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Ritrodrine: analysis, comparative maternal and fetal pharmacokinetics and pharmacodynamics in sheep Wright, Matthew Rowland 1992

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RITODRINE: ANALYSIS, COMPARATIVE MATERNALAND FETAL PHARMACOKINETICS ANDPHARMACODYNAMICS IN SHEEPByMATTHEW R. WRIGHTB. Sc. ( Pharm. )M.Sc., TheThe University of British Columbia, 1985.University of British Columbia, 1987.A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Faculty of Pharmaceutical Sciences)(Division of Biopharmaceutics and Pharmaceutics)We accept this thesis as conformingto the standard required.THE UNIVERSITY OF BRITISH COLUMBIAJanuary, 1992ØMATTHEW R. WRIGHTIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of cJ 1’C.-..LThe University of British ColumbiaVancouver, CanadaDate Si_j 2, i’i z.DE.6 (2/88)•I1ABSTRACTRitodrine is a48-adrenoreceptor agonist that is used clinically inEurope, Australia and the United States as a tocolytic. Despite therelatively widespread pharmacological investigation of ritodrine in bothhuman and animal species there is very little pharmacokinetic data. Infact, there are no estimates of any fetal pharmacokinetic parameter inany species.This thesis reports: 1) the development of a capillary gaschromatographic assay method using electron capture detection for thequantitation of ritodrine from the various biological fluids of thechronically instrumented pregnant sheep, ii) the placental and fetalnon-placental clearances of ritodrine during constant rate fetalintravenous ritodrine infusion, iii) the disposition of ritodrine insome of the fluid compartments of the fetal lamb during and afterconstant rate fetal intravenous ritodrine infusion, iv) the maternaland fetal pharniacokinetics of ritodrine following maternal intravenousadministration, v) the maternal and fetal pharmacological responses toritodrine following maternal intravenous administration, vi) the fetalpharmacokinetics of ritodrine following fetal intravenous bolusadministration, vii) the fetal pharmacological responses to ritodrinefollowing fetal intravenous bolus administration, viii) the effects ofa fetal intravenous ritodrine bolus administration on the regional bloodflow distribution of the anaesthetised fetal lamb, ix) the comparativematernal and fetal pharmacokinetics of ritodrine in the sheep, x) thecomparative pharmacological responsiveness of the ewe and fetus toritodrine.111In the course of this work a gas-chromatographic assay method usingelectron capture detection was developed for the quantitation ofritodrine. The developed method was sufficiently sensitive andselective to allow the determination of ritodrine from a variety ofbiological fluids from the chronically instrumented pregnant sheep. Theminimum quantifiable concentration of ritodrine was 2.5 ng/mL from a 500uL fluid sample. This method was used to quantitate ritodrine from allthe fluid samples obtained during the pharmacokinetic studies.The developed assay method has been applied to the study of theclearance and disposition of ritodrine in the fetal lamb during andafter constant rate fetal intravenous infusion. Ritodrine appears tohave only very limited clearance across the sheep placenta (Clv 9.2 ± 2mL/min/kg; mean ± s.e.ni.). Furthermore, ritodrine could not be detectedin either maternal arterial or uterine venous plasma. There is,however, significant fetal non-placental clearance of ritodrine (Clf052.8 ± 8.0 mL/min/kg). At least part of this clearance appears to bedue to fetal drug metabolism, as evidenced by the accumulation ofglucuronide conjugates of ritodrine in the amniotic fluid. Ritodrinewas also shown to accumulate in the amniotic and fetal tracheal fluidsand persist after fetal arterial plasma ritodrine concentrations becameundetectable.The pharmacokinetics and pharmacodynamics of ritodrine were studiedin 10 pregnant ewes following the administration of a 50 mg maternalintravenous bolus dose. In all but one animal a biexponential declinein maternal arterial plasma concentrations was observed. Fetal arterialconcentrations were very low and peaked at 10 minutes followingmaternal drug administration. Fetal drug exposure as determined by theivfetal to maternal AUC ratio was very low (zO.03). Maternal total bodyclearance averaged 24 mL/min/kg. The volumes of distribution determinedwere relatively large: Vdc, 1.6 ± 0.3; Vdarea, 14.3 ± 3.5; Vd55, 10.0 ±1.9 L/kg. The maternal distributional half-life (a) averaged 0.5 hourswhile the elimination half-life () averaged 8.7 hours. The apparentfetal elimination half-life averaged 2.3 hours. Ritodrine accumulatedin both the amniotic (peak concentrations 1O ng/mL) and fetal tracheal(peak concentrations 2O ng/mL) fluids and persisted in these fluidslonger than in the fetal arterial plasma. As in the fetal infusionstudies, glucuronide conjugates of ritodrine could be detected in theamniotic fluid.A number of physiological effects were observed in the motherfollowing the maternal bolus administration. Following drugadministration there was an approximate doubling of maternal heart ratewith no apparent change in maternal arterial blood pressure. Maternalheart rate declined to control levels over a period of 4 - 6 hours. Theewe developed a metabolic acidosis which was in part mediated by lacticacid. This acidosis was partially compensated by respiratory alkalosis.There was also a decrease in uterine contractures during the first 6hours following drug administration. There were no apparent changes infetal cardiovascular or metabolic parameters during these studies.There was a direct relationship between the maternal arterialritodrine concentration and the maternal heart rate. This relationshipwas modeled using an Emax equation. Direct relationships could not befound between the maternal arterial plasma ritodrine concentrations andthe maternal metabolic effects.VThe pharmacokinetic and pharmacodynamics of ritodrine were studiedin 5 fetuses and ewes following a 2 mg fetal intravenous bolusadministration. In all animals a biexponential decline in the fetalarterial plasma concentration was observed. A mean fetal total bodyclearance of 31.7 ± 10.0 mL/min/kg (mean ± s.e.m.) was noted which wasnot significantly different from the maternal total body clearancecalculated in the maternal bolus studies but significantly less than thefetal total body clearance calculated in the fetal infusion studies. Itis felt that the effects of the profound fetal acidemia and hypoxiacreated in the fetal bolus studies may account for this difference.Similar to the maternal bolus studies the fetal volumes of distributionwere very large (Vc, 1.6 ± 0.8; Vdarea, 8.7 ± 1.8; Vd55, 8.5 ± 2.6 L/kg)but were not significantly different from the comparable maternalvolumes of distribution. The fetal distributional (cr) half-life was 0.2± 0.1 hours and the fetal elimination (40) half-life was 2.9 ± 0.7 hours(mean ± s.e.m.). The fetal distributional half-life was notsignificantly different from that of the mother. The fetal elimination(/3) half-life was significantly less than that of the mother but was notsignificantly different from the apparent fetal half-life determined inthe maternal bolus studies. Ritodrine could not be determined in eitherthe maternal arterial or uterine venous plasma further confirming thelack of placental transfer of ritodrine across the ovine placenta.There were no significant differences between either the dose (on amg/kg basis), AUC, AUMC, or concentrations achieved in the fetus duringthe fetal bolus studies and the ewe in the maternal bolus studies.In the fetus the cardiovascular responses were characterized by afall in the fetal mean arterial pressure (by z13 mm Hg) without theviimmediate sustained tachycardia seen in the maternal bolus studies. Incontrast a slow progressive increase in fetal heart rate (to 4O beatsper minute above control) was noted. The fetal tachycardia persistedfor approximately 4 hours following the fetal bolus. The fetus alsodeveloped metabolic acidosis in response to the ritodrine dose. Similarto the maternal bolus studies this acidosis was largely mediated bylactic acid. The acidosis in the fetus was much more intense andpersisted for approximately twice as long as the maternal metabolicacidosis in the maternal bolus studies. There was also a significantincrease in the fetal arterial glucose concentration during thesestudies. There was no apparent effect of the fetal ritodrine bolus onthe rate of uterine contractures. There were no apparent maternaleffects following the fetal bolus administration.There were no direct relationships between the fetal arterialplasma concentration of ritodrine and the fetal physiological effectsobserved.Two fetuses were acutely prepared for study using radioactivemicrospheres to examine fetal regional blood flows following fetal bolus(2 mg) administration. Both tachycardia and hypotension were seenfollowing the fetal bolus dose which may be the results of the acutenature of the preparation, the halothane anaesthesia, and thehypercapneic/acidotic condition of these fetuses. The fetal arterialritodrine concentrations determined in these studies were not differentfrom those seen in the fetal bolus studies. The fetal ritodrine bolusresulted in decreases in umbilical, muscle, bone, gastrointestinal tractand renal blood flows, suggesting a decrease in fetal cardiac output,but increases in flow to the perirenal brown fat and skin.viiAbstract iiTable of Contents viiList of Figures XList of Tables xvList of Abbreviations xviAcknowledgements xxiDedication xxii1. INTRODUCTION 11.1 Ritodrine 11.1.1. Ritodrine Pharmacology and Clinical Use 11.1.2. Methods of Analysis of Ritodrine 81.1.3. Ritodrine Pharmacokinetics 91.1.4. Factors Affecting Placental Transfer 121.1.5. Placental Transfer of Ritodrine 141.2. Rationale and Specific Aims 151.2.1. Rationale 151.2.2. Specific Aims 172. EXPERIMENTAL 192.1.192.1.1. 192.1.2. 212.1.3. 232.1.4.242.1.5. 252.1.6. 262.1.7. 262.1.8.272.1.8.1. 272.1.8.2. 272.1.8.3. 282.1.8.4. 292.1.8.5 292.1.8.6302.2. 312.2.1. 312.2.2.312.2.3. 322.2.4. 332.2.5. 34Table of ContentsDevelopment of a Capillary GC-ECD Method forRitodrine.MaterialsPreparation of Stock SolutionsInstrumentation and EquipmentOptimization of Derivatization of Ritodrine andInternal StandardsOptimization of GC ConditionsOptimization of Drug ExtractionGas-Chromatography Mass-SpectrometryProcedures for Ritodrine Analysis fromBiological FluidsGC Operating ConditionsDrug Extraction from Biological FluidsPreparation of Calibration CurveRecovery StudiesValidation of Assay MethodAnalysis of Glucuronide and Sulphate Conjugatesof RitodrineStandard Procedures for Animal ExperimentsAnimal HandlingSurgical Preparation for ChronicExperimentationRecording ProceduresBlood Gas AnalysisBlood Glucose Measurementviii2.2.6. Blood Lactate Measurement 342.3. Experimental Protocols 352.3.1. Fetal Infusion Studies 352.3.2. Maternal Bolus Studies 362.3.3. Fetal Bolus Studies 372.3.4. Acute Microsphere Studies followingFetal Bolus Administration 392.4. Data Analysis 402.4.1. Calculation of Pharmacokinetic Parameters 402.4.2. Fetal Arterial Pressure, Fetal Heart Rate,Amniotic Pressure 432.4.3. Fetal Breathing Movements and UterineActivity 432.4.4. Regional Blood Flows 442.4.5. Computer Fitting 462.4.5.1. Pharmacokinetic Fitting 462.4.5.2. Pharmacodynamic Fitting 472.4.6. Statistical Analyses 473. RESULTS 483.1. Development of a Capillary GC-ECD Method forRitodrine. 483.1.1. Optimization of Derivatization 483.1.2. Optimization of GC Conditions 533.1.3. Optimization of Drug Extraction 613.1.4. Gas-Chromatography Mass-Spectronietry 613.1.5. GC Operating Conditions 653.1.6. Drug Extraction from biological Fluids 693.1.7. Recovery Studies 693.1.8. Calibration Curve 733.1.9. Validation of Assay method 733.2. Fetal Infusion Studies 733.3. Maternal Bolus Studies 833.4. Fetal Bolus Studies 1063.5. Acute Microsphere Studies following Fetal BolusAdministration 1284. DISCUSSION 1404.1. Development of GC-ECD Method of Analysis 1404.2. Fetal Infusion Studies 1454.2.1. Placental Clearance of Ritodrine 1464.2.2. Non-placental Clearance and Disposition ofRitodrine 151ix4.3. Maternal Bolus Studies 1564.3.1. Pharmacokinetics and Placental Transfer. 1564.3.2. Cardiovascular Effects 1594.3.3. Metabolic Effects 1614.3.4. Uterine Contractures 1644.4. Fetal Bolus Studies 1654.4.1. Pharmacokinetics 1664.4.2. Ontogeny of 8-adrenoreceptor responses in thefetal lamb. 1724.4.3. Cardiovascular Effects 1744.4.4. Metabolic Effects 1794.4.5 Uterine Contractures 1824.4.6. Microsphere Studies 1824.5. Comparative Maternal and Fetal Kinetics andEffects 1864.5.1. Kinetics 1864.5.2. Pharmacological Effects 1894.5.3. Indices of Fetal Drug Exposure 1914.5.6. In viva Pharmacokinetic/PharmacodynamicModelling 1935. SUMMARY AND CONCLUSIONS 2016. REFERENCES 207xList of FiguresFigure 1Chemical structure of ritodrine 1Figure 2Representative polygraph recording 45Figure 3Chromatograms of ritodrine derivatized withTFAA and HFBA. 49Figure 4Detector response vs volume of HFBA added. 50Figure 5Detector response vs volume of TEA added. 51Figure 6Time Course of Derivatization. 52Figure 7Chromatogram of HFBA ritodrine derivative(indicated by the arrow) evaporated under N2 andreconstituted in 1 mL toluene. (Non-optimized GCconditions). 54Figure 8Chroniatogram of HFBA derivatized ritodrine(indicated by the arrow) neutralized with 2 ml 0.067M phosphate buffer (pH 6). (Non-optimized GCconditions). 55Figure 9Effect of injection port temperature. 56Figure 10Effect of purge time on detector response. 57Figure 11van Deemter curve for HFBA derivatized ritodrineusing H2 as carrier gas. 59Figure 12Effect of detector temperature on detectorresponse. 60Figure 13Relative recovery of ritodrine from organicsolvents (a) and mixtures of ethyl acetate tolueneisopropanol (b). Extracted from aqueous standardsusing pH 9.5 carbonate buffer. 62xiFigure 14Effect of the addition of TEA to the extractionsolvent (ethyl acetate). 63Figure 15Effect of extraction time on the relative recoveryof ritodrine. Extracted from aqueous standardsusing pH 9.5 carbonate buffer. 64Figure 16Total ion chromatograni (top) and negativechemical ionization spectrum ofTFAA derivatized ritodrine. 66Figure 17Total ion chromatogram, A) electron impact,B) negative chemical and C) positive chemicalionization spectra of HFBA derivatized ritodrine. 67Figure 17 (cont’d)Total ion chromatogram, A) electron impact,B) negative chemical and C) positive chemicalionization spectra of HFBA derivatized ritodrine. 68Figure 18Extraction and derivatization procedure. 70Figure 19Representative chromatograms from variousbiological fluids obtained from pregnant sheep. 71Figure 20Absolute recovery of ritodrine from maternalarterial plasma (n=3, mean ± s.d.). 72Figure 21Comparison of ritodrine concentrations determinedby HPLC (n=2) (Gross et al., 1987) vs thosedetermined by the developed GC method (n=2). 75Figure 22Ritodrine concentrations (mean ± s.e.ni.) in thefetal fluids of the 8 hour infusion group (n=6). 79Figure 23Ritodrine concentrations (mean ± s.e.m.) inthe fetal fluids of the 12 hourinfusion group (n=3). 80Figure 24Ritodrine concentrations (mean ± s.e.ni.) inthe fetal fluids of the 24 hourinfusion group (n=4). 81xiiFigure 25Ritodrine concentrations (mean ± s.e.m.) infetal arterial plasma, amniotic fluid and amnioticfluid concentrations of ritodrine glucuronide(s),expressed as liberated ritodrine for the 24 hourinfusion group (n=3). 82Figure 26Representative plot of ritodrine concentration vstime in the ewe and fetus following a 50 mgmaternal intravenous bolus. 87Figure 27Relationships between maternal arterial ritodrineconcentration and time, maternal heart rate andtime and maternal heart rate and maternal arterialritodrine concentration (points connected intemporal order) in a representative ewe. 91Figure 28Representative polygraph recording following 50 mgmaternal bolus ritodrine administration, indicatedby arrow. 94Figure 29Effects on maternal blood gas parameters followinga 50 mg maternal intravenous bolus dose ofritodrine 96Figure 30Effects on fetal blood gas parametersfollowing a 50 mg maternal intravenous bolusdose of ritodrine 97Figure 31Representative plots of maternal arterial ritodrineconcentration vs time, maternal arterial wholeblood base excess vs time, and maternal arteriallactate concentration vs time, following a 50 mgmaternal intravenous bolus administration. 98Figure 32Mean lactate and glucose concentrations vs timefollowing a 50 mg maternal intravenous bolusadministration. (n = 4) 100Figure 33Mean lactate and glucose concentrations vs timefollowing a 15 mL maternal intravenous normalsaline dose. (n = 2) 101xiiiFigure 34Estimated uterine lactate and glucose fluxesfollowing saline (upper graph) and maternalritodrine bolus (50 mg) administration (lowergraph). 103Figure 35.Uterine contractures per six hour time periodfollowing maternal ritodrine (upper graph) orsaline (lower graph) administration. 105Figure 36Representative plot of ritodrine concentration vstime in the ewe and fetus following a 2 mg fetalintravenous bolus. 109Figure 37Polygraph tracing following a 2 mg fetal bolusadministration of ritodrine (a - low speedrecording, b- high speed recording). 116Figure 38Fetal heart rate recorded each minute for 10minutes prior to and 10 minutes after 2 mgritodrine fetal bolus (ewes 617, 201, 105 and 338)or control saline injection (ewes 201 and 617). 117Figure 39Average fetal heart rate (mean ± s.e.m.) over 30minute intervals before and after 2 mg ritodrinebolus (closed circles) and control saline injection(open circles). 118Figure 40Fetal arterial pressure recorded each minute for 10minutes prior to and 10 minutes after 2 mgritodrine fetal bolus (ewes 617, 201, 105 and 338)or control saline injection (ewes 201 and 617). 119Figure 41Effects on fetal blood gas parameters following a 2mg fetal intravenous bolus dose of ritodrine. 121Figure 42Effects on maternal blood gas parametersfollowing a 2 nig fetal intravenous bolusdose of ritodrine. 122Figure 43Representative plots of fetal arterial ritodrineconcentration vs time, fetal arterial whole bloodbase excess vs time, and fetal arterial lactateconcentrations vs time, following a 2 mg fetalintravenous bolus administration. 125xivFigure 44Mean lactate and glucose concentrations vs timefollowing a 2 mg fetal intravenous bolusadministration. Inset figure shows the glucoseconcentrations on an expanded scale. (n = 5) 126Figure 45Mean lactate and glucose concentrations vs timefollowing a 0.7 mL fetal intravenous normal salinedose. (n = 2) 127Figure 46Fetal fluxes of glucose and lactate followingsaline (upper graph) and 2mg fetal ritodrine bolus(lower graph) administration. 129Figure 47Uterine contractures per six hour time periodsfollowing fetal bolus ritodrine (2 mg)administration. 131Figure 48Representative polygraph tracing during acutemicrosphere study. 135xvList of TablesTable 1 Calibration curve from sheep plasma 74Table 2 Experimental details for fetal infusions. 76Table 3 Calculated ritodrine clearances 84Table 4 Experimental details for the maternal bolusexperiments 85Table 5 Pharmacokinetic parameters from maternalbolus experiments 88Table 6 Mean concentrations of ritodrine and itsglucuronide(s) (ng/mL expressed as liberatedritodrine) in the amniotic fluid during thematernal bolus experiments (n = 4). 90Table 7 Pharmacodynamic parameters describing theeffect of ritodrine on maternal heart rate 92Table 8 Maternal and fetal blood gas parameters priorto maternal bolus administration 95Table 9 Uterine contractures 104Table 10 Experimental details for the fetal bolusexperiments 107Table 11 Pharmacokinetic parameters from the fetalbolus experiments 111Table 12 Mean concentrations of ritodrine and itsglucuronide(s) (ng/mL expressed as liberatedritodrine) in the amniotic fluid during thefetal bolus experiments (n = 5). 113Table 13 Maternal and fetal blood gas values prior tofetal intravenous bolus administration. 120Table 14 Uterine contractures 130Table 15 Experimental details for the acute microsphereexperiments. 132Table 16 Fetal femoral arterial blood gas resultsfrom acute microsphere experiments. 134Table 17 Regional flows in the fetus following aritodrine 2 mg fetal bolus administration. 137Table 17 (continued) 138Table 17 (continued) 139xviABBREVIATIONSACTH adrenocorticotrophic hormoneADH antiduretic hormoneAL allantoic fluidAM amniotic fluidAMN amniotic pressureamu atomic mass unitsANOVA analysis of varianceApp. apparentAUC area under the plasma concentrationvs time curveAUMC area under the first moment curveAUTOAN decision making pharmacokineticmodelling program[A-Vj arterio-venous concentrationdifferenceBA gestational age at deliveryB.E. whole blood base excessBW birthweightbpm beats per minutedegrees CelsiusCfa fetal arterial plasma concentrationCum umbilical venous plasmaconcentrati onCL(t) blood lactate concentration attime = tClf0 fetal non-placental clearancexviiCliactate lactate clearanceClmo maternal non-placental clearanceCl placental clearanceCltb total body clearanceCNS central nervous systemCp55 apparent steady-state plasmaconcentrati onCp(t) plasma concentration at time = tC.V. coefficient of variationcAMP cyclic adenosine monophosphatedL deciliterECD electron capture detectionEC50 plasma concentration requiredto elicit a 50% ofmaximal responseEl electron impact ionizationEmax maximal pharmacological effecteV electron voltsFA fetal arterialFAP fetal arterial pressureFHR fetal heart ratefu fraction unboundg gramg acceleration due to gravityGA gestational ageGC gas chromatographyh hourxviiiH2 hydrogen gasHb haemoglobinHETP height equivalent to a theoreticalplateHFBA heptafluorobutyric anhydrideHPLC high performance liquidchromatographyHz HertzI number of radioactive counts1.0. internal diameterI.M. intramuscularmt Tr P integrated tracheal pressureI.U. international unitsi.v. intravenouskg kilograminfusion rateL literlamdan terminal elimination rate constantM molarMA maternal arterialMANOVA multivariate analysis of varianceMAP maternal arterial pressuremeq milliequivalentsMFA mean fetal arterial pressureMHR maternal heart ratemg milligrammm minutexixmL milliliterMLCK myosin light chain kinasemm Hg millimeters of mercurymmol millimolarMS mass spectrometrym/z mass to charge ration number of theoretical platesn number of samplesn shape factorN normalN2 nitrogen gasNAD nicotinamide adenine dinucleotideNCI negative ion chemical ionizationN.D. not detectableng nanogramNONLIN pharmacokinetic modelling program02 sat. oxygen saturationPCI positive ion chemical ionizationpCO2 partial pressure of carbon dioxidepg picogrampH negative logarithm (base 10) of thehydrogen ion concentrationP02 partial pressure of oxygenp.s.i. pounds per square inchPTFE p0] ytetrafl uoroethyl eneQ blood flow rateum umbilical venous blood flow ratexxr correlation coefficientRIA radloimmunoassayr.p.m. revolutions per minutes.d. standard deviationsec seconds.e.m. standard error of the meana2 varianceSmax sigmoid Emax modelt1/2 half-lifeTEA triethylamineTFAA trifluoroacetic anhydrideTR tracheal fluidTr F tracheal flow rateTRIS tris(hydroxymethyl )aminomethaneTr P tracheal pressureUT uterine veinUV umbilical venousV volume of the central compartmentVd55 steady-state volume of distributionVdfl apparent volume of distributionmicrolitermicrometerxxiAcknowl edgementsI would like to sincerely thank my supervisors Dr. James E. Axelsonand Dr. Dan W. Rurak for their guidance, friendship and interest inthese studies. I thank you both also for your encouragement andunderstanding during many of the initial difficulties in conducting thiswork. Finally, I am extremely grateful for the examples of constructivecriticism, attention to detail and deeper understanding that you bothhave set for me.Thanks to my committee members, Dr. Frank Abbott, Dr. GrahamMcMorland and Dr. John Sinclair for their interest and guidance over theperiod of these studies. Many thanks also to Dr. K. Wayne Riggs for hisencouragement and many helpful discussions during my time as a graduatestudent. Also, I very much appreciate the assistance of Dr. TimStratton in the statistical analysis of many of these experiments.I also express my sincere appreciation to Ms. Marlene van derWeyde, Ms. Sandy Taylor, Ms. Caroline Hall and Mr. Eddie Kwan for theirassistance with the experimental studies and for their friendship.Many thanks to my colleagues in the laboratory, Mr. KrishnaswamyYeleswaram, Mr. George Tonn, Ms. Jing Wang, Ms. Judit Orbay and Dr.Andras Szeitz for their friendship and many constructive suggestions.Last, but not least, my special thanks to my wife, Monika, for hermany suggestions, discussions, patience and encouragement during thecourse of this work.This project was made possible by grants received form the MedicalResearch Council of Canada and the University of British Columbia. Ialso gratefully acknowledge the receipt of my studentship from theMedical Research Council of Canada.DedicationThis thesis is dedicated to my wife, Monika, and to my parents, Derekand Virginia Wright, for their love, support and patience.xxii11. INTRODUCTION1.1 RitodrineRitodrine hydrochloride (erythro-p-hydroxy-a-[1-[(p-hydroxyphenethyl )amino]ethyl ]benzyl alcohol hydrochloride) (YutoparR) isa potent 13-sympathomimetic drug with its predominant actions upon the82-adrenoreceptors of the uterus (Barden et al., 1980). Ritodrine wasthe first tocolytic approved by the Food and Drug Administration in theUnited States (1980). In addition ritodrine has been registered for usethroughout Europe (1970-1979) and Australia (1978) (Barden et al.,1980).HCH HFigure 1: Chemical structure of ritodrine1.1.1. Ritodrine Pharmacology and Clinical UseA. Mechanism of ActionRitodrine and other B-mimetic agents that have been used toterminate premature labor (salbutamol, terbutaline, fenoterol) elicituterine relaxation through interaction with the t32-adrenoreceptors ofthe myometrial cells (Chez, 1984; Nuwayhid and Rajabi, 1987). The2commonly proposed mechanism is that stimulation of the myometrial f2-adrenoreceptor results in the formation of cyclic adenosinemonophosphate (cAMP) by the enzyme adenylate cyclase (Chez, 1984). Therise in intracellular cAMP is generally believed to result in myometrialrelaxation via a number of mechanisms including 1) activation of cAMP-dependent kinase (protein kinase A) and subsequent phosphorylation ofmyosin light chain kinase (MLCK) which inactivates MLCK, 2) reduction ofintracellular Ca2+ via stimulation of calcium uptake and extrusion, 3)cell membrane hyperpolarization, 4) decreasing the permeability of gapjunctions (Chez, 1984; Nuwayhid and Rajabi, 1987). Although these cAMPdependent mechanisms have been widely proposed and accepted (Huszar andRoberts 1982), there is evidence to suggest that fl-agonists can causemyometrial relaxation at concentrations below those required to increaseintracellular cAMP levels (Diamond, 1990). In spite of the large bodyof literature describing the role of cAMP in myometrial relaxation, theavailable evidence is compatible with the existence of both a cAMP-dependent and a cAMP-independent mechanism (Carsten and Miller, 1987;Diamond, 1990). It has been suggested that the cAMP-independentmechanism may be of primary importance (Diamond, 1990).B. Clinical Use and EfficacyPreterm birth is the most significant contributor to infantmorbidity and mortality in North America (McCormick, 1985; Creasy andResnik, 1989). In spite of the availability of tocolytic agents andmore aggressive approaches to the management of tocolysis, the overallincidence of preterm delivery has remained constant (King et al., 1988).3Although there are large numbers of clinical trials examining theefficacy of fl-mimetics as tocolytics, there has been little definitiveproof of efficacy. The interpretation of these clinical trials has beencomplicated