemoral vascular resistance and a significant and prolonged increase in femoralblood flow.3.6.3. Metabolic EffectsThe arterial and venous blood gas changes are shown in Fig 46. The baseexcess and pCO2 showed significant decreases while the pH showed a decreasingtrend. The arterial and venous oxygen contents are shown in Fig 47. While atrend towards an increase is seen in both the arterial and venous oxygen contents,only the latter was statistically significant. Significant hyperglycemia and lacticacidosis were also observed (Fig 48). The calculated arterio-venous fluxes ofglucose and lactate across the hind limb are shown in Fig 49. A net uptake ofglucose over the initial 2 h of the infusion is evident from the significant positiveflux which, however, decreases quickly. The flux reversed before the end ofinfusion, signifying net release of glucose, and the negative flux was maintainedfor up to 18 h post-infusion. Similar changes were also seen in the case of lactate,which shows sustained net output from the hind limb beyond 2 h. The calculatedarea under the lactate flux curve, which corresponds to the net amount of lactatereleased from the hind limb is equal to 9.2 ± 3.12 g (0.102 ± 0.035 mol) of lacticacid. The mean oxygen consumption by the hind limb almost doubledimmediately following labetalol administration, but the change was not significant(Fig 47).3.6.4. Intra-arterial Norepinephrine StudiesIntra-arterial administration of NE caused decreases in femoral blood flowin a dose dependent manner (Fig 50), but the NE dose-response curve was not116,or^T0 \ (.4011'D 6—o„-,-o^T^o'—'00 \6,0\• •—0^/I\I —• • -^ ,1 1Ot?D 120.090.00.,^60.0 •30.0Llo 40.035.030.045.01.0– 2.07.0cr^4.00^ °ARTERIAL • •VENOUS150.0ifQD T^L I T ,_ zj .,..--- 0e VJeri)),^1 1 If) 6 0 o____,__10 0^\I /I^1,\ , 0 0_,1• , 0 \o,•1 ,- I,,0^,•__,_T:1•,\ e __• —T1ill I ri 1 Ii7.500 -7.450 -Q-4 7.400 -7.3507.300 -T,•\Ill I^•^▪^•we I NT T^i/• --0^•/\* ,I l l^ 0/1 II 0-0^ioi^N o_..9^9 11 1^* – 5.0^ 25.0^–12^5^8^11^24^–1 2^5^8^11^24TIME (h) TIME (h)FIG 46: Changes in femoral arterial and venous blood gas parameters during andafter continuous infusion of labetalol in adult nonpregnant sheep (mean ±SEM). (Asterisks denote significant difference from control values; solidline indicates infusion period).6005^60 ^-14002118o — 0 ARTERIAL^• — • VENOUSTIME (h)FIG 47: Oxygen homeostasis in adult nonpregnant sheep before, during and aftercontinuous infusion of labetalol (mean ± SEM). (Asterisks denotesignificant difference from control values; solid line indicates infusionperiod).•Go1190^ 0 ARTERIAL^• -• VENOUSIII^I1!1-14(1 .4•10 OrIP \ TI\ ••N^•09 006.003.000 . 0 012.009.006.003.00^—1 2^5^8^11^24TIME (h)FIG 48: Effect of Labetalol on Arterial and Venous Blood Glucose and LactateConcentrations (mean ± SEM). (solid line indicates infusion period;Arterial and venous blood lactate concentrations were significantlydifferent from control between 30 min and 2 h post-infusion; arterialand venous glucose concentrations were significant between 3 h and 2 hpost-infusion.).GO10120, cp-0„.o_.0o--o oil1 ' 'I1....0—10 ^—110-2 .^I5^8^. 11 -^11 2.4^01\ /?`(17- /^1 I^00,^01 I.^,• 1.1 •^#2^5^ 24'^I^•^6^.TIME (h)FIG 49: Hind limb arterio-venous glucose and lactate flux before, during and afterlabetalol infusion in adult nonpregnant sheep (mean ± SEM). (Asterisksdenote significant difference from control values; dashed line indicatesinfusion period).0^ 0 CONTROL^•^ • LABETALOL60.0 - 40.0 -20.0 -0.0 ^0.01 0.10^ 1.00NOREPINEPHRINE DOSE (ug)FIG 50: Intra-arterial norepinephrine dose-response curve before and duringlabetalol infusion (mean ± SEM).122significantly shifted during labetalol steady-state. The calculated EC50 values forNE in the control period and during labetalol steady-state were 0.204 ± O. 041 and0.172 ± 0.028 j.tg/kg, respectively, and were not statistically significant (paired t-test, p<0.05).3.7. Intra-arterial Labetalol Studies3.7.1. Studies in Adult Non-pregnant SheepDirect responses to labetalol were studied by injections of the drug via thefemoral artery. Direct intra-arterial injection of labetalol caused dose-dependentvasodilation and no significant changes were seen in the response followingphentolamine or propranolol infusion (Fig 51A and B). Also, the vasodilationcaused by intra-arterial labetalol was virtually instantaneous, with the peak effectbeing achieved within 20 sec of injection (Fig 52). Injection of control saline hadno effect on the flow.3.7.2. Studies in Fetal SheepDirect responses to labetalol in the fetal lamb were studied by injections viathe external pudendal epigastric artery (HLA) (Fig 2). No consistent changes wereseen in the HLA blood flow and no meaningful dose-response relationship couldbe established (Fig 53).3.8. Fetal Labetalol Bolus Studies3.8.1. Experimental Details123000—'90-30-OA80 -E- 60-O'4 40-zrx4• AFTER PHENTOLAMINE20-OHO01E-4^1E-3^1E-2^1E-1LABETALOL DOSE (mg)150- • AFTER PROPRANOLOL100_0•01E-4^1E-3^1E-2^1E- 1^1LABETALOL DOSE (mg)FIG 51: A.: Intra-arterialsheep hind limbB.: Intra-arterialsheep hind limblabetalol dose-response relationship in adult nonpregnantbefore and after phentolamine administration (E# 543).labetalol dose-response relationship in adult nonpregnantbefore and after propranolol administration (E# 105).1 minTIME^I 1 I—400I 241-01-240i-200 .1, ,1^.1.^111'41 , 1611 1 1 1 I I I 14 14. 11' 10,111111 I11,1, 1 I I^1 1 1 1 1 .1 , 111.1 0A l2r 400Lo—240—60200lilt^I It^ , , 0111 I1 1l I ,^I—0As1,41,414,11411/2Qh:P-AirtidLIJJL,...Lbrit , p,•1"-e'v„^, 1,1 NI l it^ , 0 1 1 , ,^..111,11,^11,11, „I I^Ltd IE1 d3E3—400•0—20001101101011,111011KINOSIMPOWINAtte—0r-2402 -2I 1^I—60FIG 52: Polygraph tracings showing the time course of hemodyamic changescorresponding to intra-arterial administration of A.: labetalol, B.:norepinephrine and C.: control saline. Vertical arrow on the time scalecorresponds to the time of injection.[1: Femoral Blood Flow (mL/min), 2: Mean Arterial Pressure (mm Hg)and 3: Heart Rate (bpm)].330 -0 •4-4 Q) 20 -10-0• •••••1 E — 2^lE — 1^1^10^100^1 000LABETALOL DOSE (ug)FIG 53: Intra-arterial (HLA) injection of labetalol in the fetal lamb (E#1154).126Table 9 lists the particulars about the animals used in the study. Thegestational age of the ewes at the time of experiment in the labetalol group was129.4 ± 2.2 days while in the control group it was 131.8 ± 1.5 days. The meanbirth weight of the operated fetus was 3309 ± 296 g in the labetalol group and3294 ± 364 g in the control group. The fetal and maternal cardiovascular andacid-base status were within the normal range at the beginning of each experiment.The mean pre-experiment values are listed in Table 3.3.8.2. PharmacokineticsThe disposition of labetalol in various sampling sites following a 4 mg fetalintravenous bolus administration is shown in Fig 54. The decline of labetalolconcentration in fetal arterial plasma was best described by either a biexponentialequation Ae-at + Be-ftt or a triexponential equation of the typePert + Ae -at + Be-flt. The weighting factor chosen was 1/C 2 . The estimates of thefetal pharmacokinetic parameters obtained by nonparametric analysis of thearterial plasma concentration data are listed in Table 10. Labetalol appears in thetracheal fluid instantaneously and the concentrations are consistently higher thanthat in fetal plasma over the entire sampling period. In contrast, a gradualaccumulation is seen in the amniotic fluid and beyond 3 h, the concentrations inthe amniotic fluid were higher than that in fetal plasma. Labetalol was alsodetected in the maternal arterial plasma but at much lower concentrations. Thepeak maternal arterial plasma labetalol concentration (4.17 ± 0.18 ng/mL) wasroughly 1/50th of that seen in fetal plasma (207.39 ± 27.66 ng/mL) and thematernal to fetal AUC ratio was 0.031 ± 0.002. The apparent terminal eliminationhalf-life of labetalol in maternal plasma was 4.75 ± 0.15 h. The maternal and fetalTABLE 9: FETAL BOLUS STUDIES: EXPERIMENTAL DETAILSEWE#BODYWT.(kg)GEST^# OFAGE^FETUS(days)TERM(days)BIRTH WT.OF FETUS(g)LABETALOL EXPERIMENTS116 80.0 130 2 139 3206*, 4162489 88.9 125 2 138 3923*, 4235540 75.1 136 2 142 3960*, 4494608 89.3 124 2 132 2360*, 4957722 71.1 132 2 139 3096*, 3330CONTROL EXPERIMENTS116 80.0 136 2 139 3206*, 4162201 72.9 129 2 143 3910*, 4350338 92.4 133 2 137 3500*, 3950608 89.3 129 2 132 2360*, 4957Weight of operated fetus.1270 ^0 FA A 6' AM •^ A TR, ^ MA- A100OH7'4OlE -A A'012^24000T IMFIG 54: Disposition of Labetalol in Pregnant Sheep following a 4 mg Fetal IntravenousBolus Administration (concentrations are mean ± SEM).(FA: Fetal Arterial Plasma; AM: Amniotic Fluid; TR: Tracheal Fluid and MA:Maternal Arterial Plasma).TABLE 10: Pharmacokinetics of labetalol in the fetal lamb and ewefollowing a 4 mg fetal i.v. bolus (mean ± SEM; n=5).129PARAMETER ESTIMATEA. FETUSCLf (mL/min/kg)a 50.45 ± 1.37 *CLfp (mL/min/kg)a 7.27 ± 2.11 *CLfa (mL/min/kg)a 43.18 ± 3.72*t to 0'0 4.35 ± 0.33 *MRT (h) 4.72 ± 0.33 *Vd„ (L/kg)a 14.28 ± 0.95*Vdarea (L/kg)a 19.01 ± 1.52*[FAUC/MAUCIC 0.144 ± 0.015Apparent t 1/2/3 (h)B. EWE4.75 ± 0.18MAUC/FAUC 0.031 ± 0.002CLmp (mL/min/kg)a 23.40 ± 8.99CLmp (mL/min/kg)b 29.84 ± 17.3CLm (mL/min/kg)b , c 30.80 ± 3.83b/w (h)c 2.79 ± 0.66MRT (h)c 1.80 ± 0.25Vd„ (L/kg)b , c 3.02 ± 0.18Vdarea (L/kg)b , c 6.49 ± 0.72a^Normalised to an average fetal body weight of 3 kg.b^Normalised to the average maternal body weight of 73.8 kg.c^From maternal bolus administration studies.*^Significant difference from corresponding maternal values followingmaternal bolus administration.130transplacental and nonplacental clearances were calculated using data obtainedfrom this study and the maternal bolus study and are included in Table 10.Estimates of fetal total body, placental and nonplacental clearances, eliminationhalf-life and apparent volume of distribution were found to be significantly higherthan the corresponding values for the ewe.3.8.3. MetabolismConjugates of labetalol were analyzed by enzyme incubation of amnioticfluid samples. The results are shown in Fig 55. No sulfate conjugates could bedetected, but a significant proportion of the dose was present as glucuronideconjugate. Fetal arterial plasma and amniotic fluid samples (up to 2 mL volume;obtained by pooling samples) were also analyzed for the presence of 3-APB (Fig29), before and after incubation with glucuronidase and sulfatase preparations, butthe metabolite could not be detected in any of the samples.3.8.4. Hemodynamic EffectsThe changes in fetal heart rate, mean arterial pressure, femoral blood flowand calculated hind limb vascular resistance ([mean arterial pressure-hind limbvenous pressure] hind limb flow) following the administration of labetalol areshown in Figs 56 & 57. The heart rate tends to decrease over the first 2 h,followed by a gradual and significant increase. No significant changes were seenin arterial pressure, femoral blood flow and vascular resistance.3.8.5. Metabolic EffectsU9TIME (h)3FREE^L FREE+ GLUCURONIDE1200900zO600 -300 -zC)^00a 12^24FIG 55: Concentrations of labetalol and its glucuronide conjugate in the amniotic fluidfollowing a 4 mg fetal intravenous bolus administration (mean ± SEM).^ W132140T^--1°10 I/1 1‘0^0-°I T/1 I0^I \I 0^I 16 -°-?I 0.0/ 1I i180160120— 1 2^5 8^1160.0T-,1- 1.C2? 0\Ij^I ,o j T 0I ,TT^so,oil^T^0-6o I I 0 1\0 _0 T ^6/I 0 sI 1^II 1‘ o" \p^0012^5^8^1130.0 ^—1TIME (h)FIG 56: Effect of a 4 mg fetal intravenous bolus on mean fetal heart rate and arterialpressure (mean ± SEM). Asterisk denotes significant difference.45.00 30.0 -T0I-01T.0 °J.I.60.0 -133TT^T -00, I T-44'1°-1 I LI °I-°11^11" ° I CI, 0. TI 0'0/ 1 0^1-0-7^I 1^O'l 01I IT/1^I I I0 .02^5^8^11T01^,/, )-s- -0.1. T^P, T^0,T-R -0-0 /\T T I °0^ \T T T1 i l^y-Y 1 ? -? -0-6'I ? -6 ,a,c ,Y I ? -6l'el3^yi1,0-1^2 5^8^11TIME (h)FIG 57: Effect of a 4 mg fetal intravenous bolus on mean fetal hind limb blood flow andvascular resistance (mean ± SEM).134Changes in the fetal arterial blood gas parameters following bolusadministration of labetalol are shown in Fig 58. A gradual decrease in pH andbase excess over the initial 4 h was seen, suggesting the development of metabolicacidosis. A maximum decrease of 0.096 ± 0.015 in pH and 5.7 ± 1.3 mEq/L inbase excess were seen at 4 h after labetalol administration. No significant changeswere seen in p02 and pCO2 . Blood oxygen content showed an apparent (-50%)decrease, which, however, did not reach statistical significance.  