by the relatively poor quality of scientific evidenceobtained during these studies (King et al., 1985). In a thoughtfulanalysis of the relatively well conducted trials, King et al. (1988)have demonstrated that the main benefit of tocolytic therapy is to allowfor the short-term delay of delivery such that other therapies may beinstituted to improve the chance of survival of the premature infant(e.g. corticosteroid administration). Regardless of existence ofdefinitive proof of fl-mimetic efficacy, or evidence for the safety andbenefit of tocolytic therapy these agents are standard therapy in thecountries where they are available for use as tocolytics (Keirse, 1984;Diamond, 1990).C. Maternal and Fetal EffectsIn terms of its therapeutic use the most relevant pharmacologicaleffect of ritodrine is its ability, in common with other fl2-agonists, torelax uterine smooth muscle (Barden et al., 1980). Silmes and Creasy(1976) have demonstrated that this uterine relaxant property wasprevented by the co-administration of propranolol. Subsequently it hasbeen demonstrated in sheep (Casper and Lye, 1986; Lye et al., 1987; Wardet al., 1988; Caritis et al., 1991) that continuous administration butnot pulsatile dosing of ritodrine results in the desensitization of theuterus and the reemergence of uterine contractions. These observationsare consistent with observations of desensitization of other tissues4following continuous stimulation by other $-agonists (Hausdorf et al.,1990).Due to the ubiquitous distribution of J3-adrenergic receptors andthe only relative selectivity of l2-agonists forfl2-adrenoreceptors,ritodrine and other -agonists have effects in a wide variety of organsand physiological systems (Chez, 1984; Nuwayhid and Rajabi, 1987). Manyof these effects are evident with ritodrine and observed in both motherand fetus.D. Maternal EffectsA number of studies have reported on the maternal effects ofritodrine administration in both humans and animals (Ferguson et al.,1989; Wilkins et al., 1988; Witter et al., 1988; Essed et al., 1987;Bassett et al., 1985; Main et al., 1985; Hankins et al., 1983; Caritiset al., 1983; Lippert et al., 1980; Schreyer et a7., 1980; Siimes andCreasy, 1980; Barden et al., 1980; Erkkola et a7., 1979; Siimes andCreasy, 1979; Siimes and Creasy, 1976; Kleinhout and Veth, 1975;).Although it has not been reviewed separately, ritodrine appears to havesimilar pharmacological properties to other2—adrenoreceptor agonists(Nuwayhid and Rajabi, 1987; Chez, 1984; Benedetti, 1983).Ritodrine and other fl-adrenergic agonists may act both directly orindirectly on the cardiovascular system to produce tachycardia,increased cardiac output, decreased arterial pressure and redistributionof cardiac output in both humans and animals (Ferguson et a7., 1989;Caritis et al., 1985; Caritis et al., 1983; Lippert et al., 1980; Siimesand Creasy, 1979; Siimes and Creasy, 1976; Kleinhout and Veth, 1975).5Uteroplacental blood flow has been reported to increase in pregnanciescomplicated by preeclampsia, hypertension or growth retardation butremains unchanged in normal pregnancies (Brettes et al., 1976; Sunio etal., 1978; Brotenek and Brotanek, 1981; Suonlo, 1982; Joupilla et al.,1985).A relatively few patients complain of hyperventilation, dyspnea,chest pain and tightness; however, the most serious pulmonarycomplication is the development of pulmonary edema in 5% of patients(Pisani and Rosenow, 1989).Ritodrine has also been shown to increase the activity of thematernal renin-angiotensin-aldosterone system (Erkkola et al., 1979) andthis along with increased ADH secretion, and changes in glomerularfiltration rate common to other -agonists (Nuwayhid and Rajabi, 1987)may predispose patients to fluid overload.In both humans (Schreyer et al., 1980) and sheep (Siimes andCreasy, 1980) short term maternal infusion of ritodrine causeshyperglycemia. Intravenous ritodrine therapy has been demonstrated tosignificantly impair glucose tolerance tests in pregnant women in theearly third trimester (Main et al.,, 1980). Both lactic- andketoacidosis have been reported but occur rarely in patients (Richardset al., 1983; Lenz et al., 1979). Essed et al. (1987) have shown thatritodrine treatment increased the thyroid hormone concentration in 17women treated for tocolysis.In studies involving either short (1 hour) or long-term infusions(48-120 hours) of ritodrine to pregnant ewes, increases in maternallactate, glucose, insulin, and pyruvate plasma concentrations have been6observed (Siimes and Creasy, 1980; Basset et al., 1985). In addition,Basset et al. (1985) have shown that fetal glucose, insulin and pyruvateplasma concentrations parallel the changes in maternal metaboliteconcentrations. Interestingly these metabolic parameters, with theexception of lactate, returned to control values in the ewe within 72hours despite continued ritodrine infusion (Basset et al., 1985). Therewere no statistically significant changes in fetal blood gas parametersduring the maternal infusions (Basset et al., 1985). Block et al.(1989) have recently suggested decreases in both fetal umbilical venousand descending aortic oxygen content during maternal ritodrine infusion.E. Fetal EffectsFetal effects of ritodrine have been reported in both clinicalstudies (human) and experimental studies (animal) following eithermaternal or fetal administration.Most human fetuses appear to be able to tolerate the effects ofritodrine exposure, however, there have been many reports of fetal andneonatal complications following maternal ritodrine therapy (Katz andSeeds, 1989). These complications include hypoglycemia, cardiacdysrhythmias, thickening of the intraventricular septum, and myocardialinfarction (Kazzi et al., 1987; Katz and Seeds, 1989).In studies where ritodrine has been infused to the fetus in thechronically instrumented sheep model a variety of effects have beenobserved. During fetal ritodrine infusion, fetal heart rate increasesand this tachycardia persists for 12 to 24 hours (Siimes et al., 1978;Basset et al., 1989). The fetal heart rate, however, returns to control7values within 48 hours despite continued ritodrine infusion (Basset etal., 1989). Short term ritodrine infusion (1 hour) to the fetus resultsin a redistribution of fetal cardiac output although cardiac outputitself remained unchanged (Siimes et al., 1978; Siimes and Creasy,1979). Umbilical blood flow has been shown to be unaffected whileuterine blood flow has been shown to decrease during fetal ritodrineinfusion (Ehrenkranz et al., 1976; Siimes and Creasy, 1979).Fetal ritodrine infusion for 24 hours results in a 6.9 folddecrease in tracheal fluid flow, an increase in lung surfactant content,and improvement in lung stability (Warburton et al., 1987a). Further,accelerated pulmonary glycogen depletion and increased availability ofsurfactant phospholipid to the alveoli have also been reported(Warburton et al., 1987b).Long-term (48-80 hours) fetal infusion of ritodrine causes similarfetal metabolic effects, in terms of increases in lactate, glucose, andinsulin plasma concentrations (Basset et al., 1989), to those observedin the ewe during maternal administration. Again the metabolicparameters returned to control values within 72 hours despite continuedinfusion (Basset et al., 1989). In these experiments (Basset et al.,1989) there was a significant fall in fetal arterial°2 (by 5.1 mm Hg)as has been reported elsewhere (Warburton et al., 1987a). Recently thishypoxemia has been demonstrated to be the result of an increase in fetaloxygen consumption that is not compensated for by increases in eitheruterine or umbilical oxygen delivery (van der Weyde et al., 1990, 1991;van der Weyde, 1990). These metabolic effects may put the fetus atrisk, particularly in situations where fetal oxygen delivery is alreadyreduced.8With the exception of the studies by van der Weyde (van der Weydeet al., 1990, 1991; van der Weyde, 1990), fetal ritodrine concentrationshave not been determined in any other study.1.1.2. Methods of Analysis of RitodrineThomas et al. (1982) reported the development of a radioimmunoassayfor ritodrine. While this method displays reasonable sensitivity ( 0.1ng/mL from 100 itL serum samples) the precision of measurement is poor(C.V. > 10% below 2.5 ng/mL) and the kinetic parameters obtained instudies using this assay have shown large intersubject variability (Linet al., 1984). This variability has been ascribed to cross-reactivityof the anti-sera with endogenous substances (Lin et al., 1984).Lin et al. (1984) and Kuhnert et al. (1983) developed high-performance liquid chromatography (HPLC) methods which employelectrochemical detection of the oxidation of the phenolic hydroxylgroups of ritodrine. Although both methods show acceptable selectivity,the sample volume required in both instances ( 1 ml) is impractical forrepetitive sampling where sample volume is limited.Gross et al. (1987) have recently reported an HPLC method using thenative fluorescence of ritodrine. Although the selectivity of thismethod is excellent, it requires a relatively large sample volume forextraction (>1 ml plasma) and a small reconstitution volume (100 ,iL)which does not allow for multiple injections of the same sample.91.1.3. Ritodrine PharmacokineticsIn spite of the relatively large numbers of pharmacological andclinical studies involving ritodrine, there are few reports of ritodrinepharmacokinetics in either humans or in animal species. One of thecontributing factors to the lack of pharmacokinetic information is theextremely low plasma concentrations of -agonists that occur following“therapeutic” doses (Morgan, 1990).Ritodrine appears to be rapidly absorbed following oraladministration (Gandar et al., 1980; Caritis et al., 1989). Followingadministration of 10 mg ritodrine hydrochloride to 4 healthy malevolunteers peak plasma concentrations were observed between 20 and 40minutes (Gandar et al., 1980). Following oral administration of 20 mgritodrine hydrochloride, peak concentrations were observed at 30 (13.4ng/mL) and 58 minutes (10.8 ng/mL) in non-pregnant and pregnant women,respectively (Caritis et al., 1989). In this study, the time to peakwas significantly different between pregnant and non-pregnantvolunteers, however, the peak concentrations were not different (Caritiset al., 1989). Additionally, there was no apparent effect of food oneither the time to peak or peak concentration in pregnant women (Caritiset al., 1989). Gander et al. (1980) have estimated the bioavailabilityof ritodrine as 31 %. Also, dose linearity of AUC was reported in non-pregnant female humans following oral doses of 10, 20 and 30 mg ofritodrine (Caritis et al., 1989).In all studies examining ritodrine kinetics, very low plasmaconcentrations of ritodrine have been determined which indicates a largevolume of distribution for this drug. Estimates of the volumes of10distribution in humans show that not only is the volume very large butalso extremely variable (V 0.55 ± 0.5 1/kg (mean ± s.d.) pregnant women(Caritis et al., 1990)) (Vdp 6.95 ± 3.95 1/kg (mean ± s.d.) pregnantwomen (Caritis et al., 1990)) (Vd55 4.38 ± 2.89 1/kg, 2.8 ± 0.5 1/kgpregnant women (Caritis et al., 1990; Gross et al.. 1987); Vd 4.8 ±1.7 1/kg non-pregnant women (Gross et al. . 1987)). Also, in rhesusmonkeys following intravenous infusion, the volume of distribution(Vd55) is larger in non-pregnant animals (4.75 ± 0.9 1/kg; mean ± s.d.)than in pregnant animals (1.99 ± 0.94 1/kg; mean ± s.d.) (Caritis etal., 1988). Thus, it would be expected that the affinity of ritodrinefor tissue is quite large.The protein binding of ritodrine in humans is quite small with thefree fraction (fu) being 0.64, 0.68, and 0.75 in non-pregnant women,pregnant women and umbilical venous plasma, respectively (Gross andBrown, 1985).Following ritodrine administration, the AUC values determined arerelatively small suggesting a large total body clearance. In pregnantwomen, estimates of total body clearance are 2.2 ± 0.3 and 1.94 ± 0.71L/min (mean ± s.d. (Caritis et a!., 1990; Gross et a!.. 1987)) (30 ± 0.6and 31 ± 1.1 mL/min/kg respectively) and 1.4 ± 0.2 1/mm (26 ± 0.5mL/min/kg) in non-pregnant women (Gross et a!.. 1987). In rhesusmonkeys, non-pregnant animals were shown to have a higher total bodyclearance than pregnant animals (12.7 1/h vs. 9.6 1/h) (Caritis et al.,1988). At least some of the total body clearance may be accounted forby sulphate and glucuronide conjugation and subsequent renal excretionas well as renal elimination of intact drug (Brashear et al., 1988;Kuhnert et al., 1986). Recently, the structure of the glucuronides and11suiphates present in maternal urine have been determined (Brashear etal., 1990). In humans ritodrine forms two glucuronide and two sulphateconjugates, each with a single conjugating group at a terminal phenolicposition (Brashear et al., 1990). In women treated with the drug 45 %,35 % and 11 % of the dose are excreted in maternal urine as thesulphate conjugates, glucuronide conjugates and free drug, respectively,whereas in their newborns 66 %, 20 % and 10 % of the total ritodrineexcreted in the neonatal urine were in these forms, respectively(Brashear et al., 1988). A similar pattern of metabolism and excretionwas observed in urine obtained from a single baboon treated withritodrine (Borrisud et al., 1985). In samples obtained from in vitrorat liver perfusion experiment, both glucuronide and sulphate conjugatescould be identified in the bile (Borrisud et al., 1985). However, inthe urine of rats orally gavaged with ritodrine, only free drug andglucuronide conjugates were observed (Borrisud et al., 1985). Thestructure of the conjugates in the animal studies have not beenelucidated.Following the attainment of peak drug levels ritodrine declines bymultiexponential kinetics and a terminal elimination half-life of from 2- 17 hours has been reported (Caritis et al., 1990, 1989; Gross et al.,1987; Kuhnert et al., 1986). The large variability in the reportedhalf-lives may stem from the extremely low plasma concentrationsencountered in these studies. This may result in an underestimation ofthe true elimination half-life (Gibaldi and Weintraub, 1971). In rhesusmonkeys the terminal elimination half-life has been reported as 1.8 ±0.4 hours and 3.3 ± 0.4 hours in pregnant and non-pregnant animals,12respectively (mean ± s.d.) and are significantly different from oneanother (Caritis et al., 1988).1.1.4. Factors Affecting Placental TransferThe movement of substances (e.g. nutrients, metabolic wasteproducts) between mother and fetus is of obvious significance to fetalmaturation and survival. In general, substances may move across theplacenta by active transport, facilitated diffusion, passive diffusion,pinocytosis or via filtration and bulk flow (Reynolds and Knott, 1989).It is well recognized that of these processes, passive diffusion is themost important mechanism of drug movement between mother and fetus(Rurak et al., 1991). Despite the anatomic differences between theplacentas of various species, the limiting layer of fetal tissue isessentially a continuous lipid membrane (Reynolds and Knott, 1989) andmovement across this membrane is governed by the rules of simple lipiddiffusion (Reynolds and Knott, 1989).Virtually all drugs will cross the placenta, although the rate andextent of transfer is dependent on the physicochemical properties of thedrug. The factors governing the rate and extent of placental transferand which ultimately determine the drug concentration in the mother andfetus have been thoroughly reviewed (Rurak et al., 1991; Reynolds andKnott, 1989; Besunder et al., 1988; [fill and Abramson, 1988; Mitani etal., 1987; Mucklow, 1986; Symonds, 1985; Mihaly and Morgan, 1984; Yurth,1982; Levy, 1981; Waddell and Marlowe, 1981; Green et al., 1979; Levyand Hayton, 1973; Meschia et al., 1967).13The factors determining the drug concentration in the mother,placental transfer and drug concentration in the fetus may be summarizedas follows:1) Maternal plasma concentration of drug is dependent on: a) the dose,route and formulation of drug, b) distribution, which in turn isdependent on hemodynamics, tissue affinity, and protein binding, c) therate and extent of drug metabolism, d) the rate of excretion of intactdrug and metabolites.2) Placental transfer is controlled by a) the physicochemicalproperties of the drug including, polarity, molecular weight, degree ofionization, and protein binding, b) the concentration gradient of freedrug across the placenta, c) the magnitude and relative orientation offetal and maternal blood flows, d) the maternal-fetal pH gradient, e)the stage of placental development, f) placental drug metabolism, g)differences in the extent and/or affinity of maternal and fetal proteinbinding, h) the nature of drug administration to the mother (viz. i.v.bolus, oral, i.v. infusion, single vs. multiple vs. continuous dosing).3) Fetal drug concentrations are influenced by a) distribution, whichis in turn affected by the fetal circulation geometry and shunting,tissue affinity, and protein binding, b) hepatic metabolism, c) renalexcretion, d) umbilical blood flow, e) dilution of the drug in the fetalcirculation resulting in a delay in equilibrium between fetal tissuesand blood, f) recirculation of drug from amniotic and tracheal fluids.141.1.5. Placental Transfer of RitodrineIt is clear from studies examining maternal and umbilical cordblood samples at delivery that ritodrine crosses the human placenta(Fujimoto et al., 1990; Fujimoto et al., 1986; Gross et al., 1985; vanLierde et al., 1982; Gandar et al., 1980). Furthermore, ritodrine hasbeen detected in both the plasma and urine of neonates in whose mothersritodrine tocolysis had failed (Brashear et al., 1988; Kuhnert et al.,1986). Umbilical cord blood ritodrine concentrations range from 5 - 282ng/mL with fetal to maternal concentration ratios ranging from O.3 -1.3 (Fujimoto et al., 1990; Fujimoto et al., 1986; Gross et al., 1985;van Lierde et al., 1982; Gandar et al., 1980). The wide range of fetalto maternal concentration ratios is probably reflective of the fact thatthe value of this measure is entirely dependent on the time of samplingrelative to the time of maternal drug administration (Anderson et al.,1980c). The concentration of ritodrine in amniotic fluid has also beendetermined and is apparently higher than that in umbilical cord blood atdelivery (Fujimoto et al., 1990; Fujimoto et al., 1986; van Lierde etal., 1982). This observation may suggest that the human fetus has someability to eliminate ritodrine. None of these studies, however, canestimate the full magnitude of exposure of the human fetus to ritodrinefollowing maternal administration.The sheep is the only animal species in which placental transfer ofritodrine has been examined in two studies, both of which indicatedminimal maternal/fetal transfer of the drug (Kleinhout and Veth, 1975;Fujimoto et al., 1984). However, the true extent of fetal ritodrineexposure cannot be estimated from these studies as they are essentially15single point determinations with neither the mother or fetus maintainedat steady-state. Further indirect evidence for the limited placentaltransfer of ritodrine in sheep can be inferred from the studies ofBassett et al. (1989, 1985) where pharmacological effects due toritodrine were not observed on the side of the placenta opposite that towhich the drug was administered. The apparent difference between thehuman and sheep in terms of the extent of placental transfer ofritodrine probably relates to the structural differences between theplacentae (Faber and Thornberg, 1983).1.2. Rationale and Specific Aims1.2.1. RationaleAlthough a number of studies have examined the pharmacologicaleffects of ritodrine, very few studies have measured ritodrineconcentrations in fetal or maternal blood. The assay methods availablefor ritodrine are impractical for use due to the large biological fluidsample volumes required and in some cases the unacceptable variabilityof the assay method. Although several studies have examined thepharmacokinetics of ritodrine in pregnant women, there is no descriptionof the pharruacokinetic behavior of this drug in the fetus. It is clearfrom single point concentration determinations in maternal and umbilicalcord blood that the human fetus is exposed to ritodrine in utero.Further, it is clear from many clinical reports that newborns which havebeen exposed to ritodrine exhibit physiological effects characteristicof fl-agonist stimulation. The extent of the true exposure, both16pharmacokinetic and pharmacological, of the human fetus to ritodrinefollowing maternal administration cannot, however, be determined fromthese studies.The chronically instrumented sheep has been extremely useful inaddressing both the effects and kinetics of drugs in the in uteromammalian fetus (Van Petten et al., 1978). While the fetal lamb is moremature at birth than the human infant, both species show similar fetalbehavioral states (fetal breathing movements, limb and body movements,electrocortical activity patterns, eye movements) towards the end ofgestation (de Vries et al., 1982; de Vries et al., 1985; Nijhuis et al.,1982) as well as similar fetal biochemistry and physiology (Comline andSilver, 1974; Szeto et al., 1978; van Petten et al., 1978).In addition, a number of studies in sheep have demonstrated avariety of pharmacological effects in the fetus following fetalritodrine administration, some of which may be detrimental to fetalsurvival. None of these studies have measured ritodrine concentrationsin fetal blood. Preliminary reports (Kleinhout and Veth, 1975; Fujimotoet al., 1984) are equivocal as to the extent of placental transfer ofritodrine across the ovine placenta. The physicochemical nature ofritodrine, however, would suggest that placental transfer of ritodrinewould be limited in the pregnant sheep.The use of the sheep model, despite the structuraldifferences between the ovine and human placentas, would allow for thedetailed study of the fetal and maternal pharmacokinetics andpharmacodynamics of ritodrine in a chronic preparation. The fetalsampling, both pharmacokinetic and pharmacological, that can beaccomplished in this model cannot be duplicated in either humans, due to17ethical and technical constraints, or in small animal models due to thelimited blood volumes of both mother and fetus. Despite the apparentdifferences between the human and sheep in terms of placental transferof ritodrine, study in the chronically instrumented pregnant sheep isrelevant since fetal drug administration is possible and thecardiovascular, hemodynamic, biochemical and behavioral responses aresimilar between the two species. Moreover, if in fact there is limitedmaternal/fetal ritodrine transfer in sheep, this would allow forindependent assessment of the pharmacodynamics of the drug in the eweand fetus without the confounding effects that could result from thepresence of drug in the other component of the maternal-fetal unit.1.2.2. Specific Aims1) To develop a gas-chromatographic assay method for ritodrine toallow quantitation of ritodrine from the biological fluidsobtained from the chronically instrumented sheep.2) To determine the extent of total body clearance of ritodrine fromthe sheep fetus and the contributions of placental and non-placental clearances during constant rate intravenous fetalinfusion.3) To examine the distribution of ritodrine within the fluidcompartments of the fetal lamb during and after constant rateintravenous fetal infusion.4) To study the pharmacokinetics of ritodrine in the ewe and fetusfollowing maternal intravenous bolus administration.185) To examine the pharmacological effects of ritodrine in the ewe andfetus following maternal intravenous bolus administration.6) To examine the pharmacokinetics of ritodrine in the fetus and ewefollowing fetal intravenous bolus administration.7) To examine the pharmacological effects of ritodrine in the fetusand ewe following fetal intravenous bolus administration.8) To examine the relative sensitivity of pharmacological responses ofthe ewe and fetus to ritodrine in vivo.192. EXPERIMENTAL2.1. Development of a Capillary GC-ECD Method for Ritodrine.2.1.1. MaterialsRitodrine hydrochloride (erythro-p-hydroxy-cr[l-(phydroxyphenethyl )amino]ethyl ]benzyl alcohol hydrochloride) (>98.5%pure) (Lot #056553); ritodrine free base (erythro-p-hydroxy-[1-(p-hydroxyphenethyl)amino]ethyl]benzyl alcohol) (>95 % pure) (Lot # WHK8O9-001);isoxuprine hydrochloride (>99.6% pure ) (Lot #056766) (Duphar B.V.,Weesp, The Netherlands); propafenone hydrochloride; 5-hydroxypropafenone hydrochloride (Knoll Pharmaceuticals Canada Inc., Markham,Ontario); nylidrin hydrochloride; 1-phenylephrine hydrochloride (SigmaChemical Co., St. Louis, MO); ritodrine injectable 10 mg/mL (YutoparR)(Bristol-Myers Squibb Canada, Montreal, Quebec); thiopental sodiuminjectable 1 g /vial (PentothaiR); sodium chloride for injection USP(Abbott Laboratories, Montreal, Quebec); ampicillin injectable 250mg/vial (PenbritinR), and halothane (FluothaneR, Ayerst Laboratories,Montreal, Quebec); gentamicin sulphate injectable 40 mg/vial(GaramycinR, Schering Canada Inc., Pointe Claire, Quebec); atropinesulphate injection 0.6 mg/mL (Glaxo Laboratories, Montreal, Quebec);heparin 1000 U/mL (Organon Canada Ltd., West 1-lill, Ontario); 85Sr, 57Cr,l25, 46Sc and ‘41Ce (0.5 mCurie/5 mL) labelled microspheres 15 jtmdiameter (3M Company, St. Paul, MN).Heptafluorobutyric anhydride, trifluoroacetic anhydride, andtriethylamine (Sequanal Grade) (Pierce Chemical Co., Rockford, IL); ACS20reagent grade potassium dihydrogen orthophosphate (monobasic), disodiumhydrogen orthophosphate (dibasic), potassium carbonate, sodiumbicarbonate, ammonium carbonate, sodium acetate, andtris(hydroxymethyl)aminomethane (TRIS free base) (BDH Chemicals,Toronto, Ontario). -glucuronidase/arylsulfatase (GlusulaseR, Type H-2,Helix pomatia, G-8076), arylsulfatase (from Aerobacter aerogenes, 5-1629), fl-glucuronidase (GlucuraseR, from bovine liver, G-4882), glucosestandard solution 1 mg/mL, zinc sulphate 0.3 N, barium hydroxide 0.3 N,o-dianisidine dihdyrochloride, PGO capsules (500 I.U. glucose oxidase,100 Purpurogallin units peroxidase and buffer salts), lactatedehydrogenase, glycine buffer, nicotinamide adenine dinucleotide (NAD),and antipyrine (2,3-dimethyl-1-phenyl-3-pyrazolin-5-1-phenazone) (SigmaChemical Co., St. Louis, MO); sodium nitrate, zinc sulphateheptahydrate, and ACS reagent grade sodium hydroxide pellets (FisherScientific Co, Fair Lawn, NJ).Toluene, ethyl acetate, hexane, benzene, and dichioromethane (alldistilled in glass) (pesticide grade) and acetonitrile (HPLC grade)(Caledon Labs., Georgetown, Ontario); methanol (OmnisolvR, BDH, Toronto,Canada); perchloric acid, glacial acetic acid, and sulphuric acid(Fisher Scientific Co, Fair Lawn, NJ). Deionized, high-purity water wasproduced on-site by reverse osmosis using a Milli_QR water system(Millipore, Mississauga, Ontario).Nitrogen USP (Union Carbide Canada Ltd., Toronto, Ontario); ultrahigh purity (UHP) hydrogen and argon/methane (95:5) (Matheson GasProducts Canada Ltd., Edmonton, Alberta).Needles and plastic disposable Luer_LokR syringes for drugadministration and sample collection (Becton-Dickinson Canada,21Mississauga, Ontario); heparinized blood gas syringes (Marquest MedicalProducts Inc., Englewood, CO); heparinized VacutainerR tubes (VacutainerSystems, Rutherford, NJ); 15 mL PyrexR disposable culture tubes (CorningGlass works, Corning, NY); polytetrafluoroethylene (PTFE) lined screwcaps (Canlab, Vancouver, British Columbia); polystyrene tubes (EvergreenScientific International Inc., Los Angeles, CA); silicone rubber tubingfor catheter preparation (Dow Corning, Midland, MI); PTFE-coatedstainless steel wire for electrode preparation (Cooper Corp.,Chatsworth, CA).2.1.2. Preparation of Stock SolutionsRitodrine hydrochloride was accurately weighed and dissolved indeionized water using serial dilution to a final concentration of 100ng/mL (22.25 mg of ritodrine hydrochloride is equivalent to 20.00 mgritodrine free base).The internal standard, 5-hydroxy propafenone hydrochloride, wasaccurately weighed and dissolved in deionized distilled water usingserial dilution to a final concentration of 200 ng/mL (equivalent tobase). The solutions of ritodrine and 5-hydroxy propafenone were storedat 4°C for up to three months without evidence of degradation.During the development of the GC-ECD assay method several othercompounds, namely isoxuprine hydrochloride, nylidrin hydrochloride,propafenone hydrochloride and 1-phenylephrine, were examined forpossible use as suitable internal standards. Following accurateweighing, solutions of each were prepared in deionized water using22serial dilution to a final concentration of 100 ng/mL (equivalent tobase).Triethylamine 0.0125 M in toluene was prepared by dilutingtriethylamine with toluene. Four or five pellets of sodium hydroxidewere added to the solution.Carbonate buffer 1 M (p1-I 9.5) was prepared by dissolving potassiumcarbonate and sodium bicarbonate in deionized distilled water. The pHwas adjusted to 9.5 using either concentrated hydrochloric acid or 5 Nsodium hydroxide. The pH of the stock solution was determined dailyprior to use.Phosphate buffer 0.067 M (p1-I 6.0) was prepared by combining 90 mL0.067 M potassium dihydrogen phosphate and 10 mL 0.067 M disodiumhydrogen phosphate.Sodium acetate 0.2 M was prepared by dissolving sodium acetate indeionized water and adjusted to a final pH of 5.0 using glacial aceticacid.Tris(hydroxymethyl)aminomethane (TRIS) 0.05 M was prepared bydissolving TRIS free base in deionized water and adjusted to a final pHof 7.5 using 1 N hydrochloric acid.0-dianisidine dihydrochloride (50 mg/vial) was reconstituted in 20mL deionized, distilled water. One PGO capsule was dissolved in 100 mLdistilled deionized water to which 1.6 mL of the o-dianisidinedihydrochloride solution had been added. These solutions were stablefor up to one month at 4°C.Nicotinamide adenine dinucleotide (NAD) (50 mg) was dissolved in 20mL deionized, distilled water to which 10 mL glycine buffer and 0.5 mL23lactate dehydrogenase were added. This solution was stored at 4°C forup to two days.Antipyrine was dissolved in deionized, distilled water and usingserial dilution provided a final concentration of 5 g/mL.2.1.3. Instrumentation and EquipmentGas Chromatography: A Model 5890 Hewlett-Packard gas chromatographequipped with a 63Ni electron capture detector (ECD), split-splitlesscapillary inlet system, and an HP 9000 series GC workstation (HewlettPackard Co., Avondale, PA); 25 m x 0.31 mm I.D. crosslinked 5 %phenylmethylsilicone (Ultra 2) 0.52 im film thickness fused-silicacapillary column (Hewlett-Packard Co., Avondale, PA); 2 mm and 4 mmborosilicate glass inlet liners (Hewlett-Packard Co., Avondale, PA);ThermogreenR LB-2 silicone rubber septa (Supelco, Bellafonte, PA).High-Performance Liquid Chromatography: A Hewlett-Packard Model 1090liquid chromatograph equipped with an HP Model 1040 diode-array UVdetector and an HP 1046A programmable fluorescence detector and Model310 HP (9000 series) workstation; a 250 mm x 2.1 mm I.D. ODS (5 dp)column (all Hewlett-Packard Co., Avondale, PA).Gas-Chromatography Mass-Spectrometry: A Model 5987A quadrupole GC-MS(Hewlett-Packard Co., Avondale, PA); HP Model 2623 terminal with an HPModel 1000 computer (Hewlett-Packard Co., Avondale, PA); 25 m x 0.31 mmI.D. OV-1701 (0.25 m film thickness) (Quadrex Sci., New Haven, CT).General Experimental: vortex-type mixer (VortexGenieR, FisherScientific Industries, Springfield, MA); incubation oven (IsotempR model350, Fisher Scientific Industries, Springfield, MA); IEC model 2Kcentrifuge (Damon/IEC division, Needham Hts., MA); rotating-type mixer24(LabquakeR model 415-110, Lab Industries, Berkeley, CA); infusion pump(Harvard model 944, Harvard Apparatus, Millis, MA); IL 1306 pH/blood gasanalyzer (Allied Instrumentation Laboratory, Milan,); HemoximeterR(Radiometer, Copenhagen); Beckman R612 or Sensormedics R712 DynographRecorder (Sensormedics, Anaheim, CA); Strain-gauge manometers (StathamModel P23Db, Gould mc, Oxnard, CA); cardiotachometers (Model 9857,Sensormedics, Anaheim, CA); Gould DTX disposable transducers (SpectramedInc., Oxnard, CA); Apple lie computer and computer data aquisitionsystem consisting of Interactive Systems (Newton Square, PA), analog todigital converter and clock card (Mountain Software, Scott’s Valley,CA); Pye Unicam SP8-400 UV/VIS spectrophotometer (Pye Unicam Ltd,Cambridge, UK); AMS gamma counter (Microrad Inc., Knoxville TN).2.1.4. Optimization of Derivatization of Ritodrine and InternalStandardsSolutions of ritodrine and the potential internal standards,isoxuprine, nylidrin, 7-phenylephrine, propafenone, and 5-hydroxypropafenone, each at 500 ng/mL, were prepared in methanol. To 1 mL ofthe methanolic ritodrine solution were added 1 mL of each of thepossible internal standard solutions (triplicates of each combination).Following evaporation to dryness under a gentle stream of N2 andreconstitution in organic solvents, the following derivatizationparameters were examined:1) Derivatization by trifluoroacetic anhydride (TFAA)and heptafluorobutyric anhydride (HFBA).2) The use of TEA 0.0125 M in toluene, toluene, and25ethyl acetate as derivatization solvents.3) The concentration of TEA in toluene employed(0.003 vs. 0.0125 N).4) The time course of derivatization.5) The quantity of HFBA required (5, 20, 50 tL).6) Method of removal of excess derivatization reagentand TEA (where applicable) (hydrolysis with H20/4%ammonium hydroxide vs 0.067 M phosphate buffer(pH 6) vs evaporation under N2).Following derivatization the samples were injected in the splitlessinjection mode under the following standard GC conditions: Injectionport temperature 260°C; initial oven temperature 200°C; detectortemperature 350°C; oven temperature rate 3°C/mm; carrier gas flow (H2)2 mL/min; make-up gas flow (Argon-methane 95:5) 60 mL/min. The peakareas of each sample were evaluated to determine which parametersprovided maximal responses.2.1.5. Optimization of GC ConditionsSamples of 1 mL ritodrine in methanol and 1 mL 5-hydroxypropafenone (500 ng each) were dried under a gentle N2 stream,reconstituted in 800 JLL TEA in toluene (0.0125 N) and derivatized at55°C for 60 minutes. The excess derivatizing reagent was neutralized byvortex mixing with 2 mL 0.067 N phosphate buffer (pH 6) and removal ofthe organic layer to borosilicate injector vials. The samples were theninjected onto the GC column and the following parameters examined withrespect to peak area of the HFB derivatives of drug and internal26standard, separation of drug and internal standard, peak shape andsymmetry, and efficiency of the GC column as expressed by the number oftheoretical plates (n).1) Injector temperature: 190, 200, 210, 230, 260°C2) Purge activation time: 10, 20, 30, 45, 60, 90, 120 sec.3) Column head pressure: 5, 7.5, 10, 15, 20 p.s.i.4) Initial column temperature: 100, 145, 190, 200, 210°C.5) Column temperature rate increase: 1, 2, 5, 10 °C/min.6) Detector temperature: 300, 340, 350, 360, 375°C.7) Make-up gas flow rate: 30, 50, 60 mL/min.2.1.6. Optimization of Drug ExtractionThe following extraction parameters were examined:1) Drug extraction efficiency of solvents: hexane,toluene, benzene, ethyl acetate, dichioromethane,and mixtures of ethyl acetate/toluene/isopropanol.2) Effect of buffer pH on extraction efficiency.3) Effect of the addition of TEA to the extraction solvent.4) Extraction time: 5, 10, 20, 30 minutes.2.1.7. Gas-Chromatography Mass-SpectrometryMethanolic samples of ritodrine (1 and 100 tug) were dried under agentle stream of N2, reconstituted in 1 mL 0.0125 N TEA in toluene andderivatized with either TFAA or HFBA. Capillary GC-MS was performed inelectron impact (El), positive (PCI) and negative chemical ionization27(NCI) modes. Helium was used as the carrier gas. The MS operatingconditions were: electron ionization energy 70, 110, 130 eV for the El,PCI and NCI modes respectively; emission current, 0.3 mA; ion sourcetemperature 240°C.2.1.8. Procedures for Ritodrine Analysis from Biological Fluids2.1.8.1. GC Operating ConditionsA 78 mm x 4 mm I.D. borosilicate glass inlet liner with asmall plug of silanized glass wool, located approximately 60 mm from thetop, was used for all analyses. Temperature conditioned ThermogreenRLB-2 septa were used throughout. The operating conditions for the GCwere: injection port temperature, 210°C; purge time, 0.5 mm; split ventflow, 30 mL/min; initial column temperature, 145°C; column headpressure, 10 p.s.i.; carrier gas (H2) flow rate, 2 mL/min; temperatureprogram: 145° held for 3 minutes, 15°C/mm to 190°C held for 1 minute,5°C/mm to 220°C, 50°C/mm to 280° then held constant for 6 minutes;detector temperature 350°C; make-up gas (Argon-methane 95:5) 60 mL/min.Aliquots of 1 iL were injected onto the column for routine analysis.2.1.8.2. Drug Extraction from Biological FluidsTo a 10 ml disposable culture tube with a polytetrafluoroethylene(PTFE)-l.ined screw cap were added: 0.05 - 0.5 ml of biological fluid(either maternal or fetal plasma, amniotic fluid, allantoic fluid, orfetal tracheal fluid), 0.5 ml of the internal standard (200 ng/mL), analiquot (0.95 - 0.5 ml) of deionized water, 0.5 ml of 1 M carbonate28buffer (pH 9.5) (final pH of the aqueous phase was z9.5). The mixturewas gently vortex mixed and 6 ml ethyl acetate added. The aqueous phasewas extracted for 20 mm on a rotary shaker. The samples were thenplaced in a freezer at -20°C for 15 mm to facilitate breakage of anyemulsion formed in the extraction step. This cooling was followed by 2centrifugations of 5 mm duration at 2300 g. The organic layer wastransferred to clean 15 ml disposable tubes and evaporated to dryness ina water bath (30°C) under a gentle stream of nitrogen. The residue wasreconstituted in 0.4 ml 0.0125 N TEA in toluene. A 20 JLL volume ofheptafluorobutyric anhydride was added to each tube, the tube vortexmixed and then placed in an oven at 55°C for 60 mm. The samples wereallowed to cool to room temperature and the excess derivatizing reagentwas neutralized with 2 ml (0.067 M) phosphate buffer (pH 6) (vortexmixing for 30 sec). Following centrifugation (2300 g) for 1 mm theorganic layer was transferred to automatic sampler injection vials anddiluted to 1 ml with toluene. One microliter aliquots were injectedinto the GC.2.1.8.3. Preparation of Calibration CurveSerial amounts of the prepared ritodrine hydrochloride stocksolution (75, 50, 25, 10, 5, 2.5 ng) were added in a fixed volume (1 ml)to either 0.1, 0.25 or 0.5 ml samples of blank pregnant sheep biologicalfluid (either maternal or fetal arterial plasma, amniotic fluid,allantoic fluid or fetal tracheal fluid). A 0.5 ml aliquot of internalstandard solution (5-hydroxy propafenone, 200 ng/mL) was added and thesamples were then extracted and derivatized as previously described.All samples were prepared in duplicate and a standard curve, in the29appropriate biological fluid, was prepared daily. Determination ofritodrine concentrations was made by plotting the peak area ratios ofthe heptafluorobutyryl derivatives of ritodrine and 5-hydroxypropafenone against the known amount of ritodrine added to each sample.2.1.8.4. Recovery StudiesRitodrine free base was dissolved in distilled water and methanol,respectively, to provide solutions of 100 ng/mL. Various volumesrepresenting 75, 50, 25, 10 , 5, and 2.5 ng of ritodrine free base weretransferred into two sets of glass tubes, respectively. To the set ofaqueous ritodrine tubes was added blank maternal sheep plasma followedby extraction as outlined previously. The internal standard, 5-hydroxypropafenone, dissolved in methanol, was then added (500 ng) toboth sets of samples. The two sets of samples were then subjected tothe same derivatization reaction. A standard curve, relating the peakarea ratio of the ritodrine derivative to the internal standardderivative vs the amount of added ritodrine base, from the samplesdissolved in methanol, was then produced. The area ratios from theextracted samples were then determined and the amount of ritodrinecalculated from the standard curve.2.1.8.5. Validation of Assay MethodBlank maternal pregnant sheep plasma was spiked with aqueousritodrine solution to provide concentrations of 10, 100 and 1000 ng/mL.Aliquots of each sample were extracted by the developed GC-ECD method30and by the HPLC fluorescence detection method of Gross et a!. (1987).The concentrations determined by each method were then compared.2.1.8.6. Analysis of Glucuronide and Sulphate Conjugates of RitodrineIn order to examine whether conjugated metabolites of ritodrinewere present following drug administration, the amniotic fluid samplesfrom selected animals were split into aliquots of 0.5 mL. Thehydrolysis procedure employed in the present study was originallyemployed by Brashear et al. (1988) for the determination of ritodrineconjugates in human maternal and neonatal urine following maternal drugadministration. One aliquot was extracted, as described above, andintact ritodrine measured. To another aliquot was added 0.5 mL 0.2 Msodium acetate buffer (pH 5) and sufficient fl-glucuronidase (Type H-2,Helix pomatia, GlusulaseR, G-8076, or bovine liver, GlucuraseR, G-4882 )to provide a $-glucuronidase activity of 2500 U/mL. To a third aliquotwas added 0.5 mL 0.05 M TRIS buffer (pH 7.5) and sufficientarylsulfatase (Aerobacter aerogenes, S-1629) to give a finalarylsulfatase activity of 0.25 U/mL. Both aliquots were incubated andshaken for 20 h at 37°C in a water bath. Following incubation, thesamples were cooled to room temperature and the liberated ritodrine wasimmediately quantitated by the method previously described. Thespecificity of the enzymes for the ritodrine conjugates has beenpreviously reported (Brashear et al., 1988).312.2. Standard Procedures for Animal Experiments2.2.1. Animal HandlingPregnant ewes were brought into the animal unit at The Children’sVariety Research Center, at least 1 week prior to surgery to allow foracclimatization. The ewes were kept in groups of 2 or more in largeadjacent pens. The animals received a standard diet and were allowedfree access to water.2.2.2. Surgical Preparation for Chronic ExperimentationStudies were performed on 32 time-dated pregnant sheep (Dorset andSuffolk breeds). Surgery was performed between 118 and 128 days ofgestation (term, 145 days) with the animals under halothane (1-2%) andnitrous oxide (60%) anaesthesia following induction with 1 g intravenoussodium pentothal and intubation of the ewe.A midline abdominal incision was made in the ewe and then throughthe uterine wall, in an area free of placentomes and major bloodvessels, thus allowing access to the fetus. Silicone rubber catheters(Dow Corning, Midland, MI) were then implanted in the fetal femoralartery, lateral tarsal vein, common umbilical vein, and trachea,maternal femoral artery and vein, amniotic cavity, and in the allantoiccavity. Not all of these catheters were implanted in every animal.Also, in some animals an ultrasound transit-time flowmeter (TransonicSystems, Ithaca, NY) was placed on the fetal trachea to measure trachealfluid flow. The catheters were tunnelled subcutaneously in theabdominal wall and exited through a small incision on the maternal32flank. When not in use they were stored in a denim pouch attached tothe maternal flank. Each catheter was flushed daily with 2 mL 0.9%sodium chloride for injection containing 1.2 U heparin per mL.Ampicillin (500 mg) was administered intramuscularly to the ewe on theday of surgery and for the first three days following surgery, to thefetus via the tarsal vein on the day of surgery, and daily into theamniotic cavity. In the year 1990-1991 gentamicin was added to theantibiotic prophylactic regimen to provide a greater spectrum ofcoverage. Gentamicin was administered 40 mg I.M. to the ewe, 10 mg I.V.via the fetal femoral artery, and 20 mg to the amniotic fluid at thetime of surgery then 40 mg I.M. daily to the ewe for the first threedays following surgery and 20 mg daily into the amniotic fluid for theduration of the preparation.The ewe was kept in a holding pen with other sheep and allowed freeaccess to food and water. After allowing 3-7 days for the ewe and fetusto recover from surgery, the sheep were transferred to a monitoring penadjacent and in full view of the holding pen to permit experimentation.2.2.3. Recording ProceduresDuring the experimental period, fetal arterial, tracheal, maternalarterial, and amniotic pressures were measured with strain-gaugemanometers (see Figure 2) (Statham Model P23Db, Gould Inc., Oxnard, CA).Fetal and maternal heart rates were measured from the arterial pulse bymeans of cardiotachometers (Model 9857, Sensormedics, Anaheim, CA).Tracheal fluid flow rate was measured using a Transonic flowmeter(Transonic Systems, Ithaca, NY). All of these variables were33continuously recorded using a Beckman R612 polygraph recorder or aSensormedics R712 dynograph recorder. The analog signals of fetalarterial, maternal arterial, and amniotic pressure and fetal andmaternal heart rate and tracheal flow were converted simultaneously todigital form using an on-line computer system (Kwan, 1989). For eachvariable the sampling rate was between 2.5 and 6 Hz depending on thenumber of variables recorded. At one minute intervals, the variablemeasurements were averaged, fetal arterial pressure corrected foramniotic pressure and the variable values displayed on computer screen.Every 30 minutes the averaged values were recorded on floppy diskette.During the fetal infusion studies umbilical blood flow was measuredusing the steady-state antipyrine diffusion technique (Meschia etal.,1967; Rurak and Gruber, 1983), at the sampling times indicated,following fetal infusion of antipyrine at 4.86 mg/mm for at least 90mm prior to sampling. Umbilical blood flow was calculated as theantipyrine infusion rate divided by the umbilical arterial- umbilicalvenous difference in antipyrine concentrations. Plasma antipyrinelevels were measured using the method of Davidson and McIntyre (1956).2.2.4. Blood Gas AnalysisSamples for blood gas analysis (0.5 mL) were taken simultaneouslywith those for drug analysis. Fetal and maternal blood°2’ pCO2, andpH were measured and, base excess, and bicarbonate were calculated usingan IL 1306 pH/blood gas analyzer (Allied Instrumentation Laboratory,Milan). Oxygen saturation and haemoglobin concentration were measured,in duplicate, using a HemoximeterR (Radiometer, Copenhagen).342.2.5. Blood Glucose MeasurementSamples for whole blood glucose concentration were drawnsimultaneously with those for drug analysis. These samples wereprepared for whole blood glucose analysis by adding 0.2 mL of wholeblood to 0.9 mL distilled water. To this solution 0.55 ml of zincsulfate (0.3 N) were added, vortex mixed and allowed to stand for tenminutes. Barium hydroxide (0.3 N), 0.55 mL, was then added, vortexmixed and allowed to stand for ten minutes prior to centrifugation at4000 r.p.m. for 15 minutes. The supernatant was removed to newpolystyrene tubes, covered with ParafilmR and refrigerated untilanalysis. All samples were assayed in duplicate within two weeks usinga Sigma Glucose assay kit (Sigma Chemical Co., St. Louis, MO). Thismethod employs enzymatic oxidation of glucose to produce gluconic acidand hydrogen peroxide. The hydrogen peroxide then oxidizes odianisidine (in the presence of peroxidase) to a product which can bequantitated colorimetrically.2.2.6. Blood Lactate MeasurementSamples for whole blood lactate analysis were drawn simultaneouslywith those for drug analysis. The samples for whole blood lactateanalysis were prepared by adding 0.3 mL of whole blood to 0.6 mLperchloric acid (8 %) followed by centrifugation at 4000 r.p.m. for 15minutes. The supernatant was removed to clean polystyrene tubes,covered with ParafilmR and refrigerated until analysis. The lactatesamples were analyzed in duplicate within 3 weeks using a Sigma Lactate35assay kit (Sigma Chemical Co., St. Louis, MO). This method employs theenzymatic conversion of lactate in the presence of nicotinamide adeninedinucleotide (NAD) to pyruvate and NADH. The NADH concentration is thendetermined spectrophotometricaly and is equivalent, in molar terms, tothe amount of lactate originally present.2.3. Experimental Protocols2.3.1. Fetal Infusion StudiesA minimum of 24 h before each experiment the ewe was placed in amonitoring cage, in full view of companion ewes, and given free accessto food and water. Ritodrine, diluted to 40 g/mL with isotonic saline,was infused into the fetal tarsal vein at a rate of 2.6 g/min (0.0648mL/min) using a Harvard Apparatus Infusion Pump. Ritodrine was infusedto the fetus for a period of 8, 12, or 24 h. As ritodrineadministration in the fetal lamb is associated with acidemia (Bassett etal., 1985; Bassett et al., 1989; Warburton et al., 1987b; van der Weydeet al., 1990) the infusion duration was determined by the acid-basestatus of the fetus. The infusion was stopped before the 24 h maximum(i.e. at 8 or 12 hours) if the fetal pH and whole blood base-excess fellbelow 7.30 and -1.0 meq/L, respectively.Samples (3.5 mL) were taken from the fetal femoral artery,umbilical vein, fetal trachea, maternal femoral artery, uterine vein,and amniotic cavity. The blood samples were placed in heparinized tubesand gently mixed. An aliquot of whole blood (0.7 mL), was taken andcentrifuged at 3,000 x g for 15 minutes and the supernatant transferred36to clean glass tubes. The remaining portion of the blood sample wasused for the measurement of blood gas values,°2’ glucose and lactate.These values have been reported separately (van der Weyde et al.,1990;van der Weyde 1990). The amniotic and tracheal fluid samples wereimmediately transferred to clean glass test tubes. All plasma and fluidsamples were frozen at -20°C until analysis. Control samples were takenat -48, -24 and -1 h before the infusion began. Upon commencement ofthe ritodrine infusion, samples were taken at 1.5, 8, 12, and 24 h(depending on the duration of the infusion). Following the terminationof the ritodrine infusion, samples were taken at 1.5, 8, 24, and 48 hpost-infusion. The volume of fetal blood withdrawn during sampling wasreplaced, via the tarsal vein, with an equal volume of maternal blood ateach sample time.2.3.2. Maternal Bolus StudiesA minimum of 24 h before each experiment the ewe was placed in amonitoring cage, in full view of companion ewes, and given free accessto food and water. In 10 animals (experimental group) a 50 mg dose ofritodrine hydrochloride (YutoparR, Bristol Myers-Squibb, Montreal,Quebec) was injected with isotonic saline (total volume 15 mL) via thematernal femoral vein over a period of approximately 30 seconds. In 4animals (control group) an isotonic saline injection (volume 15 mL) wasgiven over a 30 second period via the maternal femoral vein. Thecatheter was then flushed with 10 mL heparinized isotonic saline. Bloodsamples were simultaneously withdrawn from the maternal femoral artery(3.0 mL), uterine vein (3.0 mL), fetal femoral artery (1.5 mL), and37umbilical vein (1.5 mL) at -15, 5, 10, 15, 20, 30, 45, 60, and 90minutes, 2, 3, 4, 6, 8, 10, 12, 24, 30 and 36 hours. In additionsamples of amniotic (3.0 niL) and fetal tracheal fluids (3.0 niL) werecollected simultaneously with the maternal and fetal blood samples.Samples of maternal arterial (0.5 niL) and fetal femoral arterial bloodwere drawn at -15, 10, 15, 30, 45, 60, and 90 minutes, 2, 3, 4, 6, 8,12, 24, 30 and 36 hours for blood gas analysis. In six animals (4experimental and 2 control) 0.5 mL of each blood sample was used forwhole blood lactate (0.3 mL) and whole blood glucose (0.2 mL)determination. The volume of fetal blood withdrawn during sampling wasreplaced, via the tarsal vein, with an equal volume of maternal blood ateach sample time. The blood samples for drug analysis were transferredto heparinized tubes, centrifuged at 4000 r.p.m. for 15 minutes and thesupernatant transferred to new borosilicate glass culture tubes with apolytetrafluoroethylene (PTFE) lined screw cap. The plasma was thenfrozen and kept at -20°C until analysis. The amniotic and trachealfluid samples were transferred to new borosilicate glass culture tubeswith a polytetrafluoroethylene (PTFE) lined screw cap and kept frozen at-20°C until analysis. Ritodrine concentrations in all biological fluidswere determined by the electron-capture capillary gas chroniatographicmethod developed.2.3.3. Fetal Bolus StudiesA minimum of 24 h before each experiment the ewe was placed in amonitoring cage, in full view of companion ewes, and given free accessto food and water. In 5 animals (experimental group) a 2 mg dose of38ritodrine hydrochloride (YutoparR, Bristol Myers-Squibb, Montreal,Quebec) was injected with isotonic saline (total volume 0.5 mL) via thefetal tarsal vein over a period of approximately 30 seconds. In 2animals (control group) an isotonic saline injection (volume 0.5 mL) wasgiven over a 30 second period via the fetal tarsal vein. The catheterwas then flushed with 3 mL heparinized isotonic saline. Blood sampleswere simultaneously withdrawn from the maternal femoral artery (3.0 mL),uterine vein (3.0 mL), fetal femoral artery (1.5 mL), and umbilical vein(1.5 mL) at -15, 5, 10, 15, 20, 30, 45, 60, and 90 minutes, 2, 3, 4, 6,8, 10, 12, 24, 30 and 36 hours. In addition samples of amniotic (3.0mL) and fetal tracheal fluids (3.0 mL) were collected simultaneouslywith the maternal and fetal blood samples. Samples of maternal arterial(0.5 mL) and fetal femoral arterial blood were drawn at -15, 10, 15, 30,45, 60, and 90 minutes, 2, 3, 4, 6, 8, 12, 24, 30 and 36 hours for bloodgas analysis. In all animals 0.5 mL of each blood sample was used forwhole blood lactate (0.3 mL) and whole blood glucose (0.2 mL)determination. In addition, in two animals, lactate and glucoseconcentrations were also determined in the amniotic fluid. The volumeof fetal blood withdrawn during sampling was replaced, via the tarsalvein, with an equal volume of maternal blood at each sample time. Theblood samples were transferred to heparinized tubes, centrifuged at 4000r.p.m. for 15 minutes and the supernatant transferred to newborosilicate glass culture tubes with a PTFE lined screw cap. Theplasma was then frozen and kept at -20°C until analysis. The amnioticand tracheal fluid samples were transferred to new borosilicate glassculture tubes with a polytetrafluoroethylene (PIFE) lined screw cap andkept frozen at -20°C until analysis. Ritodrine concentrations in all39biological fluids were determined by the electron-capture capillary gaschromatographic method developed.2.3.4. Acute Microsphere Studies following Fetal Bolus AdministrationAcute surgery was performed on two animals under halothane (1-2%)and nitrous oxide (60%) anaesthesia following induction with 1 gintravenous sodium pentothal and intubation of the ewe.A midline abdominal incision was made in the ewe and thenthrough the uterine wall, in an area free of placentomes and major bloodvessels, thus allowing access to the fetus. Silicone rubber catheters(Dow Corning, Midland, MI) were then implanted in both fetal femoralarteries, a lateral tarsal vein, fetal brachial artery, and amnioticcavity. Femoral arterial and the amniotic catheters were then attachedto strain-gauge manometers and a cardiotachometer to allow the recordingof intrauterine and fetal arterial pressures and fetal heart rate.In order to determine the effects of ritodrine on thedistribution of cardiac output, i.e. regional blood flow, radiolabelledmicrosphere injection was employed. This technique involves theinjection of radiolabelled (85Sr, 57Cr, l25, 46Sc and ‘41Ce (0.5mCurie/5 mL) labelled