The maternalarterial blood gas parameters did not show any significant change.The effects of labetalol on glucose and lactate concentrations in HLA, HLVand amniotic fluid are shown in Fig 59 & 60. The mean glucose concentrationsbefore the administration of labetalol in HLA, HLV and amniotic fluid were 0.87± 0.11, 0.65 ± 0.05 and 0.13 ± 0.03 mM, respectively, while the mean lactateconcentrations were 1.58 ± 0.14, 1.62 ± 0.16 and 1.71 ± 0.08 mM, respectively.Marginal increases in the glucose concentration lasting up to 5 h were observed inall three sampling sites. The changes in lactate concentrations, however, weremore prominent, with peak concentrations in HLA and HLV representing roughlya four-fold elevation over control values. Significant accumulation of lactic acidwas seen in the amniotic fluid. Lactate concentrations returned to pre-treatment(control) values in HLA and HLV by 24 h, while it remained significantlyincreased in the amniotic fluid. A transient and marginal increase in maternalarterial lactate concentration was also observed, while there were no significantchanges in the maternal glucose concentrations (Fig 61). No significant changesin glucose and lactate concentrations were seen in the control experiments (Fig62). The changes in the net arterio-venous flux of labetalol, glucose and lactateacross the fetal hind limb were calculated by the application of the Fick principle[(HLA concentration - HLV concentration) * flow] (Fig 63). Net release ofC 0.0cr)0 ^, ,_. 0^0^.Cuii;\u -2.0U^ .,-.*--1I 7,cilw -4.0^.1\T.N...,,I--- Icn 1111a—6.0^*^I0:155.00.0-r50.0CCV 45.0 •I11,^4:3,AI^•^41,14110, 0,0_..0 I^1/?\ /I^III9 T•- •^•123.0 I I11^249.012.06. . •,.---T•0 1^II^I^11• •0. 100 I^I^I ITr!^/ N I _ 7 ,_ 1^•40 02^ 8^II^24• •LABETALOL 0—oCONTROL30.07.3757.325-T-r)7.2757.225• 0TI \. /I^I27.0Q1.124.0E 21.0ONQ. 18.015.012.0II:NI1 re\e^•>‹4;1401 °"'?'?10TIME (h) TIME (h)FIG 58: Effect of labetalol and control (saline) fetal bolus administration on fetal arterialblood gas parameters (mean ± SEM). Asterisks denote significant difference.1.0 -00.5 H0^ 0 FA^• —• HV^z—z\ AM—1^2^5^8^11^24TIME (h)FIG 59: Effect of a 4 mg fetal intravenous labetalol bolus on glucoseconcentrations (mean ± SEM). (FA: Fetal arterial blood; HV: Hind limbvenous blood and AM: Amniotic fluid).86 -DL42U0^ oFA^•^ •HV^L^ LAM—1^2^5^8^11^24TIME (h )FIG 60: Effect of a 4 mg fetal intravenous labetalol bolus on lactic acidconcentrations (mean ± SEM). (FA: Fetal arterial blood; HV: Hind limbvenous blood and AM: Amniotic fluid). FA and HV concentrations werestatistically significant between 90 min and 12 h and AMN concentrationsbetween 2-24 h.T T• •3 . 02 . 00UCf)OU 1 . 000 . 0OTOI• I.-^ T4411144.11 Ic)HoOzo- 1 . 00.02^5^8^11—1- 3 . 0- 2 . 00^ 0 GLUCOSE^•^ • LACTATETIM H ice )FIG 61: Glucose and lactate concentrations in maternal arterial blood following a 4 mgfetal intravenous bolus of labetalol (mean ± SEM).TYTTT^T^ I AT! INAT I •139•^ • FA A - A HV 0-0 MA A ^ A AMN2.0z0z1.0z01.4<4<40.0ill^I4, --A 1 0 •-01^ •A AA4iAA -A -A -A^A^ A^L^-06 0^---0^0^0 010 1,00 i0-.03124 2.0E-■ 111 I I 1 111I001 . 0C.11C/)00 0 0 A^A A A A A- 1 8^11^24TIME (h )FIG 62: Changes in glucose and lactate concentrations following control (saline) fetalintravenous bolus administration (mean ± SEM). (FA: Fetal arterial blood; HV:Hind limb venous blood; MA: Maternal arterial blood and AM: Amniotic fluid).140TIME (h)TIME (h)FIG 63: Effect of a 4 mg fetal intravenous bolus of labetalol on hind limb arterio-venouslabetalol, glucose and lactate fluxes (mean ± SEM). Asterisks denote significantdifference from control values.141labetalol from the hind limb in the first 2 h, with a momentary net uptake at 15min was observed. But beyond 2 h, there was no appreciable net uptake or releaseof labetalol. The mean control values for hind limb glucose and lactate uptakeswere 0.570 ± 0.224 mmol/h and 0.085 ± 0.227 mmol/h. The net uptake of glucoseby the hind limb was unchanged following the administration of labetalol. Whilethere was also net uptake of lactate by the hind limb prior to and for the first 4 hpost-dose, a significant change resulting in net release was observed thereafter.The total lactate release from the hind limb, calculated as the AUC of the releasecurve from 4 to 24 h, averaged 3.85 ± 2.05 g (0.043 ± 0.023 M). There were nochanges in hind limb glucose and lactate fluxes in the control experiments.4. DISCUSSION4.1. Development of a Microbore HPLC Assay with Fluorescence Detectionfor the Quantitation of Labetalol in Biological Fluids.Several reverse phase HPLC assay procedures using ultraviolet (Dusci andHackett, 1979; Woodman and Johnson, 1981; Hidalgo and Muir, 1984),fluorescence (Meredith et al., 1981; Oosterhuis et al., 1981; Alton et al., 1984;Luke et al., 1987; Bates et al., 1987; Ostrovska et al., 1988), electrochemical(Wang et al., 1985; Abernethy et al., 1986) and thermospray mass-spectrometric(Lant et al., 1987) detection systems have been described for the determination oflabetalol in human plasma. To date, no microbore HPLC assay has been reportedfor the trace level quantitation of labetalol. Also, there has been no report on thequantitation of labetalol in species other than humans.Successful analysis of drugs and metabolites in the various biological fluidsobtained from pregnant sheep (maternal and fetal plasma, amniotic and fetaltracheal fluid) following a single dose drug administration pose a few challenges:(a) The various biological fluids differ considerably in their composition(concentration of proteins, phospholipids, electrolytes etc.) and matrix consistency(e.g., fetal tracheal fluid is much more viscous than plasma) so that their extractionbehaviour and chromatography could be different and hence have to be treatedindividually. (b) Due to repetitive sampling from multiple sites over a shortperiod of time (see sections 2.7.1. and 2.7.5.), the sample volume obtainable ateach point is generally low (e.g. ---250^for fetal plasma). Hence, it is essentialto have an assay that could separate the analyte of interest from the endogenoussubstances and at the same time provide sufficient sensitivity and precision in142143quantitation. The method described here has been developed to fulfill theseobjectives.The octadecylsilane (C 18) stationary phase and phosphate buffer mobilephase were chosen to begin with because most of the previously published assayshave employed them. The molarity and pH of the phosphate buffer wereoptimized to provide satisfactory peak shape and retention time. The upper end ofthe molarity range studied was set at 20 mM based on the HPLC manufacturer'srecommendations for a microbore system. The higher and lower limits of pH (8.0and 2.5 respectively) used were in accordance with the range suggested by thecolumn manufacturer. The occurrence of broad peaks (peak width > 2 min) at pHvalues of the phosphate buffer greater than 4.0 is probably due to labetalol existingin both ionized and unionized form (pKa of labetalol = 7.4). Decreasing the pH ofthe buffer from 7.0 to 2.5 probably causes progressively increasing ionization oflabetalol, resulting in the elution of a mostly single moiety (the ionized form) andhence the improvement in peak shape and symmetry.Whereas both ether and ethyl acetate provided comparable extractionrecoveries (Fig 4), the latter was chosen in preference to the former based onconsiderations of safety and ease of handling. The two-step extraction (extractionwith ethyl acetate followed by re-extraction of the organic layer with dilutephosphoric acid) was found necessary to overcome interference from endogenouscomponents (Fig 5). This improved selectivity was accompanied by a slight lossof recovery, which in absolute terms, decreased from about 81% to about 75%.However, this procedure eliminates the need for drying the samples and henceshortens the time for sample preparation. The ratio of the volume injected (60 !IL)to the volume of the aqueous phase at the end of the second step of extraction (6001.11,) was 0.1, which enables multiple injections to be made from the same sample.144The primary objective of this assay is to provide adequate mass sensitivity,that would enable quantitation of a few nanograms of labetalol present in — 200-300iLtL. It has been shown that the use of a microbore column could significantlyenhance mass sensitivity (Wong, 1989) and minimum detection limit (Simpsonand Brown, 1987) and thus is ideal for applications involving restricted sample-volume. Hence, in this assay, a microbore column (2.1 mm i.d.) and connections(0.12 mm i.d.) (between injector, column and detector) were employed. Thelength of the tubing that connects the column outlet to the detector was restrictedto 100 mm, to minimize post-column diffusion. Also, the potential peak diffusionthat could occur at the detector was overcome with the use of low volume flowcell (5 fit capacity). The use of an emission cut-off (370 nm) filter resulted insignificant reduction in baseline noise at emission wavelength equal to 412 nm.Further optimisation of the signal/noise ratio was achieved with the use of a 2 mmwide excitation slit and 4 mm wide emission slits instead of the standard 1 mmand 2 mm wide slits, respectively. Even though the wider slits increase thebandwidth of excitation and emission wavelengths and thus compromiseselectivity, no interfering peaks were found under the conditions employed.4.2. Development of a GC-MSD Assay for the Identification andQuantitation of 3-APB, an Oxidative Metabolite of Labetalol in theBiological Fluids of SheepThe possibility that labetalol undergoes oxidative biotransformation wasfirst suggested by Gal et al., 1988. They identified 3-APB and its p-hydroxyderivative in urine sample from a patient on labetalol therapy using GC-MS withnegative chemical ionization after pentafluoro-propionyl derivatization.Subsequently, the same group of authors reported a quantitative stereoselective145assay for 3-APB, which involves negative ion CI of the (S)-a-methoxy-a-trifluoromethylphenylacetic acid derivatives (Changchit et al., 1991).The assay for 3-APB presented here has been developed with an objectiveto identify and quantitate this compound in biological fluids using a bench-top GasChromatograph with Mass Selective Detector (GC-MSD) using electron impactionization.Satisfactory chromatography in terms of peak shape and resolution betweenthe analyte (3-APB) and the internal standard (1-methyl-2-phenoxyethylamine)derivatives has been obtained by "cold trapping" of the sample, produced by a 100°C gradient between the injection temperature and initial column temperature.While underivatized 3-APB shows good chromatography, derivatization isessential to overcome interference from the parent compound (labetalol), whichundergoes decomposition to yield trace levels of 3-APB under the GC conditionsemployed. No such interference could be seen following HFBA derivatizationpossibly because labetalol is not readily derivatized. The EI mass spectra of theHFBA derivatives of 3-APB and the internal standard (Fig 13B & 13C,respectively) show that the m/z 254 fragment is much more intense in the massspectrum of the internal standard, possibly due to the presence of an ether linkagein 1-methyl-2-phenoxyethylamine (structure shown in Fig 13A). Despiteextensive fragmentation of the 3-APB derivative (Fig 13B), the molecular ion (m/z345) could still be detected in the total ion chromatogram, thus enhancing thediagnostic utility of the assay. Two ions for each of the two compounds (m/z 345and 132 for 3-APB and m/z 347 and 134 for internal standard) were selected forthe quantitative SIM mode. The molecular ions were included in preference to themost intense ions (m/z 117 in the case of 3-APB and 254 with the internalstandard) to enhance the selectivity of the method. While the abundance of m/z117 in 3-APB mass spectrum was roughly 20% higher than that of m/z 132, the146latter provided a better signal-to-noise ratio in the SIM mode and hence waschosen as the second ion. The dwell time chosen for each group of ions was 200msec so that each peak (width = 6 sec) is sampled about 12 times. The sensitivityof quantitation was appreciably improved by tuning with target ions of mass 69,218.95 and 264 of PFTBA and also by selecting the optimal fractional mass of theion, correct to a decimal place. It should be noted that the MSD used in thismethod is not truly a high resolution mass spectrometer and that specifying afractional mass simply helps to narrow the scan window, which in some cases canlead to an appreciable increase in sensitivity. Thus m/z 345.4 provided the bestsignal/noise ratio for 3-APB derivative. Hexane was chosen as the extractionsolvent over ether, primarily because of safety considerations while sample pHadjustment with 5N sodium hydroxide provides sufficient alkalinization ofbiological fluid sample (1 mL) with 200-300pL volume, thus providing nearoptimal aqueous/organic phase ratio (roughly 1:4). Triethylamine was added tothe reconstitution solvent (toluene) to act as sequestering agent and minimizeadhesion of 3-APB to glass surfaces. The absolute recovery of 3-APB wasconsistently high (range: 90-107%), over the concentration range studied.4.3. Maternal Bolus StudiesLabetalol is used in the treatment of various hypertensive disorders inpregnancy (Ashe et al., 1987; Goa et al., 1989), but there is little information inthe literature regarding the in utero fetal exposure and fetal effects of labetalol.The maternal bolus studies were conducted to obtain this vital information in thepregnant sheep model.4.3.1. Pharmacokinetics147There are no reports in the literature of any detailed pharmacokinetic-pharmacodynamic investigations of labetalol in pregnant animals. The chronicallyinstrumented pregnant sheep model offers several advantages including repeatedsampling from the mother, fetus and other fluid compartments in utero as well ascontinuous physiological and biochemical monitoring.Labetalol shows a triexponential or biexponential decline in the maternalplasma with a high total body clearance of 135.86 ± 16.93 L/h (30.8 ± 3.83mIlmin/kg), a value that approximates the hepatic blood flow, which in pregnantsheep is about 150-300 L/h (Katz and Bergman, 1969). This value of clearancealso represents about 27% of the normal cardiac output in near-term ewes(Rosenfield, 1977). The rapid distribution seen in the first 15 min could beexplained by the high apparent volume of distribution (VD area = 477.5 ± 53.0 Lor 6.48 ± 0.72 L/kg and VD ss = 222 ± 12.98 L or 3.02 ± 0.18 L/kg). Table 5compares the pharmacokinetics of labetalol in pregnant sheep with the reportedvalues (Rubin et al., 1983; Michael, 1979) for pregnant women. With theexception of fetal drug exposure, where the indices (AUC vs concentration ratio atthe time of delivery) are different, the pharmacokinetic parameters show goodagreement, which suggests that the pregnant sheep is a suitable model, at least forthe maternal pharmacokinetics of labetalol. However, it should be noted that thestudies by Rubin et al., 1983 and Michael, 1979 were conducted in pre-eclampticwomen while normal, healthy sheep were used in this study. The fetal/maternalplasma labetalol AUC ratio in the pregnant sheep was much lower than the cordblood/maternal venous plasma concentration ratio reported in humans (0.144 vs0.5). This could be due to one or more of the following reasons. Firstly, theepitheliochorial sheep placenta differs from the hemochorial human placenta inhaving a lower permeability to hydrophilic polar substances (Faber and148Thornberg, 1983). Secondly, the human data are based on a single pointdetermination of fetal-to-maternal drug concentration ratio, which changesconstantly with respect to the time of dosing in non steady-state conditions(Anderson et al., 1980b). Thirdly, the extensive maternal and fetal hemodynamicchanges that occur during and immediately after delivery may affect thedisposition of the drug in the mother and/or the fetus (Hamshaw-Thomas et al.,1984) and hence extrapolation of the concentration ratio at delivery to theantepartum intrauterine conditions may be erroneous. Finally, the pathologicalchanges in the placental structure and function that are known to occur in pre-eclampsia (Dadak et al., 1984) may affect placental transfer processes.Transfer of labetalol across the sheep placenta was rapid as evidenced bythe peak concentration in the fetal plasma at 3 min (Fig 16). The significantlylonger apparent elimination half-life of labetalol in the fetus when compared to theewe could be due to extensive binding of labetalol to and consequent slow releasefrom proteins and/or tissues in the fetus or recirculation of labetalol from the fetaltracheal, amniotic and allantoic fluid compartments back to the fetus. In a clinicalcase study, Haraldsson and Geven, 1989, have reported the apparent half-life oflabetalol in a premature infant, whose mother had received oral labetalol for tenweeks prior to delivery, to be approximately 24 h. From this study, it is clear thatlabetalol accumulates in the amniotic and tracheal fluids and persists much longerin these sites (24-48 h) than it does in the maternal or fetal plasma. Similarobservations have been made with other drugs in the pregnant sheep followingmaternal administration (Rurak et al., 1991), but in each case with different fetal-to-maternal half-life relationships. The disposition half-life in the fetus wassignificantly higher than that in the ewe for metoclopramide, the same withdiphenhydramine and significantly lower in the case of ritodrine. Thus, otherfactors like the kinetics of protein and tissue binding may be involved.4.3.2. Hemodynamic EffectsThe hypotensive action of intravenous labetalol is generally believed to bemediated primarily by peripheral alpha s -blockade (van Zwieten, 1990), but thehemodynamic response to labetalol and in particular the preponderance of eitheralpha or beta-antagonism can vary considerably in different experimentalsituations depending on the balance of autonomic influences (Brittain and Levy,1976). In our study, no consistent maternal cardiovascular changes wereobserved. The two animals that showed a trend towards hypotension andsimultaneous tachycardia were ENTG and E 105. Since ENTG had a low bodyweight (Table 2) and hence received a higher labetalol dose/kg (2.47 mg/kg whilethe mean value is 1.42 ± 0.13 mg/kg), it is possible that the maternalcardiovascular changes are dose dependent. In the study of Eisenach et aL, 1991in pregnant sheep, a statistically significant change in maternal mean arterialpressure was seen with a dose of 3 mg/kg of labetalol but not with the lower dosesof 0.5 and 1.0 mg/kg, implying that a minimum dose is required to elicit anyhemodynamic response. However, the AUC and other pharmacokinetic data inour study (Table 4) suggest the lack of any labetalol concentration relatedphenomenon. In contrast to our observations and that of Eisenach et al., 1991, asignificant fall in maternal mean arterial pressure in normotensive pregnant sheepwas reported by Mohan et al., 1990, following a 100 mg infusion of labetalol over5 min. It could be that this difference is due to the mode of administration - slowinfusion as opposed to an instant bolus. The lack of any apparent change in thefetal hemodynamics in this study is similar to that reported by Eisenach et al. (at a149150dose of 1 mg/kg) and Mohan et a/.,1990. The lack of any cardiovascular responsein the fetal lamb may be at least in part due to the low fetal exposure to maternallabetalol.4.3.3. Metabolic EffectsThe metabolic effects of labetalol, unlike its hemodynamics, have notreceived much attention. In particular, the effect of labetalol on carbohydrate andlipid metabolism in pregnancy has not been studied in detail. Our results suggestthat labetalol exerts very significant metabolic effects in the pregnant sheep. Inboth the mother and fetus, the decrease in arterial blood pH is accompanied by aconcomitant change in base excess, implying that the acidosis is of metabolicorigin. Maternal pH and base excess show only marginal changes, with the valuesquickly returning to control state. This is probably a result of respiratorycompensation in the form of hyperventilation, which in the adult, is a fairly robustcompensatory mechanism. The initial fall in maternal pCO2 and the subsequentupward trend in p02 (the latter not statistically significant) is consistent withhyperventilation. The fetus, in the absence of any respiratory compensatorymechanisms, shows sustained deterioration in pH and base excess. The sharp fallin fetal oxygen saturation and content is most likely due to the Bohr effect, i.e., ashift in the oxyhemoglobin dissociation curve to the right, caused by the drop inpH.Several clinical studies of labetalol have reported variable changes in bloodglucose levels. Andersson et al., 1976, observed a significant increase in fastingblood sugar without any change in insulin levels or glucose tolerance inhypertensive men following labetalol administration, but the authors did notprovide any explanation for these observations. But, Barbieri et al., 1981,151observed only slight elevations in blood glucose level in their study and attributedthe effect to the a l -blocking action of labetalol since phentolamine, a pure a-adrenergic blocker has been shown to augment the increase in glucose duringexercise (Galbo et al., 1977) and prazosin, a selective post-synaptic a radrenergicblocker, causes an increase in glucose in hypertensive men (Barbieri et al., 1980).While the extent of a and r3-blockade caused by labetalol in this study was notinvestigated, studies in adult nonpregnant sheep (see section 4.6.4.) show thatlabetalol does not cause any significant a-blockade. On the other hand, there issubstantial evidence to show that labetalol has significant beta 2-agonist activity(Riley, 1981; Baum and Sybertz, 1983). Increases in c-AMP levels caused bylabetalol has also been reported in an in vitro study using isolated pregnant ratuterus (Chimura, 1985). Beta2-agonists are known to cause elevated maternalglucose levels in humans (Schreyer et al., 1980) and sheep (Bassett et al., 1985;van der Weyde et al., 1992). Bassett et al., 1985, observed that maternal infusionof ritodrine, a beta2-agonist, causes significant (2-fold) increase in the maternalglucose concentrations, while van der Weyde et al., 1992, found a similar effecton fetal glucose levels with fetal infusion of ritodrine. Hence, it appears that 132-agonism is responsible for the maternal hyperglycemia observed in this study,although contribution from oc i -blockade cannot be completely ruled out.The fetal hyperglycemia (Fig 20) appears to be a direct result of thematernal hyperglycemia since the changes in the fetal glucose concentrationsparalleled those in the mother. In the fetal lamb, under normoxic conditions,almost the entire supply of glucose is obtained from the maternal circulation andthere is very little glucogenesis (Hay et al., 1981). About 60% of fetal glucose isoxidized, accounting for about 30% of the net fetal oxygen uptake (Hay et al.,1983). Glucose in the fetus is also converted into lactate and fructose as well astransferred back into the maternal circulation (Prior, 1980). The transfer of152glucose across the placenta is by passive diffusion along the concentrationgradient and Hay and Meznarich, 1989, have shown that in sheep, the fetalglucose concentration shows a statistically significant linear relationship tomaternal glucose concentrations over a wide range (1-9 mM or about 20-160mg/100 mL).The observation of significant fetal acidemia, as indicated by the blood pHand base excess in our initial experiments, prompted us to monitor the lactic acidchanges in the latter experiments. The results (Fig 20) show that the fetalacidemia is largely, if not completely, due to the lactic acidosis. In the fetus, thepeak lactate concentration, the total area under the lactate concentration curve(AUC) and the incremental increase in the lactate AUC following labetaloladministration (calculated as ([Lc](ty[Lc](0)) dt) were consistent in all the fouranimals in which lactate concentrations were measured, with mean values of 5.9 ±0.26 mM, 110.4 ± 6.18 mMh and 67.8 ± 7.26 mMh, respectively. Thecorresponding values for the maternal lactate concentrations were 3.5 ± 0.77 mM,53.6 ± 1.38 mMh and 34.4 ± 2.62 mMh, respectively. Lactate in the amnioticfluid was possibly derived from fetal urine since the permeability of thechorioamnion to lactic acid is low (Britton et al., 1967). This would also meanthat similar high concentrations of lactate should be present in the allantoic fluid(not sampled in this study) as well, since the fetal urine is almost equallydistributed between the amniotic and allantoic fluid cavities (Wlodek et aL, 1988).To the best of our knowledge, there has been no report in the literature onthe effect of labetalol on lactate homeostasis in any species - pregnant ornonpregnant. There does not appear to be any simple explanation for the acutelactic acidosis that we have