microspheres 15 um diameter (3M Company, St. Paul,MN)) microspheres which are trapped in the microcirculation of the fetuson the first circulation after injection. The entrapment of themicrospheres in any given area then is in proportion to the blood flowthat the region receives.Prior to the administration of 2 mg ritodrine via the fetaltarsal vein (-60 and -30 minutes), administration of two sets of40radiolabelled microspheres was made via the fetal tarsal vein andreference samples were drawn from the femoral and brachial arteries.The reference samples were drawn at a fixed rate of 2.40 mL/min for aperiod of 2 minutes following microsphere injection using a Harvardinfusion pump. Following drug administration, microspheres wereinjected at 5, 20 and 60 minutes whereupon femoral and brachialreference samples were drawn, using a Harvard infusion pump, as werefemoral arterial samples (2 mL) for ritodrine analysis. Following thefinal microsphere administration, the ewe and fetus were sacrificed andthe fetal tissues dissected, weighed and transferred to aluminum boats.The tissues were dried over a period of days in an oven, triturated, andthen counted for radioactivity in order to determine regional bloodflows (see Section 2.3.4). In one animal a sample (300 #L) of fetalbile was obtained.2.4. Data Analysis2.4.1. Calculation of Pharmacokinetic ParametersExcept where indicated, formulae used in the pharniacokineticparameter estimation were obtained from Gibaldi and Perrier (1975,1982). All ritodrine concentrations are expressed as ritodrine freebase. The amounts of the sulphate and glucuronide conjugate(s) areexpressed in terms of the amounts of liberated ritodrine free base.The area under the plasma concentration vs time curve (AUC) wascalculated using the equation:41AUC0 = AUC0t + AUCtwhere t represents the time of the last sample and AUC0t was estimatedby trapezoidal approximation. The final term, AUCt, was determinedfrom the relationshipAUCt = Cp(t)/lambdawhere lambdan is the terminal elimination rate constant.The first moment of the area under the plasma concentration vs timecurve (AUMC0) was calculated from:AUMC0 = AUMC0t + AUMCtThe first term (AUMC0t) was calculated by the trapezoidal approximationwhile the second term (AUMC0) was estimated using the relationship(Gouyette, 1983; Yamaoka et al., 1978)AUMCt = t(Cp(t)/lambdan) + Cp(t)/(lambdan)2Total body clearance during the bolus experiments was estimated as:Clt = Dose/AUC0and during the infusion experiments as:1 — I ir’ S5CIt—where k0 is the infusion rate and CpSS is the apparent steady-stateplasma concentration.42The volumes of distribution were calculated as:Vc = Dose/Cp(O)Vdarea = Dose/(lamdan)(AUC0Vd55 = Dose AUMCC0/(AU)2Placental ritodrine clearance was estimated as:Cip= um X (Cfa - Cum)/Cfawhere Clumbil icalthe fetalumbilicalwas the placental clearance of ritodrine, um was theblood flow rate as measured by antipyrine clearance, Cfa wasfemoral arterial ritodrine concentration and Cum was thevenous ritodrine concentration at apparent steady-state.Fetal non-placental clearance (Clf0), then, was calculated as:Clf0= Cltb - ClFetal weight at the time of experimentation was determined from thebirthweight and an equation for fetal ovine growth devised by Kong etal. (1975).432.4.2. Fetal Arterial Pressure, Fetal Heart Rate, Amniotic PressureFetal and maternal heart rates, arterial and intrauterine pressureswere determined and plotted at 1 minute intervals from the computerrecords of the polygraph recordings during control and experimentalperiods.2.4.3. Fetal Breathing Movements and Uterine ActivityFetal breathing movements and uterine contractures were determinedfrom the tracheal pressure and amniotic pressure recordings respectively(Fig 2). Fetal breathing movements are presented as percentage ofbreathing like activity per unit time. Episodes of fetal breathing wereidentified by amplitude changes in the tracheal pressure trace when thebreath amplitude reached a minimum of 1 mm Hg for a period of at least10 seconds (see Figure 2). In animals with a Transonic transit timeflowmeter implanted on the fetal trachea, phasic fluctuations intracheal fluid flow were also used as an indication of fetal breathing(e.g. Fig. 2). The number of uterine contractures were determined perunit time during the control and experimental periods. Contractureswere defined in a similar manner to that of Nathanielsz et al. (1980).A contracture is a change in intrauterine pressure reaching at least 3.5mm Hg above the pre-existing basal pressure and not returning to thebaseline pressure for at least 5 minutes (see Figure 2).442.4.4. Regional Blood FlowsThe calculation of organ blood flow is similar to that outlined byRudolph and Heymann (1967). The basic calculation is as follows:Qorgan = (‘organ/Trefe ence) X referencewhere Qorgan is the organ blood flow, ‘organ is the number of counts ofa particular nuclide within the organ, preference is the blood flow in areference blood vessel (brachial artery for those organs supplied by theascending aorta, femoral artery for those organs supplied by thedescending aorta), and‘reference is the number of radioactive counts ina sample of known volume from the reference blood vessel. Flows areexpressed as mL/min/100 g of organ weight. All counts were spillovercorrected. Typically, when radioactive samples are counted, a smallrange of energy is monitored (i.e. a window is established)corresponding to the most common energies of the particles emitted.When more than one nuclide is counted, the emission spectra of thenuclides may overlap to some extent, thus, for accurate quantitation thecontribution of each nuclide to the total counts observed in aparticular window must be known. This process is known as spillovercorrection and was done using the microsphere counting software suppliedwith the gamma counter (Micrad Inc., Knoxville, TN).45nflnLLLLL!Li-L1_-1 L’JJJU_LLLL_I_LiJiL1 LL1_LL’2Sz0080U250¶ 2:25:r arsjFigure 2: Representative polygraph recording including a period offetal breathing (indicated by arrow in Jr P trace) and a uterinecontracture (indicated by arrow in AMN trace). Amniotic pressure (AMN,mm Hg), fetal arterial pressure (FAP, mm Hg), mean fetal arterialpressure (MFA, mm Hg), fetal heart rate (FHR, beats per minute),tracheal pressure (Jr P, mm Hg), mean tracheal pressure (Mean Jr P, mmHg) and tracheal flow rate (Jr F, mL/min).462.4.5. Computer Fitting2.4.5.1. Pharmacokinetic FittingPlasma drug concentration vs time data were used for initial curvefitting by the computer program AUTOAN (Sedman and Wagner, 1976) inorder to provide initial parameter estimates and the pharmacokineticmodel. These initial estimates were then analyzed by the program NONLIN(Metzler et al., 1974) to provide final kinetic parameter estimates.SELECTION OF APPROPRIATE WEIGHTING FACTOR FOR NONLIN: Data points forindividual subjects were weighted for NONLIN analysis by the method ofAlbert et al. (1974) and by inspection of the weighted sum of squaresand residuals as compared to other possible weighting procedures. Themethod of Albert et al. (1974) plots the natural logarithm of thevariance of the pooled concentrations for any time (t) against thenatural logarithm of the mean concentration at time (t) as is shown bythe following equation:in = in a + n’ inCWhere cr2 is the variance corresponding to the mean concentration (C) fora group of subjects at each sampling time following drug administrationand a and n’ are constants. The appropriate weighting factor for eachtime point is then C’ (i.e. if n’=l then the weighting scheme shouldbe the reciprocal of the observed concentration).These final parameter estimates were used in all subsequentpharmacokinetic and pharmacodynamic calculations.472.4.5.2. Pharmacodynamic FittingFollowing pharmacokinetic fitting, pharmacodynamic modelling wasaccomplished using the extended least-squares regression program MKMODEL(Holford, 1982). For observed effects where a plot of plasmaconcentration vs effect showed no hysteresis, the pharmacological modelemployed was the sigmoid Emax with the existence of a baseline effectand was of the mathematical form (Holford and Sheiner, 1981):g rflmax”____________+ LOEC50n +where E is the measured effect, Emax is the maximal observedeffect, E0 is the baseline measured effect, EC50 is the drugconcentration required to produce a 50% of maximal response, C is thedrug concentration at any time, and n is the factor describing thesteepness of the dose-response relationship (Holford and Sheiner, 1981).The appropriateness of the pharmacodynamic fitting was determined by theresidual analysis, log-likelihood and Schwartz criteria (Holford, 1982).2.4.6. Statistical AnalysesStatistical evaluations were performed on pharmacokinetic,physiologic and pharmacological parameters using the statistical testsspecified in each section. The level of significance chosen for allstatistical analyses was always a = 0.05.483. RESULTS3.1. Development of a Capillary GC-ECD Method for Ritodrine.3.1.1. Optimization of DerivatizationAlthough derivatization of ritodrine with either TFAA and HFBAproduced single, symmetrical peaks on GC analysis there were a number ofqualitative factors which favoured the choice of HFBA as thederivatizing reagent (see Figure 3). Most significantly the detectorresponse to a given concentration of ritodrine was approximately 10 foldgreater for the HFBA derivative than for the TFAA derivative. Secondly,HFBA is available in single use 1 mL ampuoles while TFAA is availableonly in multiple use bottles of volumes > 10 mL. Thus HFBA waspreferred as the derivatization reagent as it provided the potential ofmuch higher sensitivity and, in addition, the purity and activity of thereagent could be assured.The minimal volumes of HFBA and 0.0125 M TEA in toluenerequired were 20 JLL and 800 #L, respectively (Figures 4 and 5). Dataillustrating the need for TEA as a catalyst and the time course ofderivatization are shown in Figure 6. When TEA was not employed,derivatization was slow and incomplete even after 2 hours. When TEA wasemployed as a catalyst a plateau was reached at 30 minutes, however,the coefficient of variation was minimized at z60 minutes. Thus 60minutes was chosen as the time for derivatization.A number of techniques have been suggested for theneutralization and removal of excess acylating agents ( e.g. HFBA, TFAA)Figure3:ChromatogramsofritodrinederivatizedwithTFAA(1.25nginjected)(leftpanel)andHFBA(O.5nginjected)(rightpanel)andneutralizedwith0.067Mphosphatebuffer(pH6).ThepeaksindicatedbythearrowcorrespondtoTFAAderivatizedritodrineandHFBAderivatizedritodrinerespectively.4-cn F— z D 0 0 w15001000-500 0I.1.1I I0102030405060VolumeofHFBAadded(1uL)Figure4:Detectorresponse(areacounts)(mean±1s.d.;n=3)vsvolumeofHFBArequiredforderivatization.(Derivatizationoccurredin400#L0.0125MTEAintoluene)Ui15001200q) Cr) I600C-) ci) ci)3001 0I—H03006009001200jL.L0.0125MTEAinTolueneFigure5:Detectorresponse(areacounts)(mean±1s.d.;n=3)vsvolumeofTEAintoluene(0.0125M)requiredforderivatization.(Derivatizedwith20LHFBA).2E42E4LU2E42E42E4LU1E4o1E4F- o1E4LU8000060004000200000102030405060708090100110120TIME(mm)Figure6:TimeCourseofDerivatization.Detectorresponse(areacounts)(mean±1s.d.;n=2)vstimeofderivatization.Derivatizationwasaccomplishedineither800#L0.0125NTEAintoluene(0)orin800btLtoluene(•)using20iLHFBA.cii53and catalyst (e.g. TEA) such as evaporation under N2, hydrolysis withH20 followed by neutralization with 4% (or less) NH4O , andneutralization with 0.067 M phosphate buffer (pH 6) (Walle and Ehrsson,1970; Ehrsson et al., 1971). Each of these methods were tried forritodrine following HFBA derivatization in the presence of 800 4uL 0.0125M TEA in toluene. Hydrolysis and neutralization with H20 and NH4O (1 -4 %) caused the complete loss of the ritodrine HFBA derivative peak.This loss was probably the result of hydrolysis of the HFB substituentat the two phenolic sites (Ehrsson et al., 1971). Evaporation under N2caused the solvent front to become extremely large probably due to theinefficient removal of TEA (Figure 7). Neutralization with 0.067 Mphosphate buffer (pH 6) resulted in the smallest solvent front and asingle, symmetric peak corresponding to the HFBA derivative of ritodrine(Figure 8). The HFB derivative of ritodrine was found to be stable forat least 7 days at room temperature.3.1.2. Optimization of GC ConditionsIn order to obtain maximal sensitivity, the splitlessinjection mode was employed throughout. There was little effect ofinjection port temperature on the peak area ratio (HFB-ritodrine/HFBinternal standard), however, peak broadening was noted when theinjection port temperature was lowered to 200°C and there was anapparent decrease in the peak area ratio when the injection porttemperature was raised above 230°C (Figure 9). The maximum response toritodrine was observed when a purge activation time of 30 seconds orgreater was used (Figure 10). In order to minimize the transfer of60Figure7:ChromatogramofHFBAritodrinederivative(indicatedbythearrow)evaporatedunderN2andreconstitutedin1mLtoluene.(Non-optimizedGCconditions).8 7050•402468Time(mine)1214169Figure8:ChromatogramofI-IFBAderivatizedritodrine(indicatedbythearrow)neutralizedwith2mL0.067Mphosphatebuffer(pH6).(Non-optimizedGCconditions).B.7 604—50402468Time(mm.)121416Ui•1 .0000.9000.8000.7000.6000.5000.4000.3000.2000.1000.000 190200210220230240250NJECTIONTEMPERATURE(°c)Figure9:Effectofinjectionporttemperature(mean±1s.d.;n3).Qi1600-Cl)1200H z D 0 (38000--/ 9400 0II020406080100120140160180200PURGEACTIVATIONTIME(sec)Figure10:Effectofpurgetimeondetectorresponse(areacounts;mean±1.s.d.;n3)forHFBAderivatizedritodrine).58other compounds onto the GC column a purge activation time of 30 secondswas employed. The construction of van Deemter plots for ritodrinedemonstrated that maximal efficiency was obtained with column headpressures between 5 and 10 p.s.i. (Figure 11). However, the curvebetween 5 and 10 p.s.i. was relatively flat, thus a column head pressureof 10 p.s.i. (carrier gas (H2) flow rate 2 mL/min; linear velocity 80cm/sec) was considered optimal as it afforded maximal efficiency withthe smallest run time. A significant “cold-trapping” effect was notedwhen the initial column temperature was held at 190°C or lower.However, when initial temperatures of 145 or 100°C were employed thesolvent peak began to split, the chromatogram became more complex andthe run time was lengthened to impractical limits. Thus an initialcolumn temperature of 190°C was considered optimal. A columntemperature rate increase of 5°C/mm was found to provide the bestresolution, peak shape, and allowed the run to be completed within areasonable time. There was an apparent increase in detector response toHFB-ritodrine at detector temperatures above 350°C, however, there was aconcomitant increase in baseline noise at these temperatures (Figure12). At a detector temperature of 300°C there was a lowering ofresponse and the ability to maintain an uncontaminated detector wasquestionable. Therefore, a detector temperature of 350°C was chosen.There was little effect of the make-up gas (argon/methane; 95/5) flowrate on detector response, thus a flow rate of 60 mL/min was chosen asit was felt that this flow rate would allow good “sweeping” of thedetector volume.1 .0000.9000.8000.7000.6000.5000.4000.300 EE 050100150200LINEARVELOCITY(cm/sec)Figure11:vanDeemtercurveforHFBAderivatizedritodrineusingH2ascarriergas.(HETP-HeightEquivalenttoaTheoreticalPlate).U, LD1E4-9000•8000It ° 4000-C-)-3000-200010001- o.l- 300325350375400DetectorTemperature(°C)Figure12:Effectofdetectortemperatureondetectorresponse(areacounts;mean±1s.d.;n=3).C613.1.3. Optimization of Drug ExtractionThe relative extraction efficiency of organic solvents wasexamined by comparing the maximal peak areas of ritodrine HEB obtainedfollowing extraction from aqueous solution with 1 M carbonate buffer (pH9.5) (Figure 13). Ethyl acetate had the highest relative extractionefficiency. Attempts to improve the selectivity of extraction usingmixtures of ethyl acetate/toluene/isopropanol only succeeded in reducingthe relative extraction efficiency.Lin et al. (1984) had suggested that attaining an aqueousphase pH of 9.5 was essential for optimal recovery of ritodrine. Therelative recovery at pH 9.2 was =33% and at pH 10 was =60% as comparedto pH 9.5.The effects of adding TEA to the extraction solvent, ethylacetate, were examined (Figure 14). The addition of TEA did not appearto improve the extraction recovery of ritodrine above that of ethylacetate alone.Extraction times of 5, 10, 20 and 30 minutes were examinedwith 20 minutes giving the best recovery and least variability (Figure15).3.1.4. Gas-Chromatography Mass-SpectrometryGas-chromatography mass-spectrometry was performed on the TFAAand HFBA derivatives of ritodrine in the El, PCI and NCI modes. The GCMS of 5-hydroxy propafenone has previously been reported (Chan et al.,1989). Following derivatization of ritodrine with TFAA, both GC-ECD and62a 1.5001.250 ->-C-)zWC)ILLi 0.750bJId>0.500iii0.2500.000oz zId Id>- t.11W z -J IdF-C) 0uJ Id 0Cl] Fb 1 500 RELATIVE EXTRACTION EFFICIENCYOF RITODRINE(ETHYL ACETATE:TOLU EN E: I SOPROPANOL)1.250C-)zIdC)ILIL 0.750IdId>0.5000.250____Figure 13: Relative recovery of ritodrine from organic solvents (a) andmixtures of ethyl acetate:toluene:isopropanol (b). Extracted fromaqueous standards using pH 9.5 carbonate buffer.Figure14:EffectoftheadditionofTEAtotheextractionsolvent(ethylacetate)(mean±1s.d.;n=4).Extractedfromaqueous(1) H z D 0 C) LU500400300200100 0ETHYL0.5M0.1M0.05M0.01M0.001MACETATETEATEATEATEATEAstandardsusingpH9.5carbonatebufferEXTRACTIONTIME(mm)Figure15:Effetofextractiontimeothere]ativerecoveryofritodrine.ExtractedfromaqueousstandardsusingpH9.5carbonatebuffer.(Mean±s.d.;n=3).0 H w1.5001.2000.9000.6000.3000.000I TN I I III0510152025303565GC-MS demonstrate the formation of a single species (Figure 16). UnderNCI conditions this peak showed a molecular ion of m/z 671 amu whichcorresponds to a tetra-IFAA substituted derivative of ritodrine. Sincethe mass range of the quadrupole mass spectrometer employed was limitedto 0-1000 amu, it was not possible to observe a molecular or pseudo-molecular ion (m/z 1071) which would have been expected due to theformation of a similar tetra-substituted HFBA compound. Followingderivatization with HFBA, analysis by GC-ECD and GC-MS demonstrated theformation of a single species as evidenced by a single, symmetrical andsharp peak in the GC-ECD chromatogram and also by a similar total ionchromatogram (GC-MS) demonstrating one symmetrical peak (Figure 17).The fragmentation patterns of [IFBA derivatized ritodrine are shown inFigure 17. Fragmentation in both El and CI modes was extensive andcomplex due to multiple losses of all or part of the heptafluorobutyrylgroup (viz. F7C3OO, ni/z=213; F7C3O, m/z=197; F7C3, m/z=169). Althoughno molecular ion could be observed, it is proposed that the derivativeformed is tetra-substituted due to the similar size and spectrum ofchemical reactivity of TFAA and HFBA as well as the characteristicfragments formed (m/z 542 and 548) under NCI conditions.3.1.5. GC Operating ConditionsThe optimal GC conditions were: injection port temperature 210°C,initial column temperature 145°C (held for 3 mm), temperatureprogramming rates of 15°C/mm (to 190°C and then held for 1 mm),5°C/mm (to 220°C) and 50°C/mm (to 280°C and then held for 1 mm),electron-capture detector temperature 350°C, carrier gas flow rate66Fc >Mr7 -9i RIT IOONC () CLEIH L1NEP20001 C: (j iI Looj1 400400-2130-13. II jililill3.0 4.13 5.13 6.13 7.13 8.0 5.0 1L’.t’ 11.13 12.13671100 /908070a)6050< 4030 /67220100660 675m/z RatioFigure 6: Total ion chromatogram (top) and negative chemical ionizationspectrum of TFAA derivatized ritodrine.67File >tIRU3 6O.O—999O aou.r ic80El070006000soeo4 El El El3000-3 1815.40200091000 :_,s3ck I’1i’;4t:;#AA.t130 331AF7C3OOç 2r-CO C3F7• (r,c3ccZ .“‘ 31 - 317cc0 • 3 fCC036- I? (yCjCOh31003033—344z063zUi>0--,3361I ii______0- I Ij—0 100 ZOO 300 (031 300 100 700 •00(UI zFigure 17: Total ion chromatogram, A) electron impact, B) negativechemical and C) postive chemical ionization spectra of HFBA derivatizedritodrine.68•, ( I(3Oj (0 t1C30)ZI)Ill (co)t,100 ZI3(I)zUIFU-,>I—o I h0 tOO ZOO 300 400 700 OO ZOO e00 900m/zC3F7NN0tt.r 1..:ISI (9—10*1 I‘—I I(I)zU-,F2:U-,>F111/7Figure 17 (cont’d): Total ion chromatograni, A) electron impact, B)negative chemical and C) postive chemical ionization spectra of HFBAderivatized ritodrine.69(hydrogen) 2 mL/min, make-up gas flow rate (argon:methane, 95:5) 60mL/min, purge activation time 30 seconds. For optimal injection toinjection replicability 1 tL volumes were injected into the GC and a 78mm X 4 mm 1.0. borosilicate injection liner packed with a small plug ofsilanized glass wool (at z50-60 mm) was employed.3.1.6. Drug Extraction from biological FluidsThe extraction and derivatization procedure for ritodrine isshown in Figure 18. Figures 19 (A-D) show representative chromatogramsof extracts of biological fluids from the chronically instrumented eweand fetus. Both the HFBA derivatives of ritodrine and 5-hydroxypropafenone were completely resolved from endogenous compounds.3.1.7. Recovery StudiesThe absolute recovery of ritodrine by the present assay methodis 62% ± 5% which is similar to that obtained by Gross et al. (1987)(68%) but slightly lower than that reported by Lin et al. (1984) andKuhnert et al. (1983) (see Fig. 20). The absolute recovery of ritodrinemay be increased to 81% ± 5.5% by re-extraction of the aqueous phasewith 5 ml ethyl acetate for 20 minutes, followed by 2 centrifugations at2300 g for 5 mm. The organic layers were then pooled, evaporated andderivatized, as previously described. Improving the recovery in thismanner, however, resulted in an 20% increase in the coefficient ofvariation at each sample concentration measured.70Ritodrine standard Biological fluidsolution (100 ng/mL) + samples (50- 500 iiL)blank biological fluid(50- 500 L)5-hydroxy propafenone 100 rig0.5 niL I M Carbonate buffer(pH 9.5)distilled II20 q.s. 2 niL6 niL Ethyl acetatemix 20 minutesrefrigerate 10 minutescentrifuge 2 x 5 minutesAqueous layerEthyl acetate layerevaporate to drynessunder N2 at 30 Creconstitute in 400 #L0.0125 N TEA in toluene20 pL HFBAincubate at 55°C for 60minutesvortex mix with 2 niL0.067 11 phosphatebuffer (pH 6)transfer organic layerinject 1 L into GCFigure 18: Extraction and derivat’ization procedure71AB0Figure 19: Representative chromatograms from various biological fluidsobtained from pregnant sheep (blank, bottom trace; followingdrug administration, top trace): A) maternalplasma , B)fetal tracheal fluid, C) amniotic fluid. The peakslabelled 1 and 2 are the HFB derivatives of ritodrine and 5-hydroxy propafenone respectively.0 5‘r.lmo (mm.)2Ttm (mm.).C21572100.90 Y 0.612 X ± 0.6480 r=0.998U0> 6005O40II0 10 20 30 40 50 60 70 80 90 100Amount Added (ng)Figure 20: Absolute recovery of ritodrjne from maternal arterial plasma(n=3, mean ± s.d.).733.1.8. Calibration CurveThe data for a representative calibration curve used in thequantitation of ritodrine from maternal and fetal sheep plasma arepresented in Table 1. Linearity is observed over the concentrationrange studied (2.5 - 75 ng) with a coefficient of variation < 10% at allpoints. The best-fit (by linear regression) through the data points wasdescribed by the equation Y = 0.0185 X + 0.0534 with a correlationcoefficient of r = 0.997.3.1.9. Validation of Assay methodFigure 21 shows the comparison of the concentrations determined,from prepared samples of known concentration, by the HPLC fluorescencemethod of Gross et al. (1987) and the developed GC-ECD methods. Theequation describing the relationship between the concentrationsdetermined by each method is HPLC = 0.9393 GC - 2.41 with a correlationcoefficient (r) of 0.99998.3.2. Fetal Infusion StudiesTable 2 lists the ewes on which experiments were performed, thenumber of fetus present, the fetal gestational age at the time ofexperiment, the fetal birthweight and the infusion duration. Alsolisted are the types of samples collected during each experiment. Ofthe 13 experiments completed, 6 had an infusion duration of 8 h, 3 of 12h and 4 of 24 h. Fetal arterial, umbilical venous, maternal arterial74TABLE 1CALIBRATION CURVE FOR SHEEP PLASMAn = 7-8Ri todri neAdded (ng)#2.55.010.025.050.075.00.07250. 12920. 22680. 57141. 02451. 3961± 0.0075± 0.0095± 0.0200± 0.0380± 0.0340± 0.0384- Calculated as free base*- Area ratio, drug/internaldevi ationLinear Regression Statistics:Y = 0.0185 X + 0.0534r = 0.997r2 = 0.99410.307.328.896.663.302.75Area Ratio*(mean ± 1 s.d.)Coefficient ofVariation10/standard ± 1 standard100200Concentration400500byCC(ng/mL)Figure21:ComparisonofritodrineconcentrationsdeterminedbyHPLC(n=2)(Grossetal.,1987)vsthosedeterminedbythedevelopedGCmethod(n=2).HPLC=0.9393CC—2.41r=0.99998-J E C C-) -J 0 > -o 0 C E ‘2) ‘2)-D C 0 a C ‘2) C-) C 0600500400300•200100 00300determined60CLfl76TABLE 2Experimental details for the fetal infusions.EWE# # of GAa BWb DURATIONC SAMPLESdfetus239 1 129 2824 8 FA,UV,MA,UT,AM,TR264 2 128 2374 8 FA,UV,MA,UT,AM227 1 130 3735 8 FA,UV,MA,UT261 2 130 2800 8 FA,UV,MA,UT,AM,TR271 1 134 2801 8 FA,UV,MA,UT,AM74 1 129 3120 8 FA,UV,MA,UT,AM,TR274 1 127 3702 12 FA,UV,MA,UT,AM,TR77 1 134 4500 12 FA,UV,MA,UT,AM,TR270 2 143 3450 12 FA,UV,MA,UT,AM,AL251 1 128 3872 24 FA,UV,MA,UT,AM165 1 123 4522 24 FA,UV,MA,UT,AM,TR256 1 123 3735 24 FA,UV,MA,UT72 1 130 5802 24 FA,UV,MA,UT,AMa - GA - gestational age, in days, at the time ofexperimentb - BW - Birthweight of operated fetus in gramsc - Duration of fetal ritodrine infusion in hoursd - Samples collected: FA- fetal femoral artery,UV- umbilical vein, MA - maternal femoralartery, UT -uterine vein, AM - amniotic fluid,AL - allantoic fluid, TR - tracheal fluid77and uterine venous blood samples were collected in all animals.Amniotic fluid samples were collected from 11 animals, while trachealfluid samples were obtained from 6 animals. In 1 animal, allantoicfluid samples were gathered. The duration of the infusion wasdetermined by the acid-base status of the fetus as outlined in theExperimental section. In all of the animals studied, hemodynamic andmetabolic parameters were within normal limits at the start ofexperimentation. However, as reported by van der Weyde et al.(1990)there were declines in fetal arterial p02 (21.9 ± 0.6 to 17.0 ± 0.5mmHg) and pH (7.370 ± 0.004 to 7.274 ± 0.025) during the infusion.Umbilical blood flow remained constant before (206 ± 20 mL/min/kg; mean± s.e.m), during (207 ± 16 mL/min/kg) and after the infusion (182 ± 10mL/min/kg).Figures 22-24 show the ritodrine concentrations in the variousfluids samples (mean ± s.e.m.) at the sample times outlined previouslyfor the 8, 12, and 24 h infusion groups, respectively. In addition, inFig. 25 the concentration of ritodrine glucuronide(s), expressed as freeritodrine, excreted into the amniotic fluid is shown for the 24 hinfusion group along with the ritodrine concentrations in fetal arterialplasma and amniotic fluid. During the infusion period, fetal arterialritodrine concentration rose to 12.1 ± 2.6 ng/mL (mean ± s.e.m.) at 1.5h. By 8 hours the fetal arterial concentrations had increasedsignificantly (Wilcoxon signed rank test, p < 0.05) to 16.0 ± 2.2 ng/mL(mean ± s.e.m.), but thereafter, in the 12 and 24 h samples, thearterial concentration was not significantly different from the 8 hvalue. At all points the arterial concentrations were significantlydifferent than the umbilical venous concentrations, when the infusion78had reached apparent steady-state (i.e. at 8, 12, and 24 h) the meanarterio-venous difference was 0.7 ± 0.17 ng/mL (mean ± s.e.m.).Following the infusion period, fetal plasma concentrations decreased andwere not detectable by 24 - 48 h. At no point were ritodrineconcentrations measurable in either maternal arterial or uterine venousplasma. Fetal tracheal fluid ritodrine concentration increased duringthe infusion and then fell rapidly, thereafter. During the infusion,the mean tracheal fluid concentration of 64.2 ± 4.9 ng/mL (mean ± s.e.m)was significantly higher than the fetal arterial plasma concentration(17.0 ± 1.96 ng/mL; mean ± s.e.m) with a tracheal fluid to plasma ratioof 4. The value increases following termination of the infusion, asthe plasma concentrations fell more rapidly than the tracheal fluidconcentrations. There is a gradual accumulation of intact ritodrine inthe amniotic fluid, and by 8 h of infusion the amniotic fluidconcentrations equalled or exceeded those in the fetal arterial plasma(see Figures 22-24). Moreover, the drug was present in the amnioticfluid until 48 h post-infusion. In the 24 h infusion group, theglucuronide conjugate(s) of ritodrine (see Fig. 25) followed a similarpattern of accumulation. However, the concentration of the glucuronideconjugate(s) are 10 - 20 fold greater than that of intact ritodrine inthe amniotic fluid and these high conjugate concentrations are stillpresent 48 h post-infusion (see Fig. 25). There was no evidence ofsulphate conjugate(s) in the amniotic fluid. In the one animal in whichallantoic fluid was sampled ritodrine levels were similar to those inamniotic fluid (amniotic concentration (ng/mL):allantoic fluidconcentration (time h); 10.8:3.8 (8 h); 11.0:9.9 (12 h); 16.5:16.4 (1.5h post infusion); 18.8:13.9 (24 h post-infusion)).-J E C z 0 F z uJ 0 z 0 0 z cm 0 F8hInfusionsN=6100 90 80 70 60 50 40 30 20 10 0—Fetalartery(___—Umbilicalvein___—Trachealfluid7’A—Amnioticfluid1.5h8h1.5P8P24P48PFigure22:Ritodrineconcentrations(mean±s..e.m.)inthefetalfluidsofthe8hourinfusiongroup(n=6).Thex-axisscaleisinhours,Pdenotingpost-infusionsamples.-J E C z 0 F z Ui C) z 0 C) Ui z 0 F12hInfusionsN=3100 90 80 70 60 50 40 30 20 10 0—Fetalartery(n=3)__—Umbilicalvein(n=3)___—Trachealfluid(n=2p771—Amnioticfluid(n=31.5h8h12hL5P8P24P48PFigure23:Ritodrineconcentrations(mean±s.e.m.)inthefetalfluidsofthe12hourinfusiongroup(n=3).Thex-axisscaleisinhours,Pdenotingpost-infusionsamples.C2024hInfusions-jN=4E110—Fetalarte(n=4)—Umbilicalvein(n=4)c100—Trachealfluid(n1)90—Amnioticfluid(n=3)0 8070z w 0 z500 040w z30Ei.-In_1.5h8h12h24h1.5PSP24P48PFigure24:Ritodrineconcentrations(mean±s.e.m.)inthefetalfluidsofthe24hourinfusiongroup(n=4).Thex-axisscaleisinhours,Pdenotingpost-infusionsamples.12001000 800600400200 01.5h8h12h24h1.5PFigure25:Ritodrineconcentrations(mean±s.e.m.)infetalarterialplasma,amnioticfluidandamnioticfluidconcentrationsofritodrineglucuronide(s),expressedasliberatedritodrineforthe24hourinfusiongroup(n=3).Thex-axisscaleisinhours,Pdenotingpost-infusionsamples.-J E C z 0 H H z Ui C-) z 0 0 LU z 0 0 H 0:::8P24P48Pt’j83Table 3 gives the calculated clearances (mean ± s.e.m.) ofritodrine in both mL/min and mL/min/kg fetal weight. Placentalclearance (Cl) averaged 9.2 ± 2.7 mL/min/kg and accounts for 15 % ofthe average total body clearance of 62.0 ± 8.1 mL/min/kg while non-placental clearance (Clf0), 52.8 ± 8.0 mL/min/kg accounts for theremaining 85 %. The relative contribution of Cl and Clf0 to Cltb didnot change significantly with the gestational age of the fetus (r=0.16).Neither Cltb (r=0.21) nor Clf0 (r=0.25) are significantly correlatedwith the gestational age of the fetus at the time of experiment.3.3. Maternal Bolus StudiesTable 4 lists the ewes on which maternal bolus experiments wereperformed, the number of fetus present, the fetal gestational age at thetime of experiment, the gestational age at birth, and the fetalbirthweight. In the experimental group, maternal weight averaged 76.5 ±16.1 kg (mean ± s.d.) and the mean gestational age at the time ofexperiment was 131.7 ± 3.2 days (mean ± s.d.). The average number offetuses in the experimental group was 1.7 ± 0.5 (mean ± s.d.) with thegestational age at birth being 139.8 ± 4.7 days (mean ± s.d.) (term 145days) and the weight of the operated fetus at birth, 3288 ± 572 g (mean± s.d.). In the control group, maternal weight was 69.5 ± 19.5 kg (mean± s.d.) and the mean gestational age at the time of experimentation was135.8 ± 2.6 days (mean ± s.d.). The average number of fetuses in thecontrol group was 1.8 ± 0.5 (mean ± s.d.) with the gestational age atbirth being 140.3 ± 1.7 days (mean ± s.d.) (term 145 days) and theweight of the operated fetus at birth, 2908 ± 376 g (mean ± s.d.).Analysis of the maternal arterial plasma concentration vs84a) [FA]arterialb) [A-Vj - mean apparent steady-state arterioumbilical venous difference (ng/mL)c) m - mean umbilical blood flow (mL/min)d) C’l’tb - fetal total body clearance (mL/min)e) Cl - fetal placental clearance (mL/min)1) Clf - fetal non-placental clearanceg) numers in parentheses are expressed perkilogram of fetal weight at the time of experimentation.EWE #TABLE 3Calculated ritodrine clearances (mean ± s.e.m.).1FAl FA-Vl Clt Cl Clf227 24.2 0.6 318 107.7 8.2(90.3)9 (30.59) (2.3)74 21.4 0.7 580 121.3 18.1(232.9) (48.7) (7.3)264 9.4 0.1 500 277.2 3.2(229.4) (127.2) (1.5)239 18.4 1.0 578 141.3 32.0(231.2) (56.5) (12.8)165 11.0 1.8 500 235.7 81.1(155.8) (73.4) (25.3)251 8.8 0 493 295.5 0(157.0) (94.1) (0)72 13.9 0.3 921.3 187.1 19.9(201.6) (40.9) (4.4)261 29.2 0 362 89.1 0(150.8) (37.1) (0)270 8.5 1.0 688.5 305.9 83.4(206.8) (91.9) (25.1)274 15.5 0 842.5 168.0 1.6(299.8) (59.8) (0.6)256 27.1 0.6 493.1 95.9 11.3(194.9) (37.9) (4.5)271 12.8 1.5 544.0 203.0 64.6(199.3) (74.4) (23.6)77 20.8 1.1 832.5 125.1 45.7(219.7) (33.0) (12.1)99.5(28.3)103.1(41.4)273.9(125.7)109.3(43 . 7)154.6(48.2)295.5(94.1)167.2(36.6)89.1(37.1)222.5(66.8)166.3(59.2)84.6(33.4)138.4(50.7)79.5(21.0)MEAN 17.0 0.7 588.7 181.0 28.4 152.6(197.7) (62.0) (9.2) (52.8)S.E.M. 1.96 0.17 51.0 21.3 8.5 20.0(14.2) (8.1) (2.7) (8.0)- mean apparent steady-state fetalconcentration (ng/mL)85TABLE 4Experimental details for the maternal bolus experiments.EWE# Maternal # of GAb BAC BWdweiqht fetusExperimental Animals105 69.1 2 133 138 3165*, 253576 82.7 2 131 131 2620*, 265061 70.0 1 131 137 4040*64 85.0 2 134 135 3255*, 3980109 82.7 1 124 144 4082*163 76.8 2 130 147 2617*, 3970130 104.5 2 132 143 3450*, 3570248 80.0 2 133 141 2550*, 2730NT 40.5 1 133 142 3300*160 73.6 2 136 140 2701*, 3805Control Animals617 82.3 2 132 138 2873*, 2547248 80.0 2 136 141 2550*, 2730NT 40.5 1 137 142 3300*141 75.0 2 138 140 No te, 3092a - Maternal weight (kg)b - GA - gestational age, in days, at the time ofexperimentc - BA - gestational age, in days, at time ofdeliveryd - B - Birthweight of fetus in grams( indicates operated fetus)e - No wt - unsupervised delivery, fetus trampledby ewe86time profiles by AUTOAN showed that these were best described by a biexponential equation in all but two animals (triexponential). Figure 26shows the plasma concentration vs time profiles of ritodrine in thesampled fluids from a representative animal. The volume of distributionof the ewe was quite large (Vdarea 14.3 ± 3.5 L/kg; Vd 10.0 ± 1.9 L/kg) and maternal total body clearance averaged 1.5 ± 0.3 L/h/kg (24.3 ± 5niL/nun/kg). The terminal elimination half-life in the ewe was 8.7 ± 1.8h. Concentrations of ritodrine in fetal arterial and umbilical venousplasma were significantly less than those in maternal arterial oruterine venous plasma suggesting limited placental transfer of ritodrinein the pregnant sheep model . Peak concentrations in fetal arterialplasma averaged 8.7 ± 1.3 ng/mL and the apparent elimination half-lifewas 2.3 ± 0.9 h in fetal arterial plasma. Concentrations of ritodrinein fetal arterial and umbilical venous plasmas reached peak valuesbetween 10 and 15 minutes following maternal drug administration in allbut two cases where fetal concentrations peaked at 30 and 45 minutes.The AUC values for umbilical venous plasma were slightly butsignificantly (paired t-test, p < 0.05) greater than those determinedfor fetal arterial plasma suggesting fetal non-placental drugelimination. The pharmacokinetic parameters determined during theseexperiments are listed in Table 5. Ritodrine concentrations in fetaltracheal fluid were greater, peaked later, and persisted longer thanthose in fetal arterial plasma. Ritodrine accumulated slowly to maximumconcentrations of zlO ng/mL in amniotic fluid and persisted at theseconcentrations for at least 24 h post-dose. Samples of amniotic fluidtaken at -15, 10 mm, 2, 4 ,8, and 24 h and incubated with eithersuiphatase or glucuronidase did not demonstrate the presence of anyIIIII1234567891011TIME(h)Figure26:Representativeplotof’ritodrineconcentrationvstimeintheeweandfetusfollowinga50mgmaternalintravenousbolus.1000 10O•10-J E C z 0 F z LU C) z 0 C) LU z 0 0 HMaternalarterialUterinevenousA—AFetalarterialUmbilicalvenousTrachealfluidv—vAmnioticfluid10a)88Table 5: Pharmacokinetic parameters (mean ± s.e.m.; n = 10) obtainedfrom maternal intravenous bolus dosing (50 mg ritodrine hydrochloride).A. Maternal Arterial PlasmaCLf 24.3 ± 5.0(mr/mm/kg)Vd 1.6 ± 0.3(L2 kg)14.3 ± 3.5Vd 10.0 ± 1.9(L, g)AUC1 656 ± 117(ng h/mL)AUMC2 5361 ± 1854(ng h2/mL)a 0.5 ± 0.18.7 ± 1.8B. Fetal Arterial PlasmaApp.2.3 ± 0.9AUCfa 0.03 ± 0.01AUCma1. AUC (zero to infinity) determined by thetrapezoidal approximation.2. AUMC - area under the first moment of theconcentration vs time curve from zero to infinity.3. Apparent half-life in fetal plasma.Clt = Dose/AUCVdc = CP(tO’/doseVdarea = Oosé/(AUC x )Vd55 Dose x AUMC/ (AUC)289sulphate conjugate(s) of ritodrine. However, in common with the fetalinfusion experiments (section 3.2, Figure 25), there appears to beglucuronide conjugate(s) present in measurable quantities at 4 hourspost-dose. The concentration of the glucuronide conjugate(s) in theamniotic fluid during these experiments was approximately 2-7 foldgreater than those of the intact ritodrine (calculated as liberatedintact ritodrine). The glucuronide conjugate(s) persists in theamniotic fluid for at least 24 hours post-dose. The mean concentrationsof the glucuronide conjugates are shown in Table 6 at various timesduring the experiment.Following drug administration there was an approximate doubling ofmaternal heart rate in all animals (See Figure 27). The maternal heartrate then declined, to control values, over a period of 4 - 6 hours inall animals. There was no anticlockwise hysteresis in plots of theincrease in maternal heart rate vs maternal arterial plasma drugconcentration, suggesting that the site of effect of ritodrine onmaternal heart rate was within the central compartment. These plotsalso showed that the shape of the concentration-effect relationship formaternal heart rate was indeed sigmoidal and, in all cases, a maximalheart rate was reached (i.e. the sigmoidal curve reached a plateau).The relationships between maternal heart rate increase and time andmaternal heart rate increase and maternal arterial plasma ritodrineconcentration are illustrated in Figure 27 (a-c). The pharmacodynamicparameters describing these relationships are presented in Table 7. Inthe control animals, there were no significant changes in maternal heartrate. There were no apparent changes in the fetal heart rate followingmaternal drug administration. In both the experimental and control90Table 6: Mean concentrations of ritodrine and its glucuronide(s) (ng/mLexpressed as liberated ritodrine) in the amniotic fluid during the maternal bolusexperiments (n = 4).TIME: -15 m 10 m 2 h 4 h 8 h 12 h 24 hIntact 3.6 4.2 8.6 5.1 2.5 3.5()1 (1.7) (1.1) (1.5) (1.1) (1.0)Glucur- N.D.2 N.D. 1.6 12.5 17.7 22.2onide(s) (0.8) (3.4) (0.3) (8.3)Sulphate(s) N.D.2 N.D. N.D. N.D. N.D. N.D.1 - Standard error of the mean2 - not detectableE 0.II300250E V200.2 C V-C150C100501000 100-J 2 C z I; 12011E(minutes)04806007200120240360Time(minutes)150125100 75 50 25050100150200250300350400450500RITODRINECONCENTRATION(rig/mi)Figure27:Relationshipsbetweenmaternalarterialritodrineconcentrationandtime,maternalheartrateandtimeandmaternalheartrateandmaternalarterialritodrineconcentration(pointsconnectedintemporalorder)inarepresentativeewe.92Table 7 Pharmacodynamic parameters (mean ± s.e.m.; n = 9) describingthe effect of ritodrine on maternal heart rate:Emax1 (bpm) 162 ± 18EC502 (ng/mL) 118.3 ± 28.5n3 1.61 ± 0.201 - Maximal increase in maternal heart rate abovecontrol value.2 - Concentration eliciting a 50 % of maximal response.3 - 11Shape” factor describing the steepness of theheart rate-concentration relationship.93groups there were no apparent effects on either fetal or maternalarterial pressure. A representative polygraph tracing following thematernal intravenous bolus administration of 50 mg ritodrine is shown inFigure 28.Maternal and fetal arterial blood gas values prior to the maternalbolus administration are listed in Table 8. The arterial blood gasresults for the ewes and fetuses of the experimental group aresummarized in Figures 29 and 30. Following drug administration therewas a statistically significant fall in maternal arterial pH,bicarbonate, and whole blood base excess indicating the development of ametabolic acidosis. There was also a significant decline in maternalarterial over the first 15 minutes following drug administration andthen a return to control values. The metabolic acidosis persisted forapproximately four hours with its peak occurring between one and threehours after drug administration. At six hours following drugadministration all maternal blood gas parameters had returned to controlvalues. Interestingly, at 90 minutes there was a statisticallysignificant decline in maternal pCO2 probably representing somerespiratory compensation for the metabolic acidosis. There were noconsistent changes with time in the fetal blood gas parameters althoughstatistically significant differences were noted at 90 minutes for fetalarterial pH and pCO2 and at 2 h for fetal arterial pCO2. These changesin fetal blood gases probably reflect the maternal blood gas alterationspresent at these times. There does not appear to be a directrelationship between the maternal arterial drug concentrations and thechanges in maternal arterial blood gas values (Figure 31).941 mmI : : .1.:258:2:020002:::i riFigure 28:Representative polygraph recording following 50 mg maternalbolus ritodrine administration, indicated by arrow. AMN- amnioticpressure (mm Hg), FAP- fetal arterial pressure (mm Hg), FHR- fetalheart rate (beats per minute), MAP- maternal arterial pressure (mm Hg),MHR - maternal heart rate (beats per minute). The gaps in the MAP andMHR traces indicate the times at which maternal arterial blood sampleswere collected.95Table 8: Maternal and fetal blood gas parameters (mean ± s.e.m.; n = 10)prior to maternal bolus administration.pHP°2 (mm Hg)pCO2 (mm Hg){HC031 (meq/L)1B.E.2 (meq/L)[Hbj3 (g/dL)02 sat.4 (%)Maternal Arterial7.511 ± 0.011151.5 ± 6.933.3 ± 1.125.7 ± 0.54.56 ± 0.499.28 ± 0.40101.7 ± 0.2Fetal Arterial7.382 ± 0.01125.4 ± 1.944.8 ± 1.426.4 ± 0.92.11 ± 0.8511.55 ± 0.4267.8 ± 3.21 - Bicarbonate concentration2 - Whole blood base excess3 - Haemoglobin concentration4 - Oxygen saturationFigure29:Effectsonmaternalbloodgasparametersfollowinga50mgmaternalintravenousbolusdoseofritodrine(mean±s.e.m.;n=10)(*indicatessignificantlydifferentfromcontrolgroup,MANOVA,p0.05).—7.DD0—7,50100200150100 60150,-j40-307.3507.2501 : Iz 1 D U) (2 >- C90**070V00/1Ti\f/P01IJ--...I-.---------.—I,.-.---.-.01653455257051440Time(mm)I•S0__7L7L0165345 TIME(mii)525—-7051440CFigure30:Effectsonfetalbloodgasparametersfollowinga50mgmaternalintravenousbolusdoseofritodrine(mean±s.e.m;n=10)(*indicatessignificantlydifferentfromcontrolgroup,MANOVA,p0.05)40 30 20 10 0/Z7.550T7,450Tc..7.3507,250 7C’ C.) 0 (1) (.1) Li U Li.) Li.) (-I—,)—5— ±9Q-80 70-60—50‘1•c,1 0 0 0 x0 0 0 C-) c.z 0 Li’)z Li.) U**40 300165345525705Time(mm)0—0—Maternal—FetalJTI01653455257051440TIME(mm)1440980OI’:too -‘6I0 240 720 1060 1440ThLE (mm)Figure 31: Representative plots of maternal arterial ritodrineconcentration vs time, maternal arterial whole blood base excess vstime, and maternal arterial lactate concentration vs time, following a50 mg maternal intravenous bolus administration.99In order to further assess the mechanism of the observed metabolicacidosis, whole blood lactate and glucose concentrations were measuredin 4 animals following maternal drug administration. Prior to drugadministration maternal arterial blood lactate concentrations were 10.65± 2.23 mg/dL (1.18 ± 0.25 nimol) and the maternal glucose concentrationswere 56.43 ± 8.63 mg/dL (3.14 ± 0.48 mmol). The corresponding fetallactate concentrations were 11.39 ± 3.59 mg/dL (1.27 ± 0.40 mmol) fromthe fetal femoral artery and 13.02 ± 2.44 mg/dL (1.45 ± 0.27 mmol) fromthe umbilical vein. Fetal glucose concentrations were 16.66 ± 1.61nig/dL (0.93 ± 0.09 mmol) from the fetal femoral artery and 17.91 ± 4.21mg/dL (1.00 ± 0.23 mmol) from the umbilical vein. Maternal arteriallactate concentrations rose progressively as the ewe became acidotic.Peak maternal arterial lactate levels were 75.59 ± 1.35 mg/dL (8.40 ±0.15 mmol) and occurred between 2 and 3 hours following drugadministration. Thereafter maternal arterial lactate concentrationsreturned to control values. The mean incremental increase in lactateAUC was 657 ± 91 mg h/100 niL (mean ± s.e.m) (n =4). Lactate eliminationwas fit by AUTOAN/NONLIN to a one-compartment model with a meanelimination half-life of 4.4 ± 0.6 h (mean ± s.e.m.). There were noapparent changes in uterine venous lactate levels. There were noconsistent changes in maternal arterial glucose concentrations followingdrug administration. Similarly fetal lactate and glucose concentrationsremained constant. In the control animals there were no changes ineither lactate or glucose concentrations in either the ewe or fetus.The mean maternal and fetal lactate and glucose concentrations,following ritodrine administration are shown in Figure 32. The meanmaternal and fetal lactate and glucose concentrations, following saline100—S 100-j TE l o—O — Maternal artery—Uterine vein—Umbilical vein75 +Eoz50 Iii o1\fF—z IILlC-) ±‘zC-)o 25w—0FC-)-----—H---—— -- I - - -. - I 1——60 240 540 840 1140 1440 1740TIME (mm)-j J 0—0 — Maternal arteEa — Uterine veinCDç 4 0—0 — Umbilical veinTT[r T/TNz I ,‘-... 1.50o j TF(-)24C)uJ(I-)0C-)D 0 I—60 240 540 840 1140 1440 1740TIME (mm)Figure 32: Mean lactate and glucose concentrations vs time following a50 mg maternal intravenous bolus administration. (n = 4)10120— Maternal artery— Fetal arteryE •—•— Umbilical vein00EzTTT.10.TTT j TF—o—O-o_--_-z 1 11 I- 1ozIOz1bJC)z0C)LU0 l—60 0 60 120 180 240 300 360TIME (mm)100] O—O— Maternal arteryE D—D — Fetal artery— Umbilical vein0C75 TEzoT T TTT T T— To 050-i- i I..FzLUC)25-rC)LU(1)0C-)0 I I—60 0 60 120 180 240 300 360TIME (mm)Figure 33: Mean lactate and glucose concentrations vs time following a15 mL maternal intravenous normal saline dose. (n = 2)102administration (control) are shown in Figure 33. The maternal arteriovenous fluxes of glucose and lactate were estimated by multiplying thedifference between the maternal arterial and uterine venousconcentrations of glucose and lactate respectively by a value foruterine blood flow rate (1055 mL ± 131 mL/min found in other studies inthe laboratory(van der Weyde, 1990)) and are presented in Figure 34 inboth a control and experimental animal.The intrauterine pressure recording was used to determine thepresence of low amplitude rhythmic uterine contractures during theexperimental period. In those animals of the drug treated group sixanimals had recordings of sufficient quality to allow measurement ofcontractures for 24 hours. In these animals there were an average of15.2 contractures over 24 hours per animal (15.2 ± 3.0; mean ± s.e.m.; n= 6) with each contracture lasting approximately 10.4 minutes (10.4 ±0.7; mean ± s.e.m) (Table 9). There were relatively few contractures inthe first 3 hours following drug administration, however, the durationof the contractures remained relatively constant during the experiment.In the maternal bolus control group 4 records were suitable foranalysis. There were an average of 17.5 contractures per animal (17.5 ±.7; mean ± s.e.m.; n = 4) with each contracture lasting approximately94 minutes (9.4 ± 0.8; mean ± s.e.m) (Table 9). There are nosignificant differences between either the total number of contracturesor the duration of the contractures between the experimental and controlgroups (Multiple analysis of variance (MANOVA), p > 0.05). Thedistribution of the contractures during the experimental period,however,was significantly different between the control and experimentalgroups (Figure 35). During the first six hours post-drug administration1031 000 T Fetal Glucose/Lactate Fluxes E6 1 7control750 °-° — LactateI • — Glucose500250::—750—1000 I I I I I0 60 120 180 240 300 360Time (mm)MATERNAL LACTATE AND GLUCOSE FLUXES E2481E4 1000750°j O0D—DI / •__ D—D — Lactate EI •—•— Glucosei°__—2500’• \ /C’)—5000 500—7500•-z z—1E4 I I—10000 180 360 540 720 900 1080 1260 1440lime (mm)Figure 34: Estimated uterine lactate and glucose fluxes followingsaline (upper graph) and maternal ritodrine bolus (50 mg) administration(lower graph). Positive values indicate net uterine uptake of thecomponents from the circulation. Negative values represent theirrelease from the uterus into the maternal circulation.104Table 9Uterine contracturesEXPERIMENTAL GROUP1057661109163NO TAGMEAN ±GROUP617248NO TAG__________141MEAN ±CONTROLTOTAL NUMBER OFCONTRACTURES’1342121102215.2 ± 7.4DURATION OFCO NT RACTU RES7.4 ± 2.410.5 ± 3.39.9 ± 3.711.9 ± 2.610.4 ± 3.212.2 ± 3.410.4 ± 1.71 - Number of contractures per 24period.hour experimentalEWES.D.17 9.2 ± 1.618 8.2 ± 2.519 11.8 ± 3.516 8.5 ± 2.9S.D 17.5 ± 1.3 9.4 ± 1.62 - Duration of contractures (mean ± s.d.)105814(1)LiJ*F—41—zo:fffA4_0 360 720 1080 1440Time (mm)8-J6_____0 360 720 1080 1440Time (mm)Figure 35. Uterine contractures per six hour time period followingmaternal ritodrine (upper graph) or saline (lower graph) administration.(*- significantly different from control; MANOVA, p = 0.018).106there were significantly fewer contractures in the experimental group ascompared to the same time period in the control group (MANOVA, p =0.018). Thereafter there were no significant differences between thecontrol and experimental groups in the number of contractures per sixhour period.In a relatively few animals the recording of fetal intratrachealpressure and/or fetal tracheal fluid flow rate allowed the presence offetal breathing movements to be determined during the experimentalperiod. This was possible in 3 experimental and 2 control animals. Inthe experimental animals fetal breathing movements were present 27 ± 4.9% of the time (mean ± s.e.m.) while in the control group breathingmovements were present 45 ± 4 % (mean ± s.e.m.). The percentage offetal breathing time in the experimental animals was not significantlydifferent from that in the control period preceding the ritodirneadministration (30 ± 5 %; mean ± s.e.m.).3.4. Fetal Bolus StudiesTable 10 lists the ewes on which experiments were performed, thenumber of fetus present, the fetal gestational age at the time ofexperiment, the gestational age at birth, and the fetal birthweightIn the experimental group, maternal weight averaged 80.4 ± 10.2 kg (mean± s.d.) and the mean gestational age at the time of experiment was 133.8± 2.8 days (mean ± s.d.). The average number of fetus in theexperimental group was 2.2 ± 0.8 (mean ± s.d.) with the gestational ageat birth being 138.0 ± 1.9 days (mean ± s.d.) and the weight of theoperated fetus at birth, 2768 ± 970 g (mean ± s.d.). In the control107Table 10Experimental details for the fetal bolus experiments.EWE# Maternal # of GAb BAC BWdweight fetusExperimental Animals105 68.1 1 133 135 1480*617 82.3 2 136 138 2873*, 2547338 95.5 3 130 139 2318*, 2721,1400137 81.8 2 133 138 3063*, 2874201 74.5 3 137 140 4110*, 3320Control Animals105 68.1 1 130 135 1480*201 74.5 3 129 140 4110*, 3320a - Maternal weight (kg)b - GA - gestational age, in days, at the time ofexperimentc - BA - gestational age, in days, at time ofdeliveryci - B - Birthweight of fetus in grams( indicates operated fetus)108group, maternal weight was 71.3 kg (mean) and the mean gestational ageat the time of experimentation was 129.5 days (mean). The averagenumber of fetus in the control group was 2 (mean) with the gestationalage at birth being 137.5 (mean) and the weight of the operated fetus atbirth, 2795 g (mean).Similar to the maternal bolus experiments, AUTOAN best fit thefetal arterial plasma concentrations to a bi-exponential equation in allanimals. Figure 36 shows the concentration vs time profiles ofritodrine in the various sampled fluids following fetal bolusadministration in a representative animal. At no time was drugdetectable in either maternal arterial or uterine venous plasmafollowing fetal bolus administration. The volume of distribution ofritodrine in the fetus was quite large (Vdarea 8.7 ± 1.8 L/kg; Vd55 8.5± 2.6 L/kg; V 1.6 ± 0.5 L/kg; mean ± s.e.m.). None of the volumes ofdistribution in the fetus were significantly different from thoseobserved in the mother (unpaired t-test, p > 0.05). The fetal totalbody clearance Cdt) in these experiments was 31.7 ± 10.0 mL/min/kg.This value was found to be significantly different (ANOVA, p < 0.05)than the fetal total body clearance reported following fetal intravenousadministration (Clt = 62.0 ± 8.1 mL/min/kg; mean ± s.e.m.) but notsignificantly different (ANOVA, p > 0.05) from the maternal total bodyclearance reported following maternal intravenous bolus administration(Cit = 24.3 mL/min/kg). The tl/2a in these experiments was 0.2 ± 0.1 hand the terminal elimination half-life (t1/2fl) was 2.9 ± 0.7 h) (mean ±s.e.m.). The fetal tl/2a was not significantly different from thematernal tl/2a calculated following maternal bolus administration (t1/2maternal 0.5 ± 0.1; mean ± s.e.m.) (unpaired t-test; p > 0.05). The1091000— Fetal arterial— Tracheal fluid-J— Amniotic fluid.