observed, especially in the fetus, since the fetalexposure to maternal labetalol is only about 14%. Since the sheep placenta has avery low permeability to lactate (Britton et al., 1967; Sparks et al., 1982; Kitts and153Krishnamurti, 1982), the fetal lactic acidosis induced by labetalol is unlikely tohave been due to the maternal lactic acidosis. This view is supported by the fetallabetalol bolus studies (see section 4.8.4.), where there was a rise in fetal lactateconcentrations with minimal changes in the mother. The low placental lactatepermeability could also explain the persistence of this substrate in the fetus, sincetransplacental clearance is a major component of fetal elimination for a number ofcompounds (Rurak et al., 1991). The persistence of lactate in the fetal lamb forseveral hours following an initial rapid increase as observed in this study has beenreported in other studies involving physiological (hypoxia; Britton et al., 1967)and pharmacological (ritodrine administration; Bassett et al., 1985 and van derWeyde et al., 1992) perturbations. Thus, the elevation in fetal lactateconcentration following maternal labetalol administration likely resulted fromincreased production or decreased elimination of lactate or a combination of both.Studies in normoxic fetal lambs have demonstrated that circulating lactateoriginates from production by fetal tissues and by the placenta, with the latterorgan accounting for about 35% of total lactate production (Sparks et al., 1982).Fetal lactate is produced from both glucose and non-glucose (e.g. alanine) sources(Kitts and Krishnamurti, 1982). Approximately, 75% of the lactate is oxidized(Hay et al., 1983) and this accounts for about 25% of fetal oxygen consumption(Burd et al., 1975). The rate of lactate oxidation has been found to beconcentration dependent with increased oxidation occurring at higherconcentrations (Hay et al., 1983).Although hyperglycemia can lead to increased blood lactate concentrations,it is unlikely that the fetal lactic acidosis is consequent to maternal or fetalhyperglycemia since Hay and Meznarich, 1989, have found that following a four-fold increase in maternal glucose concentration caused by the glucose-clamptechnique, the fetal lactate increased only marginally (from 1.57 mM to 2.07 mM)154even though uteroplacental glucose uptake showed a three-fold increase.Sustained mild hypoxemia (fall in p02 by 5 mm Hg) in the fetus can result inelevated lactic acid levels (Towell et al., 1987). However, the modest andtransient changes in fetal p0 2 observed in this study do not suggest a similarmechanism. The role of adrenergic blockade in the metabolic status of fetal lambhas been investigated by Jones and Ritchie, 1978. They found that infusion ofphentolamine or propranolol alone does not cause any significant change in any ofthe fetal metabolic indices including lactic acid, which rules out the possibility offetal adrenergic blockade being the primary mechanism behind the lactic acidosisin this study. Labetalol has also been shown to cause significant elevations inplasma norepinephrine concentrations in hypertensive men (Lin et al., 1983;Christensen et al., 1978), which has been attributed to the intrinsicsympathomimetic activity of the compound. Exogenous norepinephrine has beenknown to cause increases in plasma glucose and lactate in adults (Himms-Hagen,1967) and in fetal sheep (Jones and Ritchie, 1978). Hence, there exists apossibility that the fetal and maternal metabolic changes are at least in part,secondary to the rise in norepinephrine concentrations, which were not measuredin this study.It is well recognized that P2-agonists like ritodrine, which are used in themanagement of premature labour, cause significant changes in the carbohydrateand lipid metabolism including elevations in glucose and lactic acid concentrationsin humans (Lenz et al., 1979) and maternal and fetal sheep (Bassett et al., 1985;van der Weyde et al., 1992). Hence it is possible that Pragonism is involved inthe pharmacodynamics of labetalol. Whether the sympathomimetic effect isderived from the intrinsic activity of labetalol (Riley, 1981) or actually mediatedthrough an active metabolite cannot be ascertained from the data obtained in thisstudy. Hence, the oxidative metabolism of labetalol was investigated in155subsequent studies involving labetalol administration in adult nonpregnant ewes(see section 4.4.) and in fetal lambs (see section 4.8.).It is possible that the release of lactate occurred from the fetal and maternaltissue and/or from the placenta since the sheep placenta is known to possess beta2-receptors (Padbury et al., 1981). The rise in lactate concentrations resulting fromthe administration of ritodrine, a 13 2-agonist, appears at least in part due toincreased placental production (van der Weyde et al., 1992; Wright, 1992).However, studies conducted in adult nonpregnant sheep and fetal lambs (seesections 4.4. and 4.8., respectively) to assess the extent of release of lactate fromthe carcass in general, and the hind limb in particular, in response to labetaloladministration, suggest that these tissues could also be involved in the increasedlactate production.A simple concentration-effect relationship does not exist between thematernal/ fetal labetalol concentrations and any of the observed effects. This isunderstandable since most of the effects (e.g. acidemia, glucose, lactateconcentration changes) presumably involve a number of events in sequence thatprecede the observed "effect". We did not attempt any pharmacokinetic-pharmacodynamic modelling in terms of an effect compartment (Holford andSheiner, 1981) or a biophase (Veng-Pedersen and Gillespie, 1988), concepts thatare applied in the minimization of the hysteresis loop, simply because themechanism(s)/events involved in the observed effects are not understood and amodel, under such circumstances, would be of dubious value. Thepharmacokinetic and pharmacodynamic data from this study underline thelimitations of using a strictly concentration-based fetal safety index (maternal/fetalconcentration or AUC ratio) since a low index of drug exposure(AUCfetal/AUCmaternal 14%) was associated with marked fetal effects (e.g.prolonged metabolic acidemia). Thus, for a given concentration-based fetal drug156exposure index, it is possible to obtain a much different fetal/maternal effect ratioand this ratio in turn could be different for different pharmacodynamic endpoints.Hence, it may be necessary to assign one or more "pharmacodynamic indices",which when applied with the AUC ratio will result in a more meaningful measureof fetal safety.The relevance of the findings in this study to clinical obstetric practiceremains to be established. Much of the data on the fetal effects of labetalol inhumans relates to uteroplacental hemodynamics (Nylund et al., 1984; Joupilla etal., 1986; Harper and Murnaghan, 1991; Pirnohen et al., 1991) and/or neonatalfollow-up (MacPherson et al., 1986; Sibai et al., 1987; Pickles et al., 1989).There are limited data on blood gas and fetal metabolic effects of labetalol in theliterature. The dose of labetalol used in the clinical management of pregnancy-induced hypertension depends on the severity of the condition, but it also variesconsiderably due to the large inter-individual variability in the hypotensiveresponse to the drug. Doses of up to 200 mg as intravenous infusion (Michael,1986; Ashe et al., 1987) and 1200 mg p.o. (Michael, 1982) have been used tocontrol hypertensive emergencies in pregnancy. Thus the labetalol dose used inthis study (100 mg or 1.4 mg/kg) is within the clinically relevant dosing range. Interns of fetal exposure to labetalol in humans, the cord blood labetalolconcentration at the time of delivery ranges from about 10-260 ng/mL (Rogers etal., 1990; Michael, 1979). In this study, following maternal administration oflabetalol, the peak labetalol concentration in ovine fetal plasma ranged from 23-66ng/mL, which suggests that the human fetus is exposed to relatively higherconcentrations of labetalol than the fetal lambs in this study. However, nosignificant deterioration of fetal acid-base or oxygenation status due to labetaloltherapy has been reported in any of the clinical studies. Pirnohen et al., 1991,observed that following a 0.8 mg/kg intravenous bolus to normotensive pregnant157women at 38 weeks gestation, the cord blood gas parameters were within thenormal range and that in all the cases, the umbilical artery pH was above 7.15 andthe umbilical venous pH was above 7.25. But, assessment of fetal blood gas statusin clinical studies is normally based on the cord blood gas parameters obtainedimmediately after delivery and cord clamping. In a recent study, Khoury et al.,1991, compared the blood gas values obtained in the intact fetal circulation justbefore elective cesarean section with those obtained immediately after deliveryand found that pH, p02 and base excess were significantly decreased while pCO2was significantly increased after delivery. This indicates that the blood gas valuesobtained at delivery do not reflect the prenatal situation. Further, if the maximumchanges in the blood gas parameters seen in the fetal lambs in this study were tobe observed in the human fetus at delivery, they would still be considered to be inthe normal range (Rurak et aL, 1987). Moreover, in the presence of pre-eclampsia, any labetalol induced fetal blood gas changes would likely be ascribedto the underlying clinical problem rather than to an effect of the drug. Furtherinvestigation to assess the metabolic consequences of maternal labetalol in thehuman fetus is thus necessary.4.4. Labetalol Bolus Studies in Nonpregnant SheepThe primary objectives of this study were to assess the contribution ofskeletal muscle and other carcass components to the lactic acidosis observedfollowing labetalol administration (section 3.4.3.) and to explore the possibility ofinvolvement of active metabolite(s) in the mediation of labetalol inducedmetabolic effects. The hind limb was chosen as a representative tissue of thecarcass due to the ease of arterio-venous catheterization and blood flowmeasurement in that region.4.4.1. PharmacokineticsThe total body clearance, apparent terminal elimination half-life and thevolume of distribution of intravenous labetalol in adult nonpregnant sheep werenot significantly different from the estimates obtained in pregnant sheep (Table 6)suggesting that pregnancy does not alter the pharmacokinetics of labetalol insheep. Similar conclusions were reached in human studies as well (Rubin et aL,1983). Also, the estimates of pharmacokinetic parameters obtained in this study(mean total body clearance equal to 29.00 ± 2.67 mL/min/kg and terminalelimination half-life equal to 2.41 ± 0.30 h) are similar to the reported values innormotensive nonpregnant women (mean total body clearance in that group was33.8 mL/min/kg and terminal elimination half-life was 2.1 h) (Rubin et al., 1983).The percentage of the dose excreted in the urine as unchanged drug was less than2%, which is similar to that reported in humans (0-5%) (Martin et al., 1976).Significant biliary excretion of the unchanged drug was also seen (Fig 25) and thebiliary concentrations were consistently higher than the correspondingconcentrations in plasma, suggesting the involvement of an active transportmechanism.4.4.2. MetabolismGlucuronidation of labetalol at both the phenolic and secondary hydroxygroups has been reported (Martin et al., 1976; Niemeijer et al., 1991). The resultsfrom enzyme hydrolysis of urine and bile samples in this study show that labetalolundergoes significant glucuronidation in the sheep (11.5%) and the cumulativeamount excreted in the urine as glucuronides was roughly ten-fold larger than the158159amount excreted as unchanged drug. In sheep, it appears that labetalol alsoundergoes sulfation, in contrast to the observations by Martin et al., 1976, in rats,rabbits, dogs and humans. These apparent differences may be species related andalso dependent upon the enzyme preparations used for conjugate hydrolysis. Incontrast to the extent of glucuronidation, sulfate conjugation was quantitativelyless significant (1.5%) and in two of the animals, the sulfate conjugates could notbe detected in the urine. Both the glucuronide and sulfate conjugates wereexcreted in the bile and the glucuronide/sulfate concentration ratio in the bile wasbetween 0.5-2.0 while it was around 8-10 in urine, suggesting preferential biliaryexcretion