‘0’.Cc 1000Czsc1)C) I o003 10•2SC-C01—0 360 720 1080 1440 1800Time (min’Figure 36: Representative plot of ritodrine concentration vs time inthe ewe and fetus following a 2 mg fetal intravenous bolus.110fetal terminal elimination half-life observed in the fetal bolusexperiments was not significantly different (unpaired t-test; p > 0.05)from the apparent fetal terminal half-life (apparent t1/2 2.3 ± 0.9;mean ± s.e.m.) observed during the maternal bolus experiments but wassignificantly different (unpaired t-test; p < 0.05)from the maternalterminal elimination half-life (maternal t1/2 8.7 ± 1.8 h). The meanfetal arterial AUC in the fetal bolus experiments was 647 ± 169 ng h/mL(mean ± s.e.m.) and was not significantly different from the maternalarterial AUC values calculated during the maternal bolus experiments(maternal arterial AUC 657 ± 117 ng h/mL; mean ± s.e.m.) (unpaired ttest, p > 0.05). Similarly, the mean fetal arterial AUMC (4709 ± 2820ng h2/mL; mean ± s.e.m.) was not significantly different from thematernal arterial AUMC observed following maternal bolus administration(5361 ± 1854 ng h2/mL; mean ± s.e.m.) (unpaired t-test, p > 0.05).Also, the mean dose that the fetus received in the fetal bolusexperiments (0.9 ± 0.2 mg/kg; mean ± s.e.m.) was not significantlydifferent from that which the mother received during the maternal bolusexperiments (0.7 ± 0.1 mg/kg; mean ± s.e.m.) (unpaired t-test, p >0.05). The pharmacokinetic parameters derived from fetal arterialplasma are summarized in Table 11.In the three animals in which tracheal fluid was sampled, drugaccumulation and persistence in the fetal tracheal fluid followed asimilar pattern to the fetal infusion and maternal bolus studies.Although measurable concentrations of ritodrine appeared in the firstsample, the peak ritodrine concentration in tracheal fluid did not occuruntil 20 - 45 minutes after the dose was given. Peak tracheal fluidconcentration was 632.9 ± 255.3 ng/mL (mean ± s.e.m.). In the fetal111Table 11: Pharmacokinetic parameters (mean ± s.e.m.; n = 5) obtainedfrom fetal intravenous bolus dosing (2 mg ritodrine hydrochloride).CLf 31.7 ± 10.0(me/mi n/kg) 1Vd 1.6 ± 0.5(17kg)8.7 ± 1.8Vdss 8.5 ± 2.6(L/kg)AUC2 647 ± 169(ng h/mL)AUMC3 4709 ± 2820(ng h2/mL)t112 a 0.2 ± 0.1(12.9 ± 0.71. Values are expressed per kilogram of in utero fetalbody weight.2. AUC (zero to infinity) determined by thetrapezoidal approximation.3. AUMC - area under the first moment of theconcentration vs time curve from zero to infinity.Cit = Dose/AUCVd =Vdarea = Dosé/(AUC x )Vd = Dose x AUMC/ (AUC)2112infusion studies (Section 3.2.) peak tracheal fluid levels were 64.2 ±4.9 ng/mL (mean ± s.e.m.). In the fetal bolus studies, ritodrinepersists in the tracheal fluid in measurable concentrations for at least24 h post-dose as was noted in the fetal infusion experiments. Theapparent elimination half-life from tracheal fluid is 2.3 ± 0.2 h (mean± s.e.m.) which is not significantly different from the half-lifeobserved in fetal arterial plasma (unpaired t-test; p > 0.05).In the amniotic fluid ritodrine appears to accumulate and persistsimilar to the fetal infusion and maternal bolus studies (albeit athigher levels). In all animals, intact ritodrine concentrations arebelow 10 ng/mL for the first two hours post-dose. Beginning atapproximately four hours there is a marked accumulation of ritodrinetoward an apparent plateau value of 165.0 ± 86.2 ng/mL (mean ± s.e.m.)which occurred between 4 and 8 hours. After this time the concentrationof intact ritodrine declined slowly and sporadically, but was stillpresent in measurable concentrations 24 hours post-dose. Samples ofamniotic fluid taken at -15, 10 mm, 2, 4 ,8, and 24 h and incubatedwith either sulphatase or glucuronidase did not demonstrate the presenceof any sulphate conjugate(s) of ritodrine. However, in common with thefetal infusion experiments (section 3.2, Figure 25) and the maternalbolus experiments (Section 3.3), there appears to be glucuronideconjugate(s) present in measurable quantities at 2 hours post-dose. Theconcentration of the glucuronide conjugate(s) in the amniotic fluidduring these experiments was approximately 3 fold greater than those ofthe intact ritodrine (calculated as liberated intact ritodrine). Theglucuronide conjugate(s) appears to persist in the amniotic fluid for at113Table 12: Mean concentrations of ritodrine and its glucuronide(s) (ng/mL expressedas liberated ritodrine) in the amniotic fluid during the fetal bolus experiments (n= 5).TIME: -15 in 10 m 2 h 4 h 8 h 12 h 24 hIntact - 4.7 31.8 70.7 168.3 170.8 35.5- -(21.1)1 (20.8) (125.6) (109.6) (19.6)Glucuronide(s) - N.D.2 14.4 252.9 352.4 603.6 379.0-(3.8) (87.0) (156.2) (235.6) (121.1)Sulphate(s) - N.D. N.D. N.D. N.D. N.D. N.D.1 - Standard error of the mean2 - not detectable114least 24 hours post-dose. The mean concentrations of the glucuronideconjugates are shown in Table 12 at various times during the experiment.During the course of the fetal bolus experiments qualitativelydifferent fetal cardiovascular responses were observed when compared tothe maternal cardiovascular responses noted after maternal bolus dosing.The most surprising observation was that there was no immediate increasein fetal heart rate following the administration of the fetal ritodrinebolus comparable to that observed in the mother during the maternalbolus experiments (Figure 37) . In some animals there was an initialtachycardia lasting for only about 2 minutes which was followed by adecrease in fetal heart rate (Figure 38). There was, however, a slowerprogressive rise in fetal heart rate to a maximum of 40 beats perminute above control values (Figure 39). Fetal heart rate remainedelevated for at least 4 hours following the fetal bolus after which timefetal heart rate declined toward control values (Figure 39).Furthermore, mean fetal arterial pressure fell significantly (paired ttest, p < 0.05) from control values of 47.6 ± 0.8 mmHg (mean ± s.e.m.)to 34.6 ± 1.96 mmHg (mean ± s.e.ni.) at 3.6 ± 0.6 minutes (mean ± s.e.m.)immediately following the fetal bolus (Figure 40). This represents amean fall in fetal arterial pressure of 13 ± 2.1 nimHg or 27 ± 0.04 %(mean ± s.e.m.). The fetal arterial pressure then returned to controlvalues at 13.2 ± 1.88 minutes following drug administration (Figure 37).In the control animals initial mean fetal arterial fetal pressure was51.7 mm Hg and this value remained constant throughout the duration ofthe experiment. There were no apparent changes in either maternal heartrate or mean arterial pressure. A representative polygraph tracing,115following the fetal intravenous administration of a 2 mg bolus ofritodrine is shown in Figure 37.The maternal and fetal blood gas values prior to fetal bolusadministration are presented in Table 13. The arterial blood gasresults for the fetuses and ewes of the experimental group aresummarized in Figures 41 and 42. Following fetal drug administration,there were statistically significant decreases in fetal arterial p1-f,bicarbonate and base excess indicating the development of a metabolicacidosis. There were also decreases in fetal arterial P02 butstatistically significant decreases were only seen at 30 and 720minutes. There were no statistically significant changes in fetalarterial pCO2. The metabolic acidosis persisted at statisticallysignificant levels for approximately 6 hours with the peak acidosisoccurring 2 hours after drug administration. Thereafter, the fetalblood gas parameters of the experimental group fetuses were notsignificantly different from those of the control group. There were nosignificant changes in any of the maternal arterial blood gasparameters. There does not appear to be a direct relationship betweenthe fetal arterial ritodrine concentrations and the changes in fetalarterial blood gas values (Figure 43).Prior to drug administration fetal arterial blood lactateconcentrations were 15.26 ± 1.64 mg/dL (1.70 ± 0.18 mmol) (mean ±s.e.m.) and the fetal arterial glucose concentrations were 12.73 ± 5.42mg/dL (0.71 ± 0.30 mmol). The corresponding umbilical venous lactateand glucose concentrations were 16.79 ± 1.87 mg/dL (1.87 ± 0.21 mmol)and 18.18 ± 2.44 nig/dL (1.01 ± 0.14 mmol). The corresponding maternallactate concentrations were 8.50 ± 1.01 mg/dL (0.94 ± 0.11 mmol) from1161 miiia 25tU1It!_Lj,LO 11111z08O8:b 2$2580IU0.24060250-*-—‘‘I20060200200I--.-—.. V-Figure 37: Polygraph tracing following a 2 mg fetal bolusadministration of ritodrine (a- low speed recording, b - high speedrecording). AMN- amniotic pressure (mm Hg), FAP - fetal arterialpressure (mm Hg), MFA - mean fetal arterial pressure (mm Hg), FHR -fetal heart rate (beats per minute), Tr P - tracheal pressure (mm Hg),MAP - maternal arterial pressure (mm Hg), MHR - maternal heart rate (mmHg). The arrows indicate the time of ritodrune administration. Gaps inthe traces of panel a indicate the times at which samples werecol 1 ected.1 mmE201ControlE617Control-E617//-E2017E10533817516315Time(minutes)Figure38:Fetalheartraterecordedeachminutefor10minutespriortoand10minutesafter2mgritodrinefetalbolus(ewes617,201,105and338)orcontrolsalineinjection(ewes201and617).225-75ci)**—‘SDI2CC-i-67Eci) a175-J7jNJJ58.5ci) L.•NTTU, C’,ci-)ci)-aIL1504J•50—-rIC&—-——-Qci)CIL.41.5<IC-4-,C*4-,LcLL1001.33—303090150210Time(mm)Figure39:Averagefetalheartrate(closedcircles)andarterialpressure(opencircles)(mean±s.e.m.)over30minuteintervalsbeforeandafter2mgritodrinebolus.(*-significantlydifferentfromcontrolvalue,p<0.05,ANOVAwithrepeatedmeasures).H H U,75E201ControlE617ControlE617-65-E201EE105E55•1) Cl) C’)4510 V354-I CL)25------t—15—12—9—6—303691215Time(minutes)Figure40:Fetalarterialpressurerecordedeachminutefor10minutespriortoand10minutesafter2mgritodrinefetalbolus(ewes617,201,105and338)orcontrolsalineinjection(ewes201and617).H H120Table 13: Maternal and fetal blood gas values prior to fetalintravenous bolus administration. (mean ± s.e.m.; n = 5)pH°2 (mm Hg)pCO2 (mm Hg)[HC03] (meq/L)1B.E.2 (meq/L)[Hbj3 (g/dL)02 sat.4 (%)Maternal Arterial7.466 ± 0.017116.0 ± 9.834.9 ± 1.824.9 ± 0.82.85 ± 0.79.6 ± 0.497.7 ± 2.0Fetal Arterial7.308 ± 0.01021.1 ± 2.049.1 ± 1.124.2 ± 0.7-1.1 ± 0.713.2 ± 0.747.8 ± 4.01 - Bicarbonate2 - Whole blood base excess3 - Haemoglobin concentration4 - Oxygen saturationconcentration-J C ci ci U x U U Cz E E 0 C 0 L ciFigure41:Effectsonfetalbloodgasparametersfollowinga2mgfetalintravenousbolusdoseofritodrine(mean±s.e.m.;n=5)(*indicatessignificantlydifferentfromcontrolgroup,MANOVA,p0.05).7.400-4*4***U.,/17.200k\t/jIi7.1001*7030•20 10 0 55503*fl**********40—-)—20—12I 8 845C- 0 a) L3501 20480660Time(mm)0io300480Time(mm)/7660380N;e •125C-’ 0’ I E 0 0 L. 0)Figure42:Effectsonmaternalbloodgasparametersfollowinga2mgfetalintravenousbolusdoseofritodrine(mean±s.e.m.;n=5).Nosignificantchangeswereobserved(MANOVA,p>0.05).150—7.6007.500j 7.400-9.-J 0 1:754IT•J/JJTT.I________ILII•I//i.111IT//\T______VTT---.1iI1\T, 11//50‘—S 6 c’ C >5 x DI00170/?Ie/N0) I E(N0 0 0 C 0)T40—35t 30— 25I,01203004806601380Time(mm)0120300480Time(mm)//660360123the maternal femoral artery and 5.98 ± 0.80 mg/dL (0.66 ± 0.10 mmol)from the uterine vein. Maternal glucose concentrations were 54.13 ±4.31 mg/dL (3.01 ± 0.24 mmol) from the maternal femoral artery and, in asingle animal, was 46.13 ± 0.17 mg/dL (2.56 ± 0.01 mmol) from theuterine vein. Fetal arterial lactate concentrations rose progressivelyas the fetus became acidotic (Figure 44). Peak fetal arterial lactatelevels were 56.01 ± 6.33 mg/dL (6.22 ± 0.70 mmol) and occurred between 1and 10 hours following drug administration. The mean peak incrementalincrease in fetal arterial lactate concentrations is smaller than thatobserved in the ewe during the maternal bolus experiments (unpaired ttest, p < 0.05). Thereafter fetal arterial lactate concentrationsreturned to control values. The mean incremental increase in fetalarterial lactate AUC was 904.3 ± 59.6 mg h/100 mL (mean ± s.e.m) (n = 5)and is significantly greater than that observed in the ewe during thematernal bolus experiments (unpaired t-test, p < 0.05). In general, thefetal arterial lactate concentrations achieved during the fetal bolusexperiments were lower but persisted for much longer than the maternalarterial lactate concentrations during the maternal bolus studies(Figure 44). There is also a complimentary rise and fall in umbilicalvenous lactate concentrations (see Figure 44) as compared with the fetalarterial lactate concentrations. Also, there is an increase in theumbilical venous-femoral arterial lactate difference which is maximal atapproximately 6 hours. Thus the incremental AUC for the umbilicalvenous lactate (1179 ± 228 mg h/dL) (mean ± s.e.m.) is larger than thatfor fetal arterial blood (904.3 ± 59.6 mg h/dL). There also seems to bea dissociation between the arterial lactate concentration and thepersistence of the fetal acidemia (see Figure 43) which is in contrast124to the observations in the maternal arterial plasma during the maternalbolus experiments (see Figure 31). There were no changes in eithermaternal arterial or uterine venous lactate concentrations (see Figure44). There was an accumulation of lactate in the amniotic fluidsuggesting that this is an important route of removal of lactate fromthe fetus (Figure 44). The lactate in the amniotic fluid persisted atmaximal concentrations for at least 48 hours (Figure 44). There werealso increases in fetal arterial glucose concentration beginning 5 - 10minutes after dosing and lasting approximately 2 hours (see Figure 44).There were no changes in glucose concentration in the umbilical vein,maternal artery, uterine vein, or amniotic fluid. There were nosignificant changes in either lactate or glucose concentrations in anyof the biological fluids sampled from the control animals (see Figure45). The umbilical (i.e. fetal) arterio-venous uptakes of glucose andlactate were estimated by multiplying the difference between the fetalarterial and umbilical venous concentrations of glucose and lactaterespectively by a value of umbilical blood flow (242 ± 28 mL/min/kg)found in a previous study in the laboratory (van der Weyde, 1990)) andare presented in Figure 46 in both a control and experimental animal.Although in the control animal there is net uptake of both glucose andlactate by the fetus, following ritodrine administration there is aperiod (4 hours) of net fetal loss of both compounds (Figure 46).Following this there appears to be net fetal uptake of lactate from theplacenta at much greater rates than in the control state. This netuptake of lactate persists until control values are reached at 48 hourspost-dose.1251000-JEc 100 0oo2NC) O—0o 10‘1)C-o.25-J0t)000oC’) /0xoz00ci,Cl)0•000-1 0 /03-00U-—15100-JE0o /°\0_o/p 0C 0.2 5O o—02 90cci,0 00C 250o‘2)c,to 0-J0 360 720 1080 1440 1800Figure 43: Representative plots of fetal arterial ritodrineconcentration vs time, fetal arterial whole blood base excess vs time,and fetal arterial lactate concentrations vs time, following a 2 mgfetal intravenous bolus administration.1261 00-0 0 — Fetal artery• —• — Umbilical VeinA — A — Maternal arteryE A — A — Uterine Veino 0—0 — Amniotic Fluid0 75-T T I 0T•• IE /lJ •oC 50 10-4-,-4-,C TP25U) C()C00 IC)U)—fl—— — S(S — —0____________________________________-4-,C-)o 0 I I 9—60 300 660 1020 1380 1740 2100 2460 2820Time (mm)25 T oo — Fetal artery• —. — Umbilicol Vein2 — Maternal arteryA — A — Uterine VeinC 0—0 — Amniotic Fk,idC0’0>1 { jiC2Ca,C,CC(-)10004z I I I IC? 5E —60 300 660 1020 1380 1740 2100 2460 28200o lime (mm)OS TTAT T T TC.2 50aL41)0255T1 r TC)0) i9--?—-----—-----0o___________—0I I I—60 300 660 1020 1380 1740 2100 2460 2820Time (mm)Figure 44: Mean lactate and glucose concentrations vs time following a2 mg fetal intravenous bolus administration. Inset figure shows theglucose concentrations on an expanded scale. (n = 5)127100 0—0 — Fetal artery— Umbilical Vein— Maternal arteryE A—A— Uterine Vein0 EJ—LJ— Amniotic Fluid0 75.-Co 500C0C-)c 25±o__________A4-,o—60 300 660 1020 1380 1740 2100 2460 2820Time (mm)100 0—0— Fetal artery— Umbilical Vein_J— Maternal arteryE A—A— Uterine Vein00 75 0—0 — Amniotic FluidEo 1C5IITTIo1_C00c 250C-)Q)(I)o0:3 O-.i°-°i I—60 300 660 1020 1380 1740 2100 2460 2820Time (mm)Figure 45: Mean lactate and glucose concentrations vs time following a0.7 mL fetal intravenous normal saline dose. (n 2)128In the experimental group all records (n = 5) were suitable for theanalysis of uterine contractures. In these animals there were anaverage of 14.6 ± 4.6 (mean ± s.e.m.) contractures during the first 24hours of the experiment with each contracture lasting an average of 8.54± 0.5 minutes (mean ± s.e.m.) (Table 14). There was no significantdifference between either the number or duration of uterine contracturesbetween the fetal bolus experimental group and the maternal boluscontrol group (ANOVA, p > 0.05). The distribution of contractureswithin the 24 period was also not significantly different from thematernal bolus control group (MANOVA, p > 0.05) (Figure 47).3.5. Acute Microsphere Studies following Fetal Bolus AdministrationTable 15 gives the experimental details of the two animals used inthese studies. Concentrations of ritodrine in the fetal arterial plasmafollowing a 2 mg fetal bolus intravenous injection were as follows(time, mean (Ewe 402:Ewe 272)): 5 minutes, 399.1 ng/mL (317.2:480.9); 20minutes, 148.2 ng/mL (144.4:152.1); 60 minutes 88.3 ng/mL (83.3:93.3).The plasma concentrations determined in these animals do not appear tobe different from those observed at the same sampling times in thechronically instrumented animals (See section 3.4). Fetal arterialblood gases were determined during the control period and at thesampling times indicated and are listed in the following table (Table16). The two fetuses used in the microsphere experiments are acidemicrelative to normal and when compared to those chronically instrumentedfetus used in the fetal bolus experiments (Section 3.4). The changes infetal arterial blood gas parameters in these experiments were similar to1291 000Umbilical Glucose and Lactate Uptakes E201750— °—° — Lactate— Glucose.2 500250 I •E 0 0x:3G —250Z—500—750—10001 I—120 240 600 960 1320Time (mm)750•150:./\o:°00C)250—50() I (3- ti 2—500 •—ioo—750 1—150—60 360 780 1200 1620 2040 2460 2880Time (mm)Figure 46: Fetal fluxes of glucose (open symbols) and lactate (closedsymbols) following saline (upper graph) and 2 mg fetal ritodrine bolus(lower graph) administration. Postive values represent fetal uptake ofcompounds from the placenta. Negative values represent fetal loss tothe placenta.130Table 14Uterine contracturesEWE TOTAL NUMBER OFCONTRACTIJRE Si-DURATION OFCONTRACIURESZEXPERIMENTAL GROUP1 - Number of contractures per 24period.hour experimental2 - Duration of contractures (mean ± s.d.).3 - Only first 17.2 hours of record useable.4 - Only first 4 hours of record useable.± S.D.617 15 9.1 ± 3.1105 8 9.4 ± 1.5338 32 8.4 ± 1.7137 11 9.2 ± 4.7201 7 6.6 + 1.4MEAN 14.6 8.5 1.1CONTROL GROUP201 9 7.6 ± 1.6105 3 7.3 ± 3.2kMEAN ± S.D 6 ± 4.2 7.5 ± 0.2± 10.2 +8-J z6cn bJ D F— C) F— z 0 C) z w F—01440Time(mm)Figure47:Uterinecontracturespersixhourtimeperiodsfollowingfetalbolusritodrine(2mg)administration03607201080H132Table 15Experimental details of animals used in acute microsphere experiments.EWE# # of GAa 8wbfetus272 3 132 2508*, 2673, 2400402 2 130 3496*, 1688a - GA - gestational age, in days, at the time ofexperimentb - BW - Birthweights of fetus in grams (*operated fetus)133those observed during the fetal bolus administration to the chronicallyprepared animals. Also similar to the chronic fetal bolus studiesdescribed previously there was a significant fall (mean decrease 24.5 mmHg at 2 minutes post-dose) in fetal arterial blood pressure followingdrug administration. However, in contrast to the chronic fetal bolusstudies, the arterial pressure did not return to control values even at60 minutes post-dose. Also unlike the chronic fetal bolus experimentsthe fall in fetal arterial pressure was accompanied by a significantincrease in fetal heart rate (mean increase 79 beats per minute abovecontrol). The heart rates were maximal 1 minute post-dose andthereafter decreased at a mean rate of 2.04 beats per minute per minute.In both cases the fetal heart rate returned to control values at 26 (Ewe402) and 55 (Ewe 272) minutes (Figure 48).A sample of fetal bile (250-300 L) was obtained from Ewe 272 at60 minutes post-dose. The intact ritodrine concentration at this timewas 488.6 ng/mL in the bile as compared to 93.3 ng/mL in the plasma.There was not sufficient fetal bile to determine the presence ofglucuronide or sulphate conjugates.The regional blood flows observed during these experiments arelisted in the following Table (Table 17) and are presented as the meanof the two animals in mL/min/100 g of organ weight. Some of the changesobserved, particularly those in the CNS, are probably the result of theprogressive acidemia observed in these fetus during the course of theseexperiments. The acidemia results both from the ritodrine and the acutenature of the preparation. The increased flow to the heart is likely areflection of the tachycardia induced by ritodrine.134Table 16Fetal femoral arterial blood gas results from acute microsphereexperiments.EWE # Parameter Control 5 mm 20 mm 60 mm402 pH 7.228 7.214 7.229 7.211pCO2 (mmHg) 48.7 51.2 48.6 50.2p02 (mmHg) 15 13 15 14B E (meq/L)1 -6.4 -6.4 -6.4 -6.9[HCO3] (meq/L) 19.9 20.2 19.9 19.7TCO2 (mmHg)2 21.2 21.6 21.2 21.1[Hbj (m/dL)3 nd5 13.8 13.4 13.502S (%)4 nd 27.8 31.9 24.8272 pH 7.226 7.257 7.206 7.203pCO2 (mmHg) 47.2 41.4 51.5 51.7P°2 (mmHg) 30 34 33 32B E (meq/L) -7.0 -7.1 -6.8 -6.9[HCO3f (meq/L) 19.2 18.1 20.0 19.9TCO2 (mmHg) 20.5 19.2 21.4 21.3[Hb] (mg/dL) 14.1 12.7 13.5 13.102S (%) 77.1 85.7 79.8 80.41 - Whole blood base excess2 - Total CO2 content3 - Haemoglobin concentration4 - Oxygen saturation5 - not determinedI Tflifl 135251 him.1 j .[1 1_ 1 1iLLLJ080Izc2.U—080LL0Figure 48:Representative polygraph tracing during acute microspherestudy. AMN- amniotic pressure (mm Hg), FAP- fetal arterial pressure(mm Hg), MFA - mean fetal arterial pressure (mm Hg), FHR - fetal heartrate (beats per minute). The time of the ritodrine injection isindicated by the arrow.36Placental blood supply as represented by cotyledonary blood flow isinitially reduced to 80 % of the control value but then tends to moveback toward control levels. These changes are mirrored in the chorioallantois, however, the flow to the amnion was extremely low, reflectingthe limited vascularization of the membrane in sheep (Steven andMorriss, 1975).In spite of initial decreases in blood supply to the left and rightkidney and adrenals, there is marked increase in flow to the perirenalbrown fat. This increase in perirenal brown fat supply is mirrored byincreases in supply to the skin of the hind limb. In contrast, flow tothe hind limb skeletal muscle and bone decreased.In general terms, there appears to be a significant fall in cardiacoutput in response to the ritodrine bolus. This may be inferred fromthe large decreases in flow to the umbilical circulation (cotyledonaryflow), kidneys, gastrointestinal tract, and fetal hind limb (femurs,biceps femoris muscles, hind skin), which is representative of overallcarcass blood flow. Together these organs and tissues account for z80 %of the fetal biventricular output. These decreases in flow are not theresult of the deterioration of the fetus from the progressive acidemiaas is demonstrated by the consistency of the flow measurements madeprior to the ritodrine administration: cotyledons 152.1:161.5, rightfemur 13.7:11.8, left femur 11.4:10.9, right biceps femoris 8.4:10.3,left biceps femoris 9.9:10.5, left kidney 121.4:127.5, right kidney118.0:127.5, omasum 22.7:21.9, abomasum 75.6:66.4, small intestine148.8:136.8 mL/min/lOOg organ weight.137TABLE 17: Regional flows in the fetus following a ritodrine 2 mg fetalbolus administration.REGION CONTROL 5 MINUTES 20 MINUTES 60 MINUTESLeft FrontalHemisphere 1675a,b 176.5 164.7 171.5Right FrontalHemisphere 174.1 182.1 171.0 181.5Left TemporalHemisphere 161.4 146.7 152.4 153.8Right TemporalHemisphere 160.6 157.2 150.8 155.4Pituitary 198.4 220.9 219.5 218.4Spinal Cord 162.5 244.1 209.5 187.2Cerebellum 272.5 309.1 280.5 314.2VentralMedulla 349.6 470.3 430.6 446.1DorsalMedulla 380.5 444.3 420.4 408.4Pons 296.0 355.1 357.4 363.2Colliculi 263.3 297.3 287.8 312.9Thalami 242.9 278.5 275.2 286.1CorpusStriatum 156.2 160.1 157.9 158.5Midbrain 227.5 219.0 221.4 230.0NuchalMuscle 7.39 8.5 8.4 7.4Left SupraspinatusMusciec 3.8 2.9 2.9 3.7Right SupraspinatusMuscle 6.2 8.0 6.7 5.8a - mean of two animalsb - flows in mL/min/100 g organ weightc - left brachial artery cannulated138TABLE 17 (continued)REGION CONTROL 5 MINUTES 20 MINUTES 60 MINUTESLe ftVentricle 3823a,b 546.0 391.6 360.3RightVentricle 327.8 523.2 334.3 322.6Interventri cul arSeptum 338.6 516.5 336.1 320.9Diaphragm 10.6 15.8 22.4 22.7Omasum 22.3 14.6 19.1 17.6Abomasum 71.0 38.8 46.7 51.1Rumen 7.83 6.04 8.15 7.18Reticulum 14.3 13.4 11.8 13.8LiverC 3.6 2.1 4.3 2.3Spleen 373.3 111.0 185.9 183.0Gall Bladder 49.1 55.1 62.4 48.4Right Kidney 120.8 70.5 87.5 89.9Left Kidney 124.5 76.7 93.8 99.6RightBrown Fat 36.3 172.5 164.7 152.6LeftBrown Fat 39.2 175.6 166.3 161.1RightAdrenal 503.8 433.2 428.9 426.8LeftAdrenal 554.3 448.9 457.5 467.5a - mean of two animalsb - flows in mL/min/100 g organ weightc - hepatic arterial flow139TABLE 17 (continued)REGION CONTROL 5 MINUTES 20 MINUTES 60 MINUTESSmallIntestine 145•6a,b 79.2 91.8 96.4LargeIntestine 60.7 60.1 77.8 66.9Right BicepsFemoris 9.4 2.4 9.4 8.6Left BicepsFemoris 10.2 2.3 9.8 9.0Right HindSkin 13.3 33.7 29.6 31.5Left HindSkin 16.5 38.3 34.6 34.7Right Femur 12.7 3.7 5.9 6.5Left Femur 11.2 3.0 4.7 5.4Cotyledons 156.8 125.6 132.7 142.7Amnion 0.2 0.2 0 0Chorioallantois 14.8 3.3 5.8 6.6a - mean of two animalsb - flows in mL/min/100 g organ weight1404. DISCUSSION4.1. Development of GC-ECD Method of AnalysisTo date, there has been no GC method reported for the quantitationof ritodrine from biological fluids. The assay method presented herehas advantages in sensitivity, selectivity and reproducibility overpreviously published techniques (Thomas et a)., 1982; Lin et a)., 1984;Kuhnert et a)., 1983; Gross et a!., 1987).The RIA method developed by Thomas et a). (1982) provides excellentsensitivity (viz. 0.1 ng/mL from a 100 #L plasma sample). However, theantiserum is not commercially available and does not appear to beavailable in quantities sufficient for pharniacokinetic studies.Furthermore, the coefficient of variation at concentrations below 5ng/mL exceeds 10 % and at least some of the variability has beenascribed to cross reactivity of the antisera (Lin et al., 1984).Both Lin et a!. (1984) and Kuhnert et al. (1983) have developedHPLC methods employing electrocheniical detection of the oxidation of thephenolic groups of ritodrine. Although both methods report goodsensitivity (viz. 200 pg injected (Lin et al, 1984); 310 pg injected(Kuhnert et al., 1983) both methods require the extraction of relativelylarge volumes of plasma/serum (1 mL) for analysis. Given that it isdesirable to conduct analyses in duplicate, such volumes would requirethe withdrawal of unacceptably large volumes of whole blood,particularly from the fetus. Further, the use of such a large volumewould only allow the determination of drug concentration and no othermetabolic or blood gas parameter. In addition, the precision of the141assay of Lin et al. (1984) is relatively poor ( CV > 10 % at 20 ng/mL).The precision of the assay of Kuhnert et al. (1983) is not presented bythe authors although they found a coefficient of variation of 3.4 % at aconcentration of 20 ng/mL.Gross et al. (1987) developed an HPLC fluorescence detection methodfor ritodrine. This method has superior reproducibility as compared tothose previously mentioned. However, this method again requires theextraction of 1 mL plasma samples making duplicate analysis difficultand the small reconstitution volume (100 iiL) and large injection volume(100 L) make duplicate injection impossible.Thus, it was decided to attempt to develop a GC method to allowquantitation of ritodrine from much smaller plasma volumes whileretaining or improving upon the sensitivity of the previous methods.Underivatized ritodrine does not possess significant electroncapture properties, therefore, derivatization is necessary to maximizedetector sensitivity. The molecular structures of both the $-agonistsand a-antagonists when derivatized often result in compounds detectablein very low amounts (Ehrsson, 1978). In the development of the presentassay both trifluoracetic anhydride (TFAA) and heptafluorobutyricanhydride (HFBA) were examined for the preparation of a suitablederivative. It has been reported for the congener of ritodrine,isoxuprine, that the TFAA derivative and HFBA derivative provide similarsensitivity and also that TFAA reacts less readily with co-extractedendogenous substances (Cova et al., 1983). HFBA was selected based onthe observations that it increased detector response 10 - 100 fold overthe TFAA derivative with no appreciable differences in reactivity withco-extracted solutes. Reaction conditions for the derivatization142reaction and removal of excess derivatizing reagent were optimizedfollowing the procedures established by Walle and Ehrsson (1970) andEhrsson et al. (1971).The structure of the HFBA derivative of the internal standard, 5-hydroxy propafenone, has been previously confirmed by El GCMS (Chan etal., 1989). Although details of the El, PCI and fast atom bombardment(FAB) mass spectra of ritodrine following isolation via HPLC have beenpreviously published (Lin et al., 1984; Sill et al., 1987), there havebeen no reports of the mass spectra of ritodrine derivatized byacylating reagents. In the development of this assay method both of thederivatives of ritodrine formed via acylation with TFAA and HFBA werecharacterized by El, PCI and NCI GC-MS. Following derivatization withTFAA both