of sulphate conjugate. Quantitative assessment of biliary excretion wasnot made since continuous collection of bile was not feasible.The oxidative metabolism of labetalol has not received much attention inthe past. Recently, Gal et al., 1988, suggested that labetalol undergoes N-dealkylation (Fig 29) to yield 3-APB and its hydroxylated derivative, which theyidentified in urine samples obtained from patients on labetalol therapy. However,the quantitative relevance of N-dealkylation or the pharmacodynamic contributionof the metabolites formed was not clarified. We developed a sensitive andselective EI GC-MSD assay for the identification and quantitation of 3-APB inbiological fluids (section 4.2.). Following positive identification of the compoundin sheep urine and bile samples, a quantitative assessment of the excretion of 3-APB in urine was made (27A & B). The rate of excretion of 3-APB in the urinesuggests rapid formation of the metabolite in the first 60 min following drugadministration. The short apparent elimination half-life of 3-APB (13.5 ± 3.8 min)prompted us to explore whether this metabolite might undergo furtherconjugative/oxidative biotransformation. Both glucuronide and sulfate conjugatesof 3-APB were found in the bile, while only the glucuronide could be detected inthe urine samples, which suggests preferential biliary excretion of sulfate160conjugate in the bile, as was observed with labetalol. It seemed surprising thatfree (unconjugated) 3-APB (MW=149) was present in the bile in relatively highconcentrations 200 ng/mL) while it remained undetectable in plasma,particularly since low molecular weight organic cations (92-236 daltons) havebeen shown to undergo insignificant biliary excretion in several species includingsheep (Abou-El-Makarem et aL, 1967). However, hydrolysis of the conjugates inbile in vivo or under the assay conditions, could not be ruled out. No evidence foroxidative metabolism of 3-APB, including p-hydroxylation (Fig 29), was found.4.4.3. Hemodynamic EffectsThe hemodynamic response obtained in nonpregnant sheep were moreconsistent than that observed in pregnant sheep study (see section 4.3.2.) andsignificant changes were seen in the mean heart rate and arterial pressure valuesafter labetalol administration. The significant increase in femoral blood flowalong with the hypotension and delayed onset of tachycardia suggest peripheralvasodilation and the development of reflex tachycardia. The tachycardia mayfurther suggest that labetalol does not cause significant f3 1 -antagonism in thesheep. Indeed, the adrenoreceptor blocking actions of labetalol have been shownto vary, depending upon the experimental situation and the balance of autonomicinfluences involved. Hypotension and reflex tachycardia with no significantchange in stroke volume were observed in conscious, chronically instrumenteddogs whereas decreases in myocardial contractility and heart rate were seen inanesthetized dogs following intravenous labetalol (Brittain and Levy, 1976). Theexact mechanism involved in peripheral vasodilation, i.e., a-receptor blockade, 13-agonism or direct vasodilation, was subsequently investigated (see sections 4.6.4.161and 4.7.1.). However, active metabolite contribution to the hemodyamic effects oflabetalol remains to be studied.4.4.4. Metabolic EffectsThe significant metabolic effects observed following labetaloladministration in adult nonpregnant ewes include hyperglycemia, lactic acidosis, afall in arterial pCO2 , rise in femoral vein p02 and oxygen content and an increasein oxygen consumption across the hind limb. Similar metabolic effects wereobserved in pregnant sheep (see section 3.4.3.) suggesting that pregnancy does notsignificantly alter the metabolic consequences of labetalol administration. Thedecrease in arterial pCO2 is probably due to acidemia induced hyperventilationwhile the increases in venous oxygen tension and content are likely a sequel to adecrease in percent oxygen extraction across the hind limb, in turn caused bysignificant increase in femoral blood flow. The increase in hind limb oxygenuptake suggests a labetalol induced rise in metabolic rate by carcass components.The initial hind limb uptake of labetalol (Fig 34) is probably a distributionphenomenon. Initial net uptake of glucose may suggest increased consumption ofglucose by the hind limb carcass while the reversal of flux may be due toglycogenolysis and sparing of glucose. The consistent and substantial net outputof lactate underlines the significant role played by the hind limb in the lacticacidosis induced by labetalol. The hind limb lactate output data can beextrapolated to the entire carcass based on carcass weight and the fraction of thecarcass weight represented by the hind limb. In sheep, each hind leg constitutesabout 15% of the total carcass weight, which in turn is roughly 50% of the wholebody weight (Gerrard, 1977). Assuming uniform distribution of cardiac outputwithin the carcass, the net lactate output from the carcass is approximately 41 g162(0.46 mol) or 0.11 g/100 g of carcass. The amount of lactate produced by the totalbody can be calculated as the product of arterial lactate AUC and the clearancevalue for lactate in sheep, based on principles of elimination kinetics (Rowlandand Tozer, 1989). Lactate kinetics in nonpregnant sheep has been studied byReilly and Chandrasena, 1978, following infusion of 14C-lactate to steady-state.Using the lactate clearance estimate from that study in conjunction with thearterial blood lactate AUC from the current investigations, a total lactateproduction of about 22 g (0.24 mol) can be calculated. This estimate assumes thatthe metabolic clearance of lactate is independent of the concentration of lactate(i.e., linearity in lactate kinetics). While both the estimates have severalassumptions and extrapolations involved in their calculations, it would nonethelessappear that the lactate output from the carcass fully accounts for the elevation inlactate concentration seen following labetalol administration. It is possible that theglucose and lactate homeostasis across the hindlimb are interrelated, but studieswith radio-labelled tracer substrates would be required to assess this.The exact mechanism involved in the metabolic effects induced by labetalolare not clear. But there are reasons to suspect the involvement of activemetabolite(s) of labetalol. Firstly, following maternal labetalol administration, thelactic acidosis in the fetal lamb was much more pronounced than that in themother, despite marginal fetal exposure to maternal labetalol (see section 4.3.3.).Secondly, the metabolic effects in both pregnant and nonpregnant sheep suggestpotent 13-agonist activity. The identification of 3-APB in the urine and bilesamples is particularly significant because of the close structural similarity of 3-APB to d-amphetamine (Fig 29), a potent central stimulant. Not much is knownabout the pharmacological effects of 3-APB, but it appears that in terms ofsympathomimetic activity, this compound is at least as potent as d-amphetamine(Larsen, 1938). The pharmacology and toxicology of d-amphetamine are well163documented and in particular, its metabolic effects in both animals and humansfollowing lethal doses have been reported (Zalis and Parmley jr., 1963; Zalis etal., 1967). The toxicity of d-amphetamine had been attributed to the developmentof a generalized hypermetabolic state, which includes hyperventilation, transienthyperglycemia, lactic acidosis, increased skeletal muscle blood flow and increasedoxygen consumption, changes which were observed in this study followinglabetalol administration. Further, coadministration of propranolol offeredprotection against acute d-amphetamine intoxication (Davis et al., 1974), whichsuggests the involvement of 13-agonism in the amphetamine-induced metaboliceffects. The systemic concentrations of d-amphetamine that were associated withthese effects are not clear, but the peak plasma concentrations observed following15-25 mg of d-amphetamine in humans (normal therapeutic dose is 10. mg; Imesand Nickerson, 1965) are reported to be in the range of 1-50 ng/mL (Beckett et al.,1969; Vree and Henderson, 1980). This along with an equal or higher potency of3-APB (Larsen, 1938) may suggest that 3-APB could be pharmacologically activeat doses that do not result in detectable plasma concentrations. Furtherexperiments involving direct administration of 3-APB will be necessary to clearlyelucidate the contribution of this metabolite towards the pharmacodynamics oflabetalol.4.5. Dilevalol Bolus Studies in Nonpregnant SheepOf the four isomers of labetalol, dilevalol, the R, R-isomer, has been themost extensively studied (Chrisp and Goa, 1990) and is also the only isomerwhich we were able to obtain in sufficient quantities to perform animal studies.However, little is known about the metabolic effects of dilevalol, whichcontributes to most of the 13 -adrenergic activities of labetalol (Gold et al., 1982).164This preliminary study described here, was undertaken to compare thehemodynamic and metabolic effects of dilevalol with that of labetalol and also toinvestigate its conjugative and oxidative metabolism in adult nonpregnant sheep.Extensive analysis of the pharmacokinetic and pharmacodynamic observationswas not possible since the data was obtained from only two animals.4.5.1. PharmacokineticsThe terminal elimination half-life and total body clearance of dilevalol inthe two animals (Table 8) were within the range observed with labetalol inpregnant and nonpregnant sheep (Tables 4 and 6, respectively). But the estimatesof volume of distribution (Vd ss and Vdarea) of dilevalol appear to be higher thanthat of labetalol. Clinical pharmacokinetic studies with dilevalol (Kramer et al.,1988; Tenero et al., 1989) have also shown the estimate of volume of distributionof dilevalol (16.6 - 24.6 L/kg) to be higher than the range reported for labetalol(2.5 - 15.7 L/kg) (Goa et al., 1989). The differences in the volume of distributionmay be related, at least in part, to the protein binding differences. In humans,about 75% of dilevalol is bound to plasma proteins while the corresponding figurefor labetalol is only 50% (Donnelly and Macphee, 1991). The fraction of dilevaloldose excreted unchanged in the urine (1.9 and 0.58%) was similar to that oflabetalol (1.61 ± 0.38%).4.5.2. MetabolismNot much is known about the metabolism of dilevalol. The p-glucuronidase and arylsulphatase enzyme incubation studies with the urine andbile samples obtained following dilevalol administration show that the isomer165undergoes both glucuronidation and sulphation (Figs 36 and 37) like labetalol.However, the extent of glucuronidation of dilevalol (4.92 and 4.34%) appears tobe roughly half of that observed in the case of labetalol (11.46 ± 2.82%). Sincelabetalol is glucuronidated at two sites - the phenolic and secondary hydroxygroups (Martin et al., 1976; Niemeijer et al., 1991), it could be that dilevalol isglucuronidated at only one of the sites, i.e., stereoselectivity in the glucuronidationof labetalol isomers. However, such a trend is not apparent in sulphation in thatboth dilevalol and labetalol seem to undergo sulphation to similar extents (about1%) .The pattern of biliary excretion of the conjugates of dilevalol appears to besomewhat different from that of labetalol in that the ratio of glucuronide tosulphate conjugate concentration was about 10 in the case of dilevalol while it wasbetween 0.6 - 3.0 with labetalol. This observation coupled with the urinaryexcretion profiles of the glucuronides of dilevalol and labetalol may suggest thatthe glucuronide conjugate of dilevalol is excreted preferentially in the bile ascompared to the glucuronide conjugate of labetalol.There has been no report to date in the literature concerning the oxidativemetabolism of dilevalol. Results from this study show that 3-APB was formed inadult sheep following dilevalol administration. It is interesting to note that theratios of the 24 h cumulative urinary excretion of 3-APB in E# 105 and E#248following dilevalol (26.87 and 2.69 mg, respectively) and following labetaloladministration (93.55 and 9.17 pg) (Table 7) were roughly equal to about 25%,i.e., the ratio of the doses administered, suggesting the lack of significantstereoselectivity. However, data from at least two other isomers are required toverify this.4.5.3. Hemodynamic Effects166Dilevalol causes hypotension, transient tachycardia and increase in femoralblood flow (Fig 39) in adult nonpregnant sheep similar to that observed withlabetalol. It is interesting to note that the magnitude of these hemodynamicchanges caused by 25 mg dilevalol bolus was similar to that caused by 100 mglabetalol bolus in adult nonpregnant sheep (Fig 30). Thus, it appears that thehemodynamic changes observed following labetalol administration are almostcompletely attributable to one isomer, i.e., dilevalol. But since dilevalol is devoidof any a-adrenergic activity (Gold et al., 1982), it would follow that theappreciable increase in femoral blood flow induced by dilevalol is caused either byits partial 13-agonism or by a direct mechanism. Further, while dilevalol isconsidered to be four times more potent in its 13-blocking activity than labetalol(Sybertz et al., 1981), the appreciable tachycardia observed in this study suggeststhat dilevalol does not exert significant p i -blockade in adult nonpregnant sheep.The progressive increase in heart rate, resulting in a distinct lag time in themaximum effect observed, may indicate that the tachycardia is mediated by areflex or other indirect mechanism and not due to the partial sympathomimeticactivity of dilevalol.4.5.4. Metabolic EffectsNotable hyperglycemia and lactic acidosis were observed followingdilevalol administration (Fig 41). The magnitude of lactic acidosis seen afterdilevalol (59.9 mMh) was similar to that observed with a 100 mg labetalol bolus inpregnant (53.6 ± 1.38 mMh) and nonpregnant sheep (42.7 ± 4.9 mMh) implyingthat the observed metabolic effects of labetalol could be totally attributed to theeffects of dilevalol. However, in contrast to the magnitude of arterial lactate167elevation, the hind limb lactate flux changes after dilevalol appear to be differentfrom that observed after labetalol. Firstly, the lactate flux in the initial 3 hfollowing dilevalol administration shows net uptake by the hind limb (cf. changeswith labetalol, section 4.4.4.). Secondly, the net release of lactate from the hindlimb (2.13 g) was roughly one-third of that observed after labetalol administrationdespite similar arterial lactate AUCs. These differences might indicate that aconsiderable amount of lactate was released in response to dilevalol administrationfrom a site other than hind limb. However, a larger sample size will be required toconfirm these differences.4.6. Labetalol Infusion in Nonpregnant SheepLabetalol is used in both short term and long term clinical management ofhypertension of various etiologies including pre-eclampsia (Goa et al., 1989), butthe metabolic consequences of continuous administration of labetalol have notbeen studied. This is especially important in the light of observations in sheepfollowing a single intravenous bolus administration, which suggest profoundmetabolic disturbances. The two main objectives of this study were (a) to assessthe hemodynamic and metabolic consesquences of continuous infusion of labetalolin adult nonpregnant sheep and (b) to determine the contribution of oc-blockade toperipheral vasodilation, which appears to be largely responsible for thehypotensive action of labetalol in adult nonpregnant sheep.4.6.1. DispositionThe priming bolus dose and the infusion rate of labetalol used in this studywere estimated from pharmacokinetic parameters obtained in the pregnant sheep168(see section 3.3.2.) to obtain average steady-state concentrations reported inclinical situations (about 300 ng/mL) following mutiple oral administrations oflabetalol (Sanders et al., 1980; McNeil et al., 1982; Chung et al., 1986). Theinfusion was continued for 6 h to accommodate NE dose ranging experiments induplicate. The mean steady-state concentration achieved (454.19 ± 111.05ng/mL) appears to be higher than the target concentration of 300 ng/mL, althoughthe difference was not significant (two-tailed, one-sample t-test). The apparenthigh steady-state concentration could be due to altered pharmacolcinetics duringinfusion. The clearance of labetalol following infusion (17.62 ± 3.02 mL/min/kg)was significantly lower than that obtained following a single bolus administration(30.80 ± 3.83 mL/min/kg) (paired t-test). Similar observations have been made inhumans (McNeil et al., 1982; Chauvin et al., 1987). The decreased clearance atsteady-state could be due to saturable first-pass hepatic extraction, a phenomenonnoted with other 13-blockers like propranolol (Evans and Shand, 1973).4.6.2. Hemodynamic EffectsThe hemodynamic effects of continuous infusion of labetalol to steady-statewere qualitatively similar to that observed following a single bolus (see section3.4.4.) and include hypotension, tachycardia and increase in femoral blood flow.However, the hypotension was sustained for up to 10 h post-infusion, with thearterial pressure remaining significantly decreased even after the femoral bloodflow had returned to control values, suggesting the possibility of a differentmechanism in the maintenance of hypotension as opposed to initiation, throughperipheral vasodilation. It is also interesting to note that the tachycardia wastransient and the heart rate gradually returns to baseline values by 4 h into theinfusion despite continued vasodilation and hypotension. It could be that the169reflex tachycardia is strictly an acute response to peripheral vasodilation and henceit could be inferred that long teim/continuous administration of labetalol in sheepwill produce sustained hypotension without causing tachycardia.4.6.3. Metabolic EffectsThe metabolic effects in this study (increase in glucose and lactateconcentrations, increase in venous oxygen content and a trend towards increasedoxygen consumption in the hind limb), which are qualitatively similar to thatobserved following bolus administration, underline the potential of labetalol tocause significant metabolic effects including lactic acidosis at therapeuticconcentrations. The net amount of lactate released from the hind limb (9.2 ± 3.12g) in response to labetalol infusion is roughly 50% higher than that observedfollowing a 100 mg i.v. bolus (see section 4.4.4.). The glucose and lactateconcentrations reached a peak during the infusion and started to decline wellbefore the end of infusion (Fig 48). This may be due to desensitization of the P-receptors (Hausdorf et al., 1990) following continuous administration, aphenomenon observed with continuous infusion of ritodrine, a f3-agonist, in sheep(Bassett et al., 1985).The implications of these results in clinical practice are not known at thisstage, but investigation of the metabolic effects of acute and chronicadministration of labetalol in humans is warranted.4.6.4. Intra-arterial Norepinephrine StudiesThe cornerstone of the observed hemodynamic effects of labetalol in sheephas been the significant increase in femoral blood flow. The peripheral170vasodilatory effect of labetalol has been previously demonstrated in dogs (Baum etal., 1981; Dage and Hsieh, 1980), rats and isolated rabbit portal vein strips(Johnson et al., 1977), but reports about the mechanism involved are conflicting.Suggested mechanisms include 132-agonism (Baum et al., 1981), a-blockade(Sweet et al., 1979) and direct vasodilation (Dage and Hsieh, 1980; Johnson et al.,1977). These differences could be at least in part due to the differences in theexperimental situations involved in the above studies (species employed anddifferences in experimental protocol). The contribution of peripheral a-blockadewas assessed in this study with intra-arterial NE, at doses that do not cause anydetectable systemic effects (an example shown in Fig 52). The transientvasoconstriction caused by NE was dose dependent as well as readily reproducible(Fig 50). However, steady-state concentrations of labetalol did not truncate orshift the dose-response relationship of NE, which implies that labetalol does notproduce any significant a-blockade in sheep. Similar conclusions were reachedfrom studies conducted in dogs by Sybertz et al., 1981.4.7. Intra-arterial Labetalol StudiesThe intra-arterial NE studies clearly show that a-blockade is not involvedin the observed femoral vasodilation, at least in adult sheep. Studies with intra-arterial administration of labetalol were conducted primarily to determine whetherlabetalol has a direct vascular effect.4.7.1. Studies in Adult Nonpregnant SheepThe immediate (Fig 52) and dose dependent vasodilation (Fig 51) causedby intra-arterial injections of labetalol suggest that labetalol has a direct171vasodilatory effect in the adult sheep hind limb. That a-blockade is not theprimary mechanism of vasodilation is also seen in the intra-arterial injectionsfollowing phentolamine infusion (Fig 51 A), which is in agreement with the resultsobtained from intra-arterial NE studies. Propranolol failed to abolish thevasodilatory response (Fig 51 B) suggesting that [3-agonism is not the principalmechansim, at least in sheep. This is in contrast to the observations by Baum etal., 1981, who reported that propranolol attenuated the vasodilatory effect of intra-arterial labetalol in dogs. Intravenous administration of ritodrine, a f3 2-agonist,does not cause any significant change in femoral blood flow in sheep (Rurak,D.W., Kwan, E., Hall, C., Wright, M.R., Szeitz, A., and Axelson, J.E.,unpublished observations). Thus it appears that the vasodilation of labetalol insheep is unrelated to its adrenergic activities. Contribution in part, from otherindirect mechanisms, such as mediation of this effect through an active metabolite(Gal et al., 1988) could not be ruled out and remains to be studied.4.7.2. Studies in Fetal SheepIn striking contrast to the observations in adult sheep, in which labetalolcauses dose-dependent femoral arterial vasodilation, direct administration oflabetalol via the external pudendal epigastric artery in the fetal lamb (0.1 to 500 Ixg) failed to produce any consistent vasodilatory effect. However, these results arein agreement with the lack of significant changes in hind limb blood flowfollowing direct fetal intravenous bolus administration (see section 3.8.4.).The disparity in the hind limb flow responses in adult and fetal sheep tointra-arterial labetalol are probably indicative of some fundamental differences inthe fetal vasculature as compared to the adult. It is unlikely that these differencesare related to receptor differentiation in the fetus, especially [3-receptors, since Van172Petten and Wiles, 1970 have demonstrated that the response to isoproterenol andpropranolol in the ovine fetus in late gestation is similar to that of the ewe in termsof changes in arterial pressure, heart rate and total peripheral resistance. A betterunderstanding of the exact mechanism involved in the labetalol inducedvasodilation in the adult sheep is necessary to identify the differentiating feature inthe fetus.4.8. Fetal Labetalol Bolus StudiesThe dose of labetalol used in the clinical management of pre-eclampsia andsubsequent fetal exposure to this drug varies considerably due to the large inter-individual variability in the hypotensive response to labetalol. Doses of up to 200mg in the form of intravenous infusion (Michael 1986; Ashe et al., 1987) and1200 mg p.o. (Michael, 1982) have been used and the reported cord bloodconcentration of labetalol at the time of delivery ranges from 10-260 ng/mL(Michael, 1979; Rogers et al., 1990). As previously mentioned, the single pointcord blood concentrations do not accurately reflect the in utero fetal exposure tothe drug (Anderson et al., 1980b) and hence it is likely that the human fetus isexposed to concentrations higher than that suggested by the cord bloodconcentrations. The present study was undertaken primarily to assess the effectsof labetalol in the fetus following exposure to clinically relevant concentrationsand to investigate the ability of the fetus to metabolise and eliminate labetalol.Direct fetal administration of labetalol was used because of the restricted placentaltransfer of labetalol following maternal administration (see section 3.3.2.). Thechronically instrumented fetal hind limb was used as a representative of the fetalcarcass to assess whether those tissues, which receive about 40% of fetal173combined ventricular output (Rudolph and Heymann, 1970), could be involved inany labetalol induced fetal lactic acidosis.4.8.1. PharmacokineticsThe disposition of labetalol in the fetal lamb was characterized by extensiveuptake in the tracheal fluid (Fig 54), a phenomenon observed with other aminocompounds like metoclopramide and diphenhydramine (Riggs et al., 1987) andritodrine (Wright et al., 1991b). Fetal tracheal fluid is formed at a rate of 4mL/h in the late gestation ovine fetus primarily by the active transport of chlorideion across the pulmonary epithelium (Harding et al., 1984). While the Na+ andK+ concentrations of tracheal fluid are similar to that of plasma, the chlorideconcentration is much higher and the protein concentration much lower than thatof plasma (Olver and Strang, 1974). In this study, the C TR/CFA ratio for labetalolwas 3.84 ± 0.21 and is similar to that of ritodrine and diphenhydramine (ratio 4),but lower than that of metoclopramide (ratio of about 15).It appears that in the adult, amphiphilic weakly basic amine drugsaccumulate and persist in lung tissue to a greater extent than neutral or basiccompounds (Philpot et al., 1977; Benford and Bridges, 1986; Okumura et al.,1978). While the exact mechanism involved in the uptake of amino drugs in theadult lung is not clear, both passive diffusion and carrier-mediated active transportprocesses across the pulmonary endothelial cells have been suggested (Philpot etal., 1977; Bend et al., 1985). The accumulation of drug in fetal tracheal fluid mayresult, at least in part, from drug uptake by the fetal lung and subsequent releaseinto the tracheal fluid (Rurak et al., 1991). In addition, compounds with 132-agonist activity cause significant decrease in the production of fetal tracheal fluidand the resorption of this fluid across the fetal lung (Warburton et al., 1987 a and174b), which in turn may increase the apparent concentration of the drug in trachealfluid (Wright, 1992). Such a mechanism is possible in the case of labetalol sincefollowing maternal or fetal administration in pregnant sheep as well as innonpregnant sheep, labetalol appears to exert potent I32-agonism (see sections4.3.3., 4.4.4. and 4.8.4.). Administration of amphiphilic amine drugs, whichaccumulate and persist in lung tissue, to animals at high doses or over prolongedperiods of time results in pulmonary phospholiposis. But the exact developmentalsignificance of accumulation of labetalol in fetal tracheal fluid and the consequentexposure of fetal lung to high concentrations of labetalol remains to be explored.The terminal elimination half-life, the body weight-normalised total bodyclearance and the volume of distribution of labetalol in the fetus were significantlyhigher than that observed in the pregnant ewe following maternal administration(see section 3.3.2.), suggesting distinct differences between the fetus and adult inthe distribution and elimination of labetalol. The longer apparent elimination half-life in the fetus (following maternal or fetal administration) could be due toaccumulation of the drug in amniotic and tracheal fluids and possible recirculationfrom these sites, although preferential binding to fetal tissues and proteins cannotbe ruled out. Placental transfer of labetalol from the fetus to the mother was rapidsince peak maternal concentrations were reached almost instantaneously (Fig 54).Although the maternal to fetal plasma AUC concentration was just 3.01 ± 0.2%,the calculated amount of labetalol transferred across the placenta (calculated as theproduct of mean maternal total body clearance obtained from maternal bolusstudies and the total maternal AUC observed in this study) is equal to 1.88 ± 0.7mg, i.e., 47.01 ± 17.5% of the fetal dose. The apparent maternal half-life oflabetalol observed in this study (4.75 ± 0.15 h) was not significantly different fromthe terminal elimination half-life in the fetus (4.35 ± 0.33 h), but significantlyhigher than that observed in the ewe following maternal administration (2.79 ±1750.66 h). This suggests that the apparent maternal elimination half-life of labetalolfollowing a fetal bolus is determined by the continuous input of labetalol from thefetal circulation at a rate equal to the rate of elimination in the fetus. From thetransplacental and nonplacental clearance estimates, it appears that the higher fetaltotal body clearance (CL f = 50.45 ± 1.37 mL/min/kg and CL m from maternalbolus study = 30.8 ± 3.83 mL/min/kg) is due to its nonplacental contribution(CLf„), which is significantly higher than that of the mother (CL„ m) (Table 10).However, unlike the case with most other drugs for which data are available(Rurak et al., 1991), the calculated CLfr, of labetalol (7.27 ± 2.11 mL/min/kg) ismuch lower than CLmp (23.4 ± 8.9 mL/min/kg), the reasons for which are notclear but may involve the fact that some of the data used to calculate the fetal andmaternal clearances (i.e., CLm, FAUC/MAUC) were obtained from a previous studyusing a different group of sheep. Labetalol induced changes inuteroplacental/fetoplacental hemodynamics or differences between maternal andfetal protein binding of labetalol could also be involved. In common with theother drugs that have been studied in pregnant sheep (Rurak et al., 1991), thepercentage of total labetalol clearance due to transplacental clearance is higher inthe fetus (14.4 ± 1.54 %) than in the ewe (3.1 ± 0.2 %). The ovine fetaltransplacental clearance of labetalol (7.27 ± 2.11 mL/min/kg) is less than thatreported for morphine (19 ± 2 mL/min/kg), methadone (168 ± 29 mL/min/kg),metoclopramide (103 ± 13 mL/min/kg), diphenhydramine (124 ± 22 mL/min/kg)and acetaminophen (31 ± 2 mL/min/kg), but higher than that for ritodrine (5 ± 2mL/min/kg) (Rural( et al., 1991). The factors which influence the transplacentaldrug transfer, in general, include the physico-chemical properties of the drug (e.g.,molecular weight, pKa, partition coefficient), maternal and fetal protein binding,maternal and fetal placental blood flows and the extent of placental drugmetabolism (Levy and Hayton, 1973; Reynolds and Knott, 1989; Rurak et al.,1761991). However, protein binding is not likely to be the major factor since theplasma protein binding of labetalol (about 50% in humans; Martin et al., 1976) issimilar to that reported for metoclopramide (49.5% in pregnant ewe; Riggs et al.,1990) and ritodrine (38% in humans; Kuhnert et al., 1986). Among the physico-chemical properties, the molecular weights of labetalol (MW = 329),metoclopramide (MW = 300) and ritodrine (MW = 287) are quite similar,suggesting that molecular size per se is not a critical determinant of the extent ofplacental transfer for these drugs. However, the partition coefficient of thesecompounds do differ appreciably with labetalol having a value of 1.2 (Riley,1981), which is intermediate between that of metoclopramide (12.0; Okumura etal., 1978) and ritodrine (<0.01; Nandakumaran et al., 1982). Thus, the extent ofplacental transfer and transplacental clearance of drugs appear to depend markedlyon lipid solubility of the compounds (Rurak et al., 1991).4.8.2. MetabolismThe results from the enzyme incubation of amniotic fluid samples (Fig 55)shows that labetalol undergoes extensive glucuronidation in the fetal lamb, with amean glucuronide/free drug concentration ratio (over 24 h) of 11.44 ± 2.82. Theconjugates are likely of fetal origin since the ovine placenta has insignificantpermeability to glucuronides (Wang et al., 1985). Although labetalol alsoundergoes sulfate conjugation in adult sheep (see section 3.4.3.1.), no evidence forsulfation was found in the present study and this is similar to results recentlyreported for ritodrine in fetal lamb (Wright et al., 1991b). The amniotic fluidconcentration of glucuronide at 12 h (820.5 ± 77.1 ng/mL) represents roughly 20%of the administered dose (assuming a normal amniotic fluid volume of 1 L) andsince fetal urine is equally divided between amniotic and allantoic fluid cavities177(Wlodek et al., 1988), it is likely that similar concentrations were present in theallantoic fluid as well. Thus, about 40% of the fetal labetalol dose appears to beglucuronidated, which underlines the significance of conjugation in the fetalnonplacental clearance of labetalol. The oxidative metabolite of labetalol, 3-APBcould not be detected in the plasma or amniotic fluid samples. The extremelyshort apparent elimination half-life of the metabolite (estimated to be about 13.5minutes in adult sheep), the low fractional turnover (about 0.05% of labetalol dosein adult sheep) and the possible increase in amniotic fluid volume due to fetallactic acidosis (Powell and Brace, 1991) may explain the absence of 3-APB inamniotic fluid. However, quantitative or even qualitative differences in themetabolism of labetalol between the fetus and adult cannot be ruled out.4.8.3. Hemodynamic EffectsThe observed hemodynamic changes suggest that labetalol does not causeany significant cardiovascular effects in the fetal lamb unlike that in the adultsheep. The significant changes in arterial pressure and heart rate induced bylabetalol in the adult sheep appear to be secondary to the significant peripheralvasodilation (see section 4.4.3.). Thus, the lack of any significant hypotension ortachycardia in the fetal lamb after labetalol administration is likely consequent tothe absence of direct vasodilatory effect of labetalol in the fetus, the reasons forwhich are not clear. The marginal bradycardia in the initial 2 h followed by aprogressive tachycardia could be due to the lactic acidosis since exogenous lacticacid administration has been shown to cause similar changes in ovine fetal heartrate (Bocking et al., 1991).4.8.4. Metabolic Effects178The changes in the blood pH, base excess and lactic acid concentrationsindicate the development of fetal metabolic acidosis, similar to that observedfollowing maternal labetalol administration (see section 3.4.5.). And there wasalso a similar rise in fetal glucose concentration. However, in this case, there wereno significant changes in maternal glucose concentrations, indicating that the fetalhyperglycemia resulted from increased fetal glucose supply/production and/ordecreased utilization. As was discussed previously (p. 151), i3 2-adrenergicagonists like ritodrine, induce hyperglycemia in the ewe and fetus, and in thelatter, at least, this appears to be due to increased glycogenolysis (Warburton etal., 1988). Hence, the labetalol induced fetal hyperglycemia may be via the samemechanism.Concentrations of labetalol, glucose and lactate in HLA and HLV weremonitored to understand the role of skeletal muscles and other carcass constituentsin the perturbation of carbohydrate metabolism induced by labetalol. The netuptake of labetalol seen in the initial 60 min is probably due to rapid tissuedistribution of the drug. While there are no changes in glucose uptake followinglabetalol administration, the lactate flux shows a delayed net release from the hindlimb. The net lactate output from the hind limb (3.85 ± 2.05 g) can beextrapolated to the entire carcass based on fetal carcass and hind limb weights.The ovine fetal hind limb (weight —300 g) represents roughly 10% of total fetalbody weight while the entire carcass constitutes about 70% of body weight(Wilkening et al., 1988). Assuming, for simplicity, that lactate production andutilization is the same throughout the carcass, the net output of lactate from theentire carcass following labetalol administration will be about 27.0 g (0.30 moles).The total lactate produced in the body can also be calculated from the metabolicclearance value of lactate in the fetal lamb and the incremental increase in arterial179lactate AUC following labetalol administration based on basic principles ofelimination kinetics (Rowland and Tozer, 1989) as applied in adult nonpregnantsheep studies (see section 4.4.4.). An estimate of lactate clearance in the fetallamb can be calculated from the data provided by Sparks et al., 1982, and is equalto 35 mL/min/kg. The total amount of lactate produced is calculated as theproduct of the incremental increase in arterial lactate AUC following labetaloladministration (61.1 mMh) and the mean lactate clearance and it amounts to 34.4g (0.38 moles). This calculation assumes that lactate kinetics is linear over theconcentrations involved. From these calculations, it would appear that carcassplays a significant role in the development of lactic acidosis in response to fetallabetalol administration. The reason(s) for the distinct lag time (4-5 h) seen inlactate release from the hind limb (Fig 63) is not clear but may suggest an indirector mediated effect. The observed delay in hind limb flux may also suggest thatlactate release over the initial 4 h occurs from a site other than the hind limb sincearterial lactate concentrations do not exhibit a similar lag time (Fig 60). Asdiscussed earlier (p. 155), one potential site is the placenta. The marginal increaseseen in maternal arterial blood lactate concentration (net increase in AUC equal to5.31 ± 5.28 mMh) is likely due to labetalol induced release of lactate in the motherand/or increased placental lactate production since the ovine placenta is nearlyimpermeable to lactic acid (Sparks et al., 1982).Fetal exposure to labetalol in this study, determined by plasma AUC, isroughly four times higher than that observed following maternal administration oflabetalol (441.76 ± 11.52 vs 111. 