GC-ECD and GC-MS demonstrate the formation of a singlespecies. Under NCI conditions this peak showed a molecular ion of m/z671 amu which corresponds to a tetra-TFAA substituted derivative ofritodrine. Since the mass range of the quadrupole mass spectrometeremployed was limited to 0-1000 amu, it was not possible to observe amolecular or pseudo-molecular ion (m/z 1071) which would have beenexpected due to the formation of a similar tetra-substituted HFBAcompound. Following derivatization with HFBA, analysis by GC-ECD andGC-MS demonstrated the formation of a single species as evidenced by asingle, symmetrical and sharp peak in the GC-ECD chromatogram and alsoby a similar total ion chromatogram (GC-MS) demonstrating onesymmetrical peak. Fragmentation in both El and CI modes was extensiveand complex due to multiple losses of all or part of theheptafluorobutyryl group (viz. F7C3OO, m/z=213; F7C3O, m/z=197; F7C3,m/z=169). Although no molecular ion could be observed, it is proposed143that the derivative formed is tetra-substituted due to the similar sizeand spectrum of chemical reactivity of TFAA and HFBA as well as thecharacteristic fragments formed (m/z 542 and 548) under NCI conditions.The fragmentation patterns observed with the HFBA derivative weresimilar to those observed from the TFAA derivatization. Additionally,the El fragmentation patterns observed in this study closely resemblethose seen by Cova et al. (1983) in their GC-MS analysis of TFAAderivatized isoxuprine, a close congener of ritodrine. Massfragmentographic studies of another related drug, terbutaline, followingderivatization with a number of agents, including HFBA and TFAA, alsohave demonstrated similar fragmentation patterns to those observed inthese studies (Clare et al., 1979).During optimization of the extraction conditions for this assay, anumber of solvents were examined (viz., toluene, benzene, hexane,dichloromethane, ethyl acetate, methyl ethyl ketone, and 1-butanol),with ethyl acetate providing the highest relative extraction ratio ofall the tested solvents. In an effort to obtain improved selectivity ofextraction (i.e. to minimize coextraction of endogenous compounds)various mixtures of ethyl acetate:toluene:isopropyl alcohol wereexamined. None of these mixtures offered significant improvements inselectivity over ethyl acetate and all showed lower relative extractionof ritodrine. Ritodrine adsorption onto glass surfaces does not appearto be a major source of drug loss during extraction since addition ofTEA to ethyl acetate in concentrations ranging from 0.001 - 0.5 M didnot significantly improve ritodrine recovery. As suggested by Lin etal. (1984), the extractability of ritodrine is highly pH sensitive andthe attainment and maintenance of pH = 9.5 is crucial to optimal144extraction and reproducibility (the relative recovery at pH 9.2 is 33%and at pH 10 is z60%). Previously published assay methods recommendedextraction for periods of less than 20 mm (Lin et a!., 1984; Kuhnert eta!., 1983; Gross et a!.; 1987). In the present study, however,extraction for 5, 10, 15, 20 and 30 mm of uniform concentrations ofdrug and internal standard followed by derivatization showed thatoptimal recovery occurred after 20 mm. The absolute recovery ofritodrine by the present assay method was 62% ± 5% which was similar tothat obtained by Gross et al. (1987) (68%) but slightly lower than thatreported by Lin et al. (1984) and Kuhnert et al. (1983). The absoluterecovery of ritodrine could be increased to 81% ± 5.5% by re-extractionof the aqueous phase with 5 ml ethyl acetate for 20 minutes, followed by2 centrifugations at 2300 g for 5 mm. The organic layers were thenpooled, evaporated and derivatized, as previously described. Improvingthe recovery in this manner, however, resulted in an 20% increase inthe coefficient of variation at each sample concentration measured.In the present study a significant “cold-trapping11 effect wasobtained by holding the initial column temperature at 145 °C. Lowerinlet temperatures resulted in significant band spreading of the solventpeak while significantly higher temperatures (i.e. >200 °C) decreasedthe degree to which sample reconcentration was obtained. Since mostcommonly employed GC solvents expand 200 - 450 times duringvaporization, the inlet liner used must have a volume large enough tocontain the volatilized solvent (Szelewski, 1987). The use of a 4 mminternal diameter inlet liner improved both the sensitivity andreproducibility compared to that obtained with a more conventional 2 mm145internal diameter liner. In addition, the use of a 1 L injectionvolume provided the best reproducibility.Figure 17 illustrates representative chromatograms obtained fromovine biological fluids. No interference occurs from endogenoussubstances with either the drug (retention time —11.1 mm) or internalstandard (retention time —17.6 mm).The data for a representative calibration curve used in thequantitation of ritodrine from maternal and fetal sheep plasma arepresented in Table 1. Linearity is observed over the concentrationrange studied (2.5 - 75 ng) with a coefficient of variation < 10% at allpoints.In summary, the assay method presented here is simple,reproducible, selective and sensitive enough to determine ritodrineduring single dose pharmacokinetic studies. Linearity of detectorresponse was noted from 2.5 - 75 ng of added ritodrine base,representing approximately 1 - 75 pg mass at the detector. The minimumquantifiable concentration (C.V. 10%) is 2.5 ng/mL from a 0.5 mlbiological fluid sample. The presented method has been used for theanalysis of ritodrine from a variety of biological fluids obtainedduring experiments with chronically instrumented pregnant sheep.4.2. Fetal Infusion StudiesA number of studies (Siimes and Creasy, 1976; Ehrenkranz et al.,1976; Siimes et a!., 1978; Siimes and Creasy, 1979; Bassett et a!.,1985; Warburton et al., 1987a and b; Bassett et al., 1989; Block et a!.,1989) have examined the effects of ritodrine on the fetal lamb during146infusion to both the ewe and fetus. All studies noted profound fetalcardiovascular and metabolic changes typical of those elicited byadreneregic stimulation. These effects included tachycardia,hypotension, increased plasma glucose, lactate, pyruvate, insulin anddecreased plasma a-amino acid nitrogen and a fall in fetal vascularIn these studies only short term infusions (O.5-2h) were used, however.When longer infusion periods have been employed (24-96 h) (Bassett etal. 1989; Warburton et al. 1987a and b) tachyphylaxis occurred with thetachycardia, plasma glucose and insulin concentrations, which returnedto normal values after 24-48 h. Fetal lactic acidemia, however,persisted for over 48 h (Bassett et al., 1989) and there was a fall infetal vascular pO. Recently, we have found this fetal hypoxemia isassociated with an increase in fetal 02 consumption and fall in 02delivery (van der Weyde et al., 1990a and b). Only in the present studyhave either fetal or maternal ritodrine concentrations been measured.4.2.1. Placental Clearance of RitodrineThe magnitude of the placental clearance of drugs and othercompounds has been estimated in intact animal preparations by severalmethods. One involves the Fick principle which relies on thesimultaneous sampi ing of maternal arterial and uterine venous blood orfetal arterial and umbilical venous blood. Furthermore knowledge of theuterine or umbilical blood flow are required, respectively. Theplacental clearance can then be calculated as the product of blood flowrate and the arterlo-veno concentration difference divided by thearterial concentration. Another method of determining placental147clearance (Anderson et al., 1980a and b) requires the simultaneousadministration of a maternal intravenous drug infusion and a fetal bolusdose of labeled (either radio- or stable isotope label). From the fetalbolus administration the fetal total body clearance (i.e. Cltb = Cl +Clf0) may be calculated. Further, from the product of the ratio of thesteady-state fetal to maternal concentrations from the maternal infusionand the fetal total body clearance, the placental clearance may beobtained (i.e. Cl = Cltb X CSSfetal/CSSmaternal) (Anderson et al.,1980a and b). The major drawback of this method is, however, therequirement for labeled drug (either radio- or stable isotope label) andthe ability to quantitate this and the unlabeled drug in the presence ofone another. Neither labeled ritodrine nor a suitable analyticalprocedure were available at the time of this study. A third method ofdetermining placental clearance is the approach of Szeto et al. (1982)which requires paired infusions to steady-state, of drug to the motherand fetus. By sampling both fetal and maternal arterial blood duringboth infusions the transpiacental and non-placental clearances can bedetermined in both mother and fetus (Szeto et al. 1982). Although bothmethods have been used to calculate transpiacental clearances there arelimitations to these methods. The Fick principle obviously requires amore intensive surgical procedure in that in addition to cannulation ofa maternal or fetal arterial vessel a major branch of the uterine veinor umbilical vein must also be cannulated, and there must be a methodfor the determination of uterine or umbilical blood flow. In additionthe blood flowing in either the uterine or umbilical venous circulationis not totally representative of blood at the sites of drug transfersince a portion of uterine venous blood has perfused the uterine muscle148and other structures not involved in placental exchange. Similarlyumbilical venous blood has also perfused non-placental structures and aportion of umbilical venous blood (22 %) is recirculated within theplacenta (Reuss and Rudolph, 1980). Thus the venous concentrationswhich must be used practically may be slightly different from the actualvenous concentrations leaving the sites of exchange. The Fick approachalso only allows the determination of “unidirectional” placentalclearances (i.e. fetus to mother or mother to fetus) at any one time.Similarly the “Szeto Model” (Szeto et al., 1982) is not withoutproblems. In the absence of stable isotope labelled or radiolabelleddrug , the infusions must be carried out on different days separated byan adequate “washout period”. Since during late gestation the sheepfetus grows at a rate of 3 - 5 % per day and organ function is alsochanging during the washout period (Kong et al., 1975) the fetal andperhaps maternal part of the preparation are dynamic during the courseof the experiment. Further the infusions used must provide measurabledrug concentrations on the placental side opposite the infusion to allowmeaningful results to be calculated. This would be a particular problemwith drug infusion to the fetus, since a low amount of drug passingtransplacentally to the ewe would be rapidly diluted and cleared, due tothe much larger size of the mother and, hence, drug may not bedetectable in the maternal circulation. This did, in fact, occur duringthe fetal ritodrine infusion studies, The drug was not detectable ineither maternal arterial or uterine venous plasma. As result of thisand the potent physiologic and metabolic effects of the drug in thefetus even at low plasma concentrations, it appears that the Fick149principal is the most practical to obtain an estimate of the placentalclearance of ritodrine.In the present study, we have employed an infusion rate of 0.93 ±0.05 ,ig/min/kg with an apparent mean steady-state fetal arterialritodrine concentration of 16.99 ± 1.96 ng/mL being reached atapproximately 8 h from the start of the infusion. Ritodrineconcentrations measured in the cord blood of human fetuses whose mothershad received ritodrine, range from 5 - 282 ng/mL (Gandar et al.,1980;van Lierde and Thomas, 1982; Fujimoto et al., 1986; Kuhnert et al.,1986). Although the concentrations achieved in the sheep fetus are atthe lower end of this range, they are associated with profound fetaleffects. This leads us to speculate that ritodrine may have similareffects on fetal metabolism and the cardiovascular system in the humanfetus.There is a slight but statistically significant difference betweenthe ritodrine concentration in fetal arterial plasma and that ofumbilical venous plasma at apparent steady-state (mean difference 0.7 ±0.2 ng/mL; mean ± s.e.m.), indicating placental uptake of the drug.However, ritodrine placental clearance (9.2 ± 2.7 mL/min/kg; mean ±s.e.m.) is much lower than the values reported for other drugs in fetalsheep (methadone: 168 ± 29, morphine: 19 ± 2, acetaminophen: 31 ± 2,metoclopramide: 103 ± 13, and diphenhydramine: 124 ± 22 mL/min/kg; mean± s.e.m.) (Szeto et al., 1982; Wang et al., 1986; Riggs et al., 1990;Yoo, 1989). This, coupled with the fact that we were unable to detectritodrine in maternal plasma, indicates that there is only a very smalldegree of placental transfer of this compound in sheep (Table 3). Thishas also been suggested by Fujimoto et al. (1984). The results in sheep150are in contrast to the findings for the drug in humans, where fetal tomaternal concentration ratios ranging from 0.3 - 1 have been reported(Gandar et al., 1980; Fujimoto et al., 1986; Kuhnert et al., 1986). Thedifference between sheep and humans likely relates to differences inplacental structure and the high polarity of ritodrine. Although theepitheliochorial placenta of the sheep generally does not constitute asignificant barrier to non-polar substances with molecular weights lessthan 600, this does not appear to be the case for polar molecules (Faberand Thornberg, 1983). Epitheliochorial placentas show a distinct “cutoff at a molecular weight around 300 for polar molecules, a phenomenonnot observed with the hemochorial placenta. Ritodrine (molecular weight287) is a highly polar molecule with a partition coefficient < 0.01between chloroform and an aqueous buffer. This value is much lower thanthose reported for nietoclopramide and diphenhydramine, which are bothweakly basic amines with molecular weights around 300, but as discussedabove, with much higher placental clearances than ritodrine (Riggs etal., 1989; Yoo, 1989).The key assumption of this analysis method is of course thatsteady-state has, in fact, been achieved. Prior to this study therewere no estimates of the kinetics of ritodrine in the fetus of anyspecies. Thus, we had to rely on a statistical demonstration of steadystate (i.e. showing that the arteria.l concentrations at 8, 12 and 24 hwere not significantly different from one another). The statisticaldemonstration of steady-state is supported by the t1/2fl found in thefetal bolus studies (see Table 9; t1/2fl 2.9 ± 0.7 h; mean ± s.e.m.).Thus at 8 hours of constant infusion the average fetus would be at151approximately 90 % of steady-state, while at 12 and 24 hours, thecorresponding values would be 94 and 99 %, respectively.4.2.2. Non-placental Clearance and Disposition of RitodrineAlthough the fetal placental clearance of ritodrine is lower thanthat for metoclopramide and diphenhydramine, the non-placental clearancevalues for these three drugs are of similar magnitude (Clf0 ritodrine:52.8 ± 8.0; metoclopramide: 28 ± 7; diphenhydramine: 100 ± 13 mL/min/kg;mean ± s.e.ni.). Of the relatively few drugs for which placental andfetal non-placental clearances have been determined in sheep (morphine,methadone, metoclopramide, diphenhydramine, acetaminophen, indomethacin,acetylsalicylic acid, and ritodrine), ritodrine appears to be somewhatunique in that it has little placental transfer but has a relativelyhigh degree of fetal non-placental clearance (Szeto et al., 1982; Riggset al., 1990; Yoo, 1989; Wang et al., 1986; Anderson et al., 1980a andb).At present, we are not able to determine and quantitate all of thepossible routes of non-placental clearance of ritodrine from the fetallamb. However, the concentrations of ritodrine attained in the trachealand amniotic fluids are of significance. Amniotic fluid, in the lategestation fetus, may act as a reservoir from which free drug may berecirculated to the fetus through fetal swallowing or by movement of thedrug across fetal membranes. This may lead to a persistence of drug anddrug effects in the fetus. The uptake of drug from the amniotic fluidhas been studied in sheep with diphenhydramine and metoclopramide (Riggset al., 1988), digoxin (Hamamoto et al., 1990) and in humans with152ampicillin (Macaulay et al., 1966). With all these drugs, preferentialuptake by the fetus was demonstrated. In the current study, allantoicfluid concentrations of intact ritodrine were essentially equal to thosefound in the amniotic fluid in the one animal sampled. This suggeststhat the allantoic cavity may also act as a reservoir for eliminateddrug in the fetal lamb.The accumulation of drug in fetal tracheal fluid has been describedfor metoclopramide and diphenhydramine (Riggs et al., 1987).Metoclopramide attained tracheal fluid concentrations approximately 15fold those in the fetal plasma, while diphenhydramine attained a 4 foldgreater concentration. In the present study, ritodrine {TR]/[FAj ratioof 4 during the infusion period is similar to that of diphenhydramineand although it is lower than for metoclopramide, it may be significantdue to the known potent effects of2-agonists on fetal lung developmentand function (Warburton et al., 1987a and b; Ekelund et al., 1983).Fetal tracheal fluid is formed at a rate of 4 mL/h in the lategestation fetus primarily by the active transport of chloride ion acrossthe pulmonary epithelium (Harding et al., 1984). The fetal trachealfluid resembles an ultrafiltrate of plasma with regards to Na+ and K+concentrations, however the concentrations of C1 and HC03 aresignificantly different from that of fetal plasma. In addition, theprotein content of fetal tracheal fluid (0.3 g/L) is much lower thanthat of fetal plasma (62.7 g/L) (Olver and Strang, 1974). Recently theaccumulation and persistence of the amine drugs, metoclopramide,diphenhydramine and labetalol, in fetal tracheal fluid have beenreported (Riggs et al., 1987;Rurak et al., 1991). The accumulation ofdrug in fetal tracheal fluid may result, at least in part, from drug153uptake by the fetal lung and subsequent release into the tracheal fluid(Rurak et al., 1991). The concentrations of ritodrine in fetal trachealfluid are much higher than in fetal plasma, peak later and persist afterthe disappearance of drug from the fetal arterial plasma. Although theaccumulation of drugs in the fetal tracheal fluid has been noted formetoclopramide and diphenhydramine (Riggs et al., 1987), the presence ofritodrine in the fetal tracheal fluid may be more complicated than asimple accumulation. Ritodrine and $-agonists in general are associatedwith a significant decrease in the production of fetal tracheal fluidand the resorption of this fluid across the fetal lung (Warburton etal., 1987 a and b; Berthiaume et al., 1987). Thus, ritodrine may “selfconcentrate” in fetal tracheal fluid by diminishing the volume of fetaltracheal fluid. The higher concentrations of drug in fetal trachealfluid than fetal plasma may reflect a small volume of fluid rather thana large amount of drug. Regardless of the mechanism(s) of drugaccumulation in fetal tracheal fluid, the implication of thisaccumulation in that the developing fetal lung may be exposed to drugconcentrations far is excess of the plasma drug concentrations.The lung has been identified as an important organ of clearance formany drugs, particularly amine compounds, in adults (Collins andDedrick, 1982; Hon et al., 1987). In part, the significance of thelung contribution to total body clearance results from the fact that,in adults, the lung receives all of the cardiac output. In the sheepfetus, the lung receives only 6 % of the biventricular output andpulmonary blood flow is only 28 niL/mm/kg (Anderson et al., 1981).This relatively low pulmonary blood flow, and the existing pulmonaryarterial-venous difference, has been shown to be sufficient to account154for the 4 fold accumulation of diphenhydramine in fetal tracheal fluidas compared to fetal arterial plasma (Yoo, 1989). Thus, despite therelatively low pulmonary blood flow in the fetal lamb, drug uptake bythe lung may be quite significant.Although drug uptake by the adult lung is a relatively wellrecognized process, the cell types (of which there are more than 40 inthe lung) responsible for drug accumulation and metabolism have not beenidentified for all drugs. The pulmonary endothelial cells lining thesmall vessels and capillaries have been shown to be an important routeof uptake for endogenous amines such as serotonin and norepinephrine(Bend et a!., 1985; Roth, 1985). In the endothelial cells, uptake byboth passive diffusion and carrier-mediated processes have beensuggested. The removal of basic amine drugs (e.g. amphetamine andpropranolol) have also been suggested by these processes (Anderson etal., 1974; Philpot et al., 1977; Bend et al., 1985; Benford and Bridges,1986). It would appear that amphiphilic weakly basic amine drugs (pKa >8) accumulate and persist in lung tissue to a greater extent than thosedrugs lacking these properties (Philpot et a!., 1977; Benford andBridges, 1986). In an isolated perfused adult rat lung preparationOkumura et al. (1978) have shown a much greater accumulation of weaklybasic amine compounds than either neutral or acidic compounds.In terms of the developmental significance of the exposure of thefetal lung to high drug concentrations, the administration ofamphiphilic amine drugs, which accumulate and persist in lung tissue, toanimals at high doses or over prolonged periods of time results in druginduced pulmonary phosphiliposis (Philpot et al., 1977; Bend et al.,1985; Haagsman and van Golde, 1985; Benford and Bridges, 1986).155Provided secondary cell damage has not occurred, this process may bereversible by drug withdrawal (Benford and Bridges, 1986).We have also identified the presence of glucuronide conjugates ofritodrine in the amniotic fluid. There are very few accounts of eitherthe metabolic routes or contribution of these routes to fetal non-placental drug elimination. Wang et al. (1985, 1986) have demonstratedthe ability of the fetus to form glucuronide and sulphate conjugates ofacetaminophen. In addition, in vitro (Dvorchik et al., 1986) and invivo (Marshall et al., 1981; Mihaly et al., 1982) studies havedemonstrated the presence and activity of drug metabolizing enzymes inthe fetal lamb. It appears, however, that the activity of these enzymesmay be lower than that of the adult animal and also there is shunting ofa portion of umbilical venous blood past the fetal liver, via the ductusvenosus (Edeistone et al., 1973). Our observations that ritodrine doesnot cross the sheep placenta to any great extent, that there issignificant fetal non-placental clearance of ritodrine and thatconjugates of ritodrine appear in the amniotic fluid would suggest thatthese conjugates are of fetal origin. If one assumes a fetal amnioticfluid volume of 1 L the concentration of glucuronide conjugate(s)determined in this study would account for approximately 11% of theadministered dose. Since the fetal lamb also excretes approximately 50% of its total daily urine production into the allantoic fluid cavity(Wlodek et al., 1988), drug and metabolite may be excreted into thisfluid compartment as well . If the glucuronide conjugate concentrationsin amniotic and allantoic fluids are similar then the excretion ofritodrine glucuronide(s) account for approximately 22 % of theadministered dose. This percentage is similar to the extent of156ritodrine glucuronide excretion in humans (maternal 38%; neonatal 23%),however, the human also excretes a very significant portion of theadministered dose as a sulphate conjugate (Brashear et al., 1988), whichwas not detected in our studies. The apparent lack of sulphateconjugates in the ovine amniotic fluid may reflect a species differencein ritodrine metabolism. Sulphate conjugates of ritodrine have onlybeen found in primate species (human, baboon) and not in other non-primate species studied (rat, sheep) (Borrisud et al., 1985).Only 35% of the total dose administered to the fetus can beaccounted for by the routes (fetal/maternal placental transfer,excretion into the amniotic fluid of parent and conjugated drug) that wehave examined in these experiments, although, urinary excretion ofintact drug and metabolites accounts for > 90% of the administered dosein adult humans (Kuhnert et al., 1986). This would suggest that otherroutes are important in the elimination of ritodrine from fetal sheep.A similar finding has been made for acetaminophen, where metabolism andurinary excretion of drug and metabolites accounted for 97% of the dosein adult sheep but only 34% of the dose in fetal lambs (Wang et al.,1986).4.3. Maternal Bolus Studies4.3.1. Pharmacokinetics and Placental Transfer.The pharmacokinetics observed in the maternal arterial plasmaclosely resemble those noted in both humans (Caritis et al.,1990;Caritis et al., 1989; Gross et al., 1987; Fujimoto et al., 1986;157Kuhnert et a!., 1986; Gross et al., 1985; van Lierde and Thomas, 1982;Gandar et al., 1980) and animal species (Caritis et al., 1988). In allcases, the plasma concentration vs. time profile showed amultiexponential decline. In all but two animals, this relationship wasbest described by a two compartment open model. The two animals whoseplasma concentrations vs. time profiles did not fit a two compartmentmodel were best described by a three compartment model. The moststriking kinetic observation from our study is the relatively largevolume of distribution (Vd 10.03 ± 1.94 L/kg) which has also beenobserved in humans (Caritis et a!., 1990). The large volume ofdistribution of ritodrine appears to agree with the general finding ofextensive distribution of most other -agonists studied (Morgan, 1990).Similar to the other studies noted the maternal total body clearance isquite large (24.3 ± 5 mL/min/kg). In common with the studies in humansis the existence of a rapid distribution phase followed by a prolongedelimination phase (Caritis et al., 1990; Caritis et a!., 1989; Gross eta!., 1987; Fujimoto et a!., 1986; Kuhnert et a!., 1986; Gross et a!.,1985; van Lierde and Thomas, 1982; Gandar et a!., 1980). The terminalelimination half-life in the ewe (8.71 ± 1.82 h) corresponds quiteclosely to that in humans where a sensitive assay procedure was used tofollow the ritodrine concentrations for up to 80 hours following dosing(Kuhnert et a!., 1986). In studies where shorter sampling periods wereemployed it is quite possible that there has been a underestimation ofthe elimination half-life (Gibaldi and Weintraub, 1971). The apparentelimination half-life in the fetus (2.28 ± 0.91; mean ± s.e.m.) is muchshorter than that of the mother and is significantly different.Interestingly, however, the peak fetal concentrations of ritodrine in158arterial plasma are reached 10-15 minutes after the maternal dose. Thisis in contrast to rnetoclopramide (Riggs et al., 1990) anddiphenhydramine (Yoo et al., 1991) where peak fetal plasmaconcentrations occurred simultaneously with those in maternal arterialplasma. This delay, in the case of ritodrine, may reflect somediffusional barrier to transfer across the sheep placenta.The pattern of accumulation of drug in the amniotic fluid followingmaternal bolus administration is similar to that noted in the fetalinfusion studies. Although the concentrations are relatively low,ritodrine is detectable in the amniotic fluid 60 minutes after maternalbolus administration. The amniotic fluid concentrations then riseslowly to maximal levels of 10 ng/mL and persist at this concentrationfor at least 24 hours. Unlike metoclopramide and diphenhydramine, theritodrine concentration in the amniotic fluid does not appear todecline. The persistence of ritodrine in the amniotic