86 ± 11.14 p.g*h/L) and the peak plasmaconcentration was about six times higher (207.39 ± 27.66 vs 33.7 ± 5.8 ng/mL).But, the magnitude of changes in the various pharmacodynamic parameters(decrease in fetal arterial blood pH by 0.096 ± 0.015, base excess by 5.7 ± 1.3mEq/L and net increase in lactate AUC by 61.11 ± 15.59 mMh) were not180significantly different from that observed following maternal administration oflabetalol in pregnant sheep (0.100 ± 0.011, 7.0 ± 1.3 mEq/L and 67.8 ± 7.26 mMhrespectively) (two-sample t-test). This pharmacokinetic-phannacodynamicdissociation may suggest that the metabolic effects are actually mediated by activemetabolite(s) and that the governing factor is fetal exposure to the metabolite(s).It could be hypothesized that the fetal exposure to the active metabolite(s)following maternal administration of labetalol is higher than that resulting fromdirect fetal administration. It could be speculated at this stage that 3-APBcontributes to the pharmacological/toxicological effects of labetalol in the fetallamb, since this metabolite is present in adult sheep urine and bile, but furtherexperiments involving direct administration of 3-APB to the ewe and fetal lambare required to clearly elucidate the role of this metabolite in the observedmetabolic effects.1815. SUMMARY AND CONCLUSIONS5.1. Quantitation of Labetalol in Biological FluidsA sensitive assay using microbore high-performance liquid chromatographyand low-dispersion fluorescence detection, has been developed for the quantitationof labetalol in various biological fluids of the pregnant sheep (Yeleswaram et al.,1991). The method represents significant improvement over previously publishedassays in terms of volume of sample required, precision of quantitation andminimum quantitation limit The sensitivity of the method has been optimized byi) the use of microbore column and connections to minimize the deadvolume in the system,ii) reducing the baseline noise with an appropriate cut-off filter,iii) enhancing the signal intensity by using wider excitation and emissionslits andiv) by using a low-dispersion fluorescence cell.The intra-day coefficients of variation were less than 10% in all cases, themean extraction recovery of labetalol from various biological fluids was between70 to 76% and the minimum quantitation limit of the assay was 30 pg injected.5.2. Analysis of 3-amino-l-phenylbutane in Biological FluidsA sensitive and specific gas chromatographic assay with electron-impactionization and mass selective detection has been developed for the identificationand quantitation of 3-amino-l-phenylbutane, an oxidative metabolite of labetalol(Yeleswaram et al., 1992b). This method represents the first assay for thismetabolite involving mass spectrometry with electron-impact ionization. The182samples were extracted with n-hexane, derivatized with heptafluorobutyricanhydride, chromatographed on a cross-linked 5% phenylmethylsilicone stationaryphase and subjected to electron-impact ionization. Identification of the metabolitewas accomplished with an authentic standard. Quantitation was performed byselectively monitoring two ions of the derivative (m/z=345 and 132), including themolecular ion (m/z=345). The coefficients of intra-sample variation were lessthan 12% in all cases, the extraction recovery was between 98 to 103% and theminimum limit of quantitation of the assay was 2 pg (amount injected).5.3. Maternal Bolus Administration of LabetalolIn this study, labetalol was administered as a 100 mg i.v. bolus to the eweand serial samples were obtained from maternal and fetal artery as well asamniotic and tracheal fluid to characterize the disposition of labetalol. Maternaland fetal cardiovascular and blood gas parameters were monitored before and forup to 24 h following labetalol administration. Fetal exposure to labetalol,determined as fetal/maternal arterial plasma AUC ratio, was found to be 14.4 ±1.5% (n=8). Labetalol persisted in the amniotic and tracheal fluid compartmentsfor up to 24 h. The apparent elimination half-life of the drug in the fetus (3.70.5 h) was significantly higher than that in the mother (2.8 0.7 h). There wereno significant changes in maternal or fetal cardiovascular parameters (heart rateand mean arterial pressure), but marked changes were seen in the fetal blood gasparameters. The fetal blood pH, base excess and oxygen content decreasedsignificantly. Only minimal changes were observed in the maternal blood pH andbase excess. The significant blood gas changes in the initial experiments led to themonitoring of maternal and fetal glucose and lactic acid concentrations insubsequent experiments, as markers of carbohydrate metabolism. The results183indicate significant, albeit reversible, hyperglycemia and lactic acidosis in both thefetus and ewe. The metabolic effects produced by labetalol resembled that of apotent P2-agonist. Also, the elevation in lactic acid concentration was morepronounced as well as lasted longer in the fetus than in the mother despitemarginal placental transfer of labetalol, suggesting an indirect or mediated effect.5.4. Labetalol Bolus in Adult Nonpregnant SheepThis study was conducted to assess the contribution of carcass in generaland in particular the hind limb, to the development of lactic acidosis in response tolabetalol administration and to investigate the metabolism of labetalol. Followinga 100 mg intravenous bolus, labetalol, glucose and lactate concentrations infemoral artery and vein, along with femoral blood flow, heart rate and arterialpressure were monitored. Urine and bile samples were obtained for the study ofconjugative and oxidative metabolism. A significant increase in femoral bloodflow, hypotension, reflex tachycardia, hyperglycemia, lactic acidosis with asubstantial output of lactate (6.25 ± 1.35 g) from the hind limb were observed.Glucuronide and sulfate conjugates of labetalol were present in the urine and bilesamples. The cumulative urinary excretion of labetalol as unchanged drug,glucuronide and sulfate were found to be 1.61 ± 0.38, 11.46 ± 2.83 and 1.47 ±0.74% of the dose respectively. The oxidative metabolite, 3-amino-l-phenylbutane, was identified in urine and bile samples, with a cumulative urinaryexcretion equivalent to 0.044 ± 0.016% of labetalol dose. Glucuronide conjugatesof the metabolite in urine samples and glucuronide and sulfate conjugates of themetabolite in bile samples were also present. The structural similarity of 3-amino-1-phenylbutane and amphetamine on the one hand, and the close similaritybetween the reported metabolic effects of amphetamine and that observed in sheepfollowing labetalol administration on the other hand, may suggest a possibleinvolvement of the metabolite in the mediation of labetalol pharmacodynamics.However, experiments involving direct administration of the metabolite arenecessary to establish the cause-effect relationship unequivocally.5.5. Dilevalol Bolus in Adult Nonpregnant SheepThe pharmacokinetics and pharmacodynamics of dilevalol, the RR-isomerof labetalol, were studied in two animals. With the exception of the apparentvolume of distribution, which was higher, the estimates of pharmacokineticparameters of dilevalol were within the respective ranges observed with labetalol.The cardiovascular and metabolic effects of dilevalol in adult non-pregnant sheepwere also similar to those of labetalol, suggesting that the observedpharmacological effects of labetalol in sheep were actually elicited by one of thefour isomers, i.e., dilevalol. But, further experiments are required to confirm this.5.6. Labetalol Infusion to Steady-state in Adult Nonpregnant SheepThe hypothesis that the mechanism of vasodilation caused by labetalol is a-blockade was examined with intra-arterial injections of norepinephrine, before andduring continuous infusion of labetalol to steady-state. No significant shift in theintra-arterial norepinephrine dose-response relationship was caused by steady-stateconcentrations of labetalol, thus implying that a-blockade is not the primarymechanism involved in the vasodilation caused by labetalol.1845.7. Intra-arterial Administration of Labetalol185In the adult sheep hind limb, intra-arterial injections of labetalol rangingfrom 0.1 pg to 1.0 mg, resulted in spontaneous and dose-dependent vasodilation.Further, administration of phentolamine, an a-adrenoceptor blocker, orpropranolol, a 13-adrenoceptor blocker, did not cause any significant  change in thevasodilatory response to intra-arterial administration of labetalol. These resultssuggest that labetalol has direct vasodilatory effect and that a-blockade or p-agonism are not involved in the vasodilation. In the fetal lamb hind limb, infra-arterial administration of labetalol failed to produce any consistent, dose-dependent change in hind limb blood flow. This observation may suggest that thefetal vasculature is different from that of the adult.5.8. Labetalol Bolus Administration in the Fetal LambThe disposition, metabolism and pharmacodynamics of labetalol in the fetallamb were studied following exposure to clinically relevant labetalolconcentrations, achieved with a 4 mg direct intravenous fetal bolus administration.The total body clearance (50.45 ± 1.37 mL/min/kg), elimination half-life (4.35 ±0.33 h) and apparent volume of distribution at steady-state (14.28 ± 0.95 L/kg) oflabetalol in the fetal lamb were significantly higher than the corresponding valuesin the ewe following a 100 mg bolus. The ratio of maternal to fetal plasma AUCwas equal to 0.031 ± 0.002. The calculated fetal transplacental clearance was 7.27± 2.11 mL/min/kg while the fetal nonplacental clearance was 43.18 ± 3.72mL/min/kg. Glucuronide conjugates were present in the amniotic fluid samplesand the amount of the conjugates at 12 h represents about 20% of the administereddose. The oxidative metabolite, 3-amino-l-phenylbutane, could not be identifiedin fetal plasma or amniotic fluid samples. No significant changes in fetal heartrate, arterial pressure and hind limb blood flow, but significant increases in arterial186and hind limb venous blood glucose and lactate concentrations were observed.The net output of lactate from the fetal hind limb was equal to 3.85 ± 2.05 g. Themagnitude of fetal lactic acidosis (incremental increase in the area under thearterial lactate concentration-time curve) (61.11 ± 15.59 mMh) was notsignificantly different from that observed following maternal administration (67.8± 7.26 mMh), despite a roughly four-fold higher fetal exposure to labetalol (asdetermined from area under the plasma labetalol concentration-time curve values).The following conclusions could be reached from these studies:(1) Labetalol undergoes rapid but limited placental transfer in the pregnantsheep.(2) Following maternal or fetal administration, labetalol causes significantmetabolic effects in the fetus, including lactic acidosis. But the magnitude of themetabolic effects were not proportional to the extent of fetal exposure to labetalol.(3) The hind limb in particular and the carcass in general, appear to releaselactic acid in response to labetalol administration in both the adult and fetal sheep.(4) The hypotensive effect of labetalol in adult nonpregnant sheep appearsto be caused primarily by a significant peripheral vasodilation. The vasodilationseems to be through a direct effect of labetalol and it does not involve a-blockadeor P-agonism. In contrast, labetalol does not cause any significant vasodilation orhypotension in the fetal lamb following similar doses.(5) In adult nonpregnant sheep, labetalol undergoes glucuronidation,sulfation as well as oxidative metabolism to form 3-amino-l-phenylbutane, astructural analog of amphetamine. It is possible that this metabolite contributes tothe overall pharmacology/toxicology of labetalol. 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