fluid could bethe result of the relatively high polarity of ritodrine which maydecrease the apparent reabsorption of drug across the chorioallantoicmembranes as has been noted for metoclopramide, and diphenhydraminefollowing intraamniotic drug injection (Riggs et al., 1988). Similar tothe fetal infusion studies, the presence of glucuronide conjugate(s) ofritodrine was noted in the amniotic fluid. Although the concentrationof the glucuronide conjugate(s) is far lower than in the fetal infusionstudies, this is not unexpected, since the fetus receives a much smallerdose of ritodrine in the maternal bolus experiments. The presence ofthe glucuronide conjugates also suggests the ability of the fetus toeliminate ritodrine via non-renal pathways.159The extent of placental transfer, as evidenced by the fetal tomaternal AUC ratio, is quite low in these studies. A similar smalldegree of placental transfer was found in the fetal ritodrine infusionstudies and in a preliminary report by Fujimoto et al. (1984). Thefinding of little placental transfer of ritodrine in the sheep is notunexpected given the small magnitude of placental clearance determinedin the fetal infusion studies and the relatively high polarity andmolecular weight (M.W. 287) of ritodrine (see Section 4.2.1.).The lack of placental transfer observed in this and previousstudies of ritodrine in the pregnant sheep model may be an advantage inexamining the differential pharmacological sensitivity of the mother andfetus to ritodrine. Since almost no drug is transferred between motherand fetus, each is pharmacokinetically isolated from the other.Further, in studies employing fetal infusion no maternal effects werenoted. Thus the fetus and mother are pharmacokinetically andpharmacologically isolated from one another. Therefore, followingadministration of a dose of ritodrine to the sheep fetus, one would beable to examine fetal responses in the absence of maternal physiologicalor pharmacological influences.4.3.2. Cardiovascular EffectsOne of the most prominent effects observed in the present study wasthe increase in maternal heart rate following drug administration. Thedecline of maternal heart rate parallels the decline of maternalarterial drug concentrations making the concentration dependence of thiseffect readily amenable to modelling. In each of the animals a maximal160heart rate was achieved which, when plotted against plasma drugconcentration, demonstrated a sigmoidal relationship.Ritodrine has been demonstrated to increase maternal heart rate inboth human (Kleinhout and Veth, 1975; Caritis et al., 1985; Witter etal., 1988; Ferguson et al., 1989) and animal (sheep) studies (Bassett etal., 1985). Very few experimenters have, however, tried to establishrelationships between ritodrine plasma concentration and changes inheart rate. Caritis et al. (1983) have examined the relationshipbetween maternal heart rate and maternal ritodrine concentration atsteady-state during maternal ritodrine infusion in human subjects.These authors (Caritis et al., 1983) demonstrated both a linearrelationship between maternal heart rate and maternal ritodrineconcentration and the apparent development of tolerance to this effectwithin 8 hours of infusion. Further, Caritis et al. (1983) demonstrateda linear relationship between changes in fetal heart rate and maternalritodrine concentration. In pregnant sheep, van der Weyde (1990) noteda linear relationship between fetal heart rate and fetal arterialritodrine concentration during fetal ritodrine infusion. Tolerance tothe effects of ritodrine on fetal heart rate was not observed duringthese experiments (van der Weyde, 1990). Direct comparison betweenthese studies is difficult due to the different modes of drugadministration, species used, and the types of analysis performed. Onepossible comparison that can be made is provided by the regressionequations between heart rate and ritodrine concentration. The slope ofthe regression lines are approximately equal to the ratio ofEmax/(EC5O)n which can be obtained from the Emax or sigmoid Emax models.In the study by Caritis et al. (1983) the slope of the regression161between human maternal heart rate and ritodrine concentration is 28.4 at4 hours after the start of infusion, and 36 at 8 hours after the startof infusion. In the study by van der Weyde (1990) the slope of theregression between fetal ovine heart rate and ritodrine concentration is18.9. In the present study the ratio of Emax/(EC50)for maternal heartrate is 60. This may suggest that the heart rate of the ewe is moresensitive to ritodrine than either the pregnant human female or sheepfetus. However, due to the limited range of ritodrine concentrationsand heart rates obtained in the previous studies (Caritis et al.1983;van der Weyde 1990) the slopes found may only reflect the initialportion of the dose response curve and, hence, may not be entirelyreflective of the sensitivity of either the pregnant human female orsheep fetus.We did not note any significant changes in fetal heart rate duringthe present studies. In our previous study employing fetal infusions,however, where apparent steady-state fetal concentrations approximate 20ng/mL an 34 beat per minute increase in fetal heart rate was noted.The fetal arterial concentrations achieved in the maternal bolusadministrations were lower (15 ng/mL) and of much shorter persistencethan in the fetal infusion studies which may account for the differencesin fetal responses between these two studies.4.3.3. Metabolic EffectsThe changes in arterial blood gas parameters for the mother aresimilar to those noted in both clinical and animal studies of ritodrine(Caritis et al., 1985; Ferguson et al., 1989; Bassett et al., 1985;162Schreyer et al., 1980). The effects induced by ritodrine are alsotypical of -agonists as a pharmacological class (Morgan, 1990). In thepresent study we noted a progressive maternal metabolic acidemia,beginning at the first sampling point (5 minutes), peaking between 2 and3 hours and returning to control values at 6 hours post-dose.Comparison of the time course of these effects with the maternalarterial ritodrine concentration, shows that there is a large temporallag between peak maternal concentrations and the peak of the metabolicacidemia. This temporal disequilibrium may suggest that the effects ofritodrine on maternal arterial blood gas parameters occurs at a siteoutside the central compartment (i.e. an effect compartment) or thatthey are mediated by one or more intermediary substances produced inresponse to ritodrine stimulation, or a combination of both factors(Holford and Sheiner, 1981). Evidence for the role(s) of intermediarysubstances is provided by our measurement of the whole blood lactateconcentrations in 4 animals. The lactate concentrations show an inverserelationship to the whole blood base excess and standard bicarbonatevalues suggesting that the observed metabolic acidemia is at least inpart a lactic acidosis. The site of production of the lactic acid wouldappear to be the maternal carcass and/or liver since the uterine venouslactate concentrations do not change with time. The constancy of theuterine venous lactate concentrations is also suggestive of a relativelyconstant rate of placental lactate production. There is, however, anextremely large increase in the uterine uptake of lactate following theritodrine bolus (Figure 34). Also, there is an apparent reversal of thematernal flux of glucose during this period and the uteroplacenta isapparently supplying glucose to the ewe (Figure 34). These two163processes, increased lactate uptake and reversal of glucose uptake, mayreflect the increased use of lactate as a metabolic substrate by theuteroplacental structures and subsequent sparing or net production ofglucose. There does not appear to have been similar observations ofthese phenomena in pregnant sheep and this interesting apparentlyreciprocal relationship between uterine lactate and glucose fluxeswarrants further investigation.The multiple possibilities for an explanation for the observedmetabolic changes make meaningful assignment of a pharmacodynamic modelimpossible. Simply modellingthese changes via an effect compartmentneglects the probable contributions of intermediate substances and wouldset up a relationship between an estimated drug concentration and aneffect that may actually be the result of several processes. Themodelled pharmacological relationship would probably be of sigmoidalform, however, the relationships of any intermediary substances are ofunknown form in relation to the measured end effect and to one another.Thus, although we could successfully account for the time lag betweenpeak drug concentration and peak effect, the parameters derived would beentirely artificial. Comparison of the metabolic effects observed inclinical and animal studies with the present study is difficult due tothe use of different doses and modes of drug administration. Further,previous studies, both human and animal, have not employed extensiveblood sampling for ritodrine determination.In the present study we have shown that the metabolic acidosis is,at least in part, mediated through lactic acid production. It isinteresting to note that in the four animals in which lactate wasexamined, all reach a similar peak concentration (75.59 ± 1.35 mg/dL)164between 1.5 and 3 hours, that the profile of lactate concentration vstime is the inverse of the base excess, and that the apparentelimination half-life in all animals is approximately 4 hours (4.36 ±0.61 h). In examining the literature it appears that no studies haveexamined lactate kinetics by taking sequential repetitive blood samples.Most studies use combinations of isotope labelled lactate todifferentiate the various rates of turnover possible within the animalsystem under study. We have calculated the AUC for the lactate producedas:AUC = cor (CL(t)) - CL(O)) dtt=0Jwhere: CL(t) is the concentration of lactate in the maternalarterial plasma at time t and CL(O) is the maternal arterialconcentration before drug administration. Sparks et al. (1982) havestudied maternal and fetal lactate turnover in the pregnant ewe andfetus. Using the data provided in their study one can estimate anapparent maternal lactate clearance of 85 mL/min (Sparks et al., 1982).Multiplying this value by the observe AUC0 for lactate produced duringour experiments would suggest that on average 33 g of lactate wasproduced by the ewe in response to the dose of ritodrine. Thiscalculation assumes that the clearance of lactate is independent of thearterial lactate concentration, that ritodrine would only affect theproduction of and not the elimination of lactate from the ewe.1654.3.4. Uterine ContracturesThe presence of low amplitude uterine contractions, in latepregnancy, has been noted in a number of species including humans(Thorburn et a!., 1984).In sheep these low amplitude uterine contractures were firstdescribed by Hinson and Ward (1973) and typically represent a 3 - 5 mmHg rise in intrauterine pressure coupled with synchronous bursts of EMGactivity across the myometrium. These contractions are frequentlyassociated with transient decreases in uterine blood flow and reductionsin fetal arterial oxygen levels (Thorburn et al., 1984). These uterinecontractures often induce changes in fetal behavioral state from REM tonon-REM sleep (Nathanielsz et al., 1980; Thorburn et al., 1984).Harding et al. (1982) have described the temporal development of uterineactivity during late gestation until parturition.Although ritodrine and other -agonists are well recognized toterminate the uterine contractions occurring during premature labour(Morgan, 1990), Harding et al. (1983) have used maternal salbutamolinfusion to temporarily abolish uterine contractures in the pregnantsheep. The present study would suggest that ritodrine, and probablyother -agonists as well, reduces the number of normal uterinecontractures following administration of a single bolus dose. It wouldbe interesting to further determine whether tolerance develops to thiseffect as has been noted in the continued treatment of women inpremature labour with fl-agonists (Morgan, 1990).1664.4. Fetal Bolus StudiesPrior to the fetal infusion studies presented earlier (Section4.2.) there had been no estimates of pharniacokinetic parameters ofritodrine in the fetus of any species. As discussed earlier, theobservation of the extremely low degree of placental transfer ofritodrine across the sheep placenta (Sections 3.2. and 3.3.) wouldsuggest that the ewe and fetus are pharmacokinetically isolated from oneanother. Further the lack of observed physiological effects on the sideof the placenta opposite the drug administration (i.e. the ewe in thecase of fetal administration and the fetus in the case of maternaladministration) suggests that the ewe and fetus are alsopharmacologically isolated from one another. Neither fetalpharmacokinetics nor fetal physiological responses are influenced byplacental drug transfer and/or drug induced maternal physiologicalchanges. Thus fetal bolus administration provided a unique opportunityfor the in vivo comparison of both pharmacokinetics and physiologicalresponse to ritodrine between the ewe and fetus.4.4.1. PharmacokineticsThe basic pharmacokinetic behavior of ritodrine in the sheep fetusfollowing fetal intravenous bolus administration is very similar to thatobserved in the ewe following maternal bolus administration (Section3.3.). Similar to the ewe the fetal arterial plasma concentration vstime profile was best described by a bi-exponential equation. Therewere no significant differences between fetal and maternal volumes of167distribution, total body clearance, AUC, AUFIC or t1/2a determined in thefetal and maternal bolus experiments respectively. The fetal terminalelimination half-life determined in the maternal bolus experiments(Section 3.3.,Table 5) was also not significantly different from thedetermined in the fetal bolus experiments (Table 9). Further thepattern of accumulation of ritodrine in the fetal tracheal and amnioticfluid and the accumulation and persistence of the glucuronideconjugate(s) is apparently similar between the fetal bolus experimentsand both the fetal infusion and maternal bolus studies. The mostapparent differences are between the maternal and fetal t1/2 andbetween the fetal total body clearances determined in the fetal infusionand fetal bolus studies.In the fetal infusion studies the fetal total body clearance wascalculated as the ratio of the infusion rate and the apparent steady-state arterial plasma concentration (ko/Cp) and provided a value of62.0 ± 8.1 mL/min/kg (mean ± s.e.m.;n = 10) (Table 3) as an estimate offetal total body clearance. In the fetal bolus studies fetal total bodyclearance was calculated as the ratio of dose and AUC (dose/AUC) and thefetal total body clearance was estimated as 31.7 ± 10.0 mL/min/kg (mean± s.e.m.; n = 5) (Table 9). Comparison of these two values by unpairedt-test suggests that they are significantly different. Several factorsmay account for this difference. As has been stated earlier (Section4.2.1.) the key assumption during the analysis of the fetal infusionstudies was that steady-state had in fact been achieved. This wasdemonstrated statistically in that the fetal arterial concentrations at8, 12, and 24 hours were not different from one another. In the fetalbolus studies, however, the terminal half-life was estimated as 2.9 ±1680.7 h (mean ± s.e.m.; ii = 5). This would suggest that at 8 hours ofinfusion approximately 90 % of steady-state would have been reached.thus the fetal arterial concentrations measured at 8 hours may be aslight underestimate of the true steady-state concentration. This wouldresult in a slight overestimate of the total body clearance byapproximately 10 %. Another contributing factor may be that in thefetal infusion studies the duration of the infusion was determined bythe response of the fetus to ritodrine in that the infusion wasterminated when the fetal arterial pH fell below 7.30 and the wholeblood base excess fell below -1.0 meq/L. This minimized the exposure ofthe fetus to the developed acidemia which was necessary for theconcurrent physiological measurements being made (van der Weyde, 1990).In the fetal bolus studies, however, the fetuses become very much moreacidotic (whole blood base excesses of -10 meq/L and arterial pH’s of <7.20 were not uncommon) during the course of the experiment and thisextensive acidemia persisted for often up to 24 hours. In addition thefetuses of the fetal bolus group became much more hypoxemic (mean fallin arterial p02 10 mm Hg) than those in the fetal infusion group (meanfall in arterial P02 4 mm Hg). Thus the pharmacokinetics of ritodrinein the fetuses of the fetal bolus group may have been affected much moresignificantly by acidemia and hypoxemia than the kinetics of ritodrinein fetuses during the fetal infusion studies.Although the effects of hypoxia and acidemia on drug kinetics donot appear to have been well examined, several studies have addressedthe alterations in pharmacokinetics in adults and newborn humans andanimals due to hypoxia and/or acidemia. The drugs in adults studied aregenerally of low therapeutic index and include digoxin (du Souich et169al., 1985), furosemide (Babini and du Souich, 1986), theophylline(Saunier et a1., 1987), lidocaine (Marleau et al., 1987) and phenytoin(du Souich et al., 1986). In response to hypoxia and/or acidemiachanges in total body and organ clearances (du Souich et al., 1985;Babini and du Souich, 1986; du Souich et al., 1986), volume ofdistribution (du Souich et al., 1985) and tissue/plasma concentrationratio (du Souich et al., 1985; du Souich et al., 1986) have beenobserved. A number of contributing factors have been suggested toaccount for these changes including alterations in regional blood flows,protein binding, glomerular filtration rate, renal tubular function, andhepatic microsomal enzyme systems. In newborns investigation of theeffects of hypoxia and pharmacokinetics seems to have been confined toantibiotics where hypoxia-induced decreases in renal blood flow andglomerular filtration rate have been shown to prolong the half-life ofamikacin and gentamicin (Myers et al., 1977; Mirhij et al., 1978).Since hypoxia and acidemia have been demonstrated to alter the kineticsof drugs in newborns and adults it is entirely possible that theseprocesses could also affect fetal drug kinetics. In addition, acutehypoxemia has been shown to alter the distribution of cardiac output inthe fetal lamb with increased blood flow to the brain, heart andadrenals and decreased perfusion of other tissues and organs (Cohn etal., 1974). Further the activity of the hepatic microsomal enzymesystem in the lamb has been demonstrated to be much lower than that ofthe adult (Dvorchik et al., 1986) and its sensitivity to furtherreductions in arterial oxygen tension is uncertain (Jones et al., 1989).Acidemia may also contribute to changes in kinetics by decreasingtracheal fluid flow (Hooper et al., 1988) and by increasing urine flow170(Freeman et al., 1988) which could change drug excretion across the lungand kidney respectively. Thus a number of physiological processesresulting from hypoxemia and/or acidemia could alter fetal drugdisposition and could explain the lower fetal clearance of ritodrine inthe fetal bolus studies compared to those involving fetal infusion.Since ritodrine use in human pregnancy is for the treatment of prematurelabor, which may involve an increased incidence of fetal hypoxia and forfetal hypoxic distress itself, the question of hypoxia or acidemiainduced alterations in fetal ritodrine clearance warrants furtherinvestigation.Similar to the fetal infusion studies, ritodrine could not bemeasured in either the uterine venous or maternal femoral arterialplasma in spite of the very large concentration fetal to maternalgradients created during the fetal bolus studies. This observationagain confirms the general finding of the extremely low degree ofplacental transfer of ritodrine across the sheep placenta.During these studies the extensive accumulation of ritodrine wasnoted in the fetal tracheal fluid. In the fetal bolus studies thetracheal fluid ritodrine concentrations were very much higher than thoseencountered during either the maternal bolus or fetal infusion studies.To some extent this probably reflects the much higher concentrationsachieved in the fetal arterial plasma and hence a potentially greaterconcentration gradient existing between arterial plasma and the trachealfluid. It is also interesting to note that the tracheal fluidconcentration plateaus (Figure 36) between 20 minutes and 3 hoursfollowing administration. This plateau may reflect the reabsorption oftracheal fluid in response to fl-adrenergic stimulation rather than a171pseudo steady-state being achieved within the tracheal fluid. Ritodrinehas been shown, at concentrations probably far lower than thoseinitially present in these studies, to cause decreases in tracheal fluidproduction and to also increase the rate of resorption of tracheal fluid(Warburton et al., 1987). Similar to the observations made in the fetalinfusion and maternal bolus studies the fetal tracheal fluid ritodrineconcentrations are 4 - 6 times higher than those present in the fetalfemoral artery.As in the fetal infusion studies, ritodrine accumulates in theamniotic fluid. There seems to be a rapid appearance of drug in theamniotic fluid and a steep accumulation over the first 4 - 6 hoursfollowing fetal drug administration (Figure 36). Thereafter theritodrine appears to persist at the “plateau” concentration until atleast 24 hours after the fetal bolus (Figure 36). The “plateau”concentrations of ritodrine observed in the amniotic fluid during theseexperiments (Ca. 150 ng/mL) appears to be slightly higher than the peakamniotic concentration observed in the 12 hour fetal infusion group(total fetal dose 1.8 rug) and is similar to those observed in the 24hour fetal infusion group (total fetal dose 3.6 mg). The pattern ofaccumulation and persistence of the glucuronide conjugate(s) during thefetal bolus studies is apparently similar to those observed in both thefetal infusion and maternal bolus studies. In all cases theconcentration of the glucuronide conjugate(s) is between 3 and 10 foldgreater than that of the free ritodrine concentration in the amnioticfluid. As was noted in the fetal infusion studies (Section 4.2.2.) thepresence of the glucuronide conjugate(s) in the amniotic fluid suggests172that these are probably of fetal origin and that at least part of thefetal total body clearance results from fetal drug metabolism.Using similar reasoning to that outlined in the fetal infusionstudies (Section 4.2.2.) approximately 75 % of the administered fetaldose can be accounted for as free and conjugated drug in the amnioticand allantoic fluids. Amniotic and allantoic fluid volumes of 1 L(Wiodek et al. 1988) were used in this calculation and it was assumedthat the concentrations of ritodrine and its glucuronide(s) were equalin each fluid space. The reason for the large difference between thetotal dose (35 % in the fetal infusion studies vs 75 % in the fetalbolus studies) recovered in these studies as compared to the fetalinfusion studies is unclear. Part of the explanation for thisdiscrepancy may be in the assumption of the 1 L volume for both fluidspaces. In at least one animal (Ewe 105) of the fetal bolus groupamniotic fluid volume was probably much less than 1 L and in no case wasallantoic fluid volume measured. Again, however, this method onlyallows a crude estimation of the extent of dose recovered via theseroutes of fetal drug elimination.4.4.2. Ontogeny of $-adrenoreceptor responses in the fetal lamb.There is general recognition of the importance that the fiadrenoreceptor system plays significant roles in the regulation ofdifferentiation and growth (Slotkin et al., 1987), gene expression (Odomet al., 1987), the fetal in utero response to stress (Jones and Ritchie,1983) and the adaptations of the fetal heart and lungs to birth (Curtisand Zalin, 1985; McDonald et al., 1986). Consequently there is a173growing body of literature describing the ontogenesis of the fl-receptorand its signal transduction mechanisms as well as the development ofphysiological responses to fl-adrenergic stimulation (Maier et al.,1989).There is considerable evidence for the presence of fl-receptorsearly in gestation in a number of tissues and species (viz. 60 days inthe myocardium of fetal sheep; 2 days in the cardiac muscle cells ofchick embryos) (Nuwayhid et al., 1975; Lipshultz et al., 1981; Miska etal., 1984). Furthermore it has been observed that in the mouse thenumber of myocardial fl-adrenoreceptors increases throughout gestationand that fetal numbers of myocardial fl-receptors are far lower than inthe adult (Chen et al., 1979). However, in spite of the presence of fireceptors many tissues do not exhibit functional responses to fl-agoniststimulation until later in gestation (e.g. secretion of lung surfactant,increases in plasma renin activity) (Lawson et al., 1978; Rawashdeh etal., 1987). The development of responsiveness in these tissues may bedependent upon the development of adenylate cyclase (Hommes and Beere,1971; Kohrman, 1973; Whitsett and Darovec-Beckerman, 1981; Schumacher etal., 1982; Kawai et al., 1985) which is involved in the transduction ofthe signal generated upon the interaction between agonist and fireceptor.There is also considerable physiological evidence for the existenceof functionally coupled fl-receptors in the fetus. Van Petten and Willes(1970) have demonstrated that the intravenous administration ofisoproterenol in the pregnant ewe or sheep fetus elicits qualitativelysimilar responses with respect to increases in heart rate and decreasesin both arterial blood pressure and total peripheral resistance.174Further these responses were blocked by the administration ofpropranolol, a $-adrenergic blocker, (van Petten and Willes, 1970).Similarly a number of -adrenoreceptor mediated responses have beendemonstrated on the fetal renal vascular bed, renin-angiotensinaldosterone system, tracheal fluid flow and surfactant efflux (Lawson etal., 1978; Rawashdeh et al., 1988; Nakamura et al., 1987; Warburton etal., 1987a). It is also clear that the magnitude of the observedresponses differ between adult and fetus as well as between fetuses ofdifferent gestational ages (van Petten and Willes, 1970; Buckley et al.,1984; Nuwayhid et al., 1975).It has been suggested (Maier et al., 1989) that tissues may notdemonstrate adrenergic responsiveness until they are necessary forhomeostasis. If this is true it would not be unexpected, given theevident differences of adult and fetus to cope with an appliedphysiological/pharmacological stress (e.g. hypoxia, a-agonistadministration), that the fetus may respond not only differently fromthe mother but also differently during the course of development toadrenergic stimulation.4.4.3. Cardiovascular EffectsFollowing the administration of the fetal bolus of ritodrine theabsence of an immediate fetal tachycardia was most unexpected (Figure38). The absence of any immediate change in fetal heart rate is notartifactual since it occurred in all experiments, the transducers andcardiotachometers were calibrated and checked prior to each experiment,examination of the fetal arterial pressure pulses when running the175recorder at higher speeds showed that the measured heart rate was infact correct. In the ewe, following maternal bolus administration ofritodrine, an approximate doubling of maternal heart rate occurredfollowed by a decline to control heart rate over a period of 4 - 6 hoursin all animals (Section 3.3.). Instead of the anticipated immediateincrease in fetal heart rate, a slow progressive increase in fetal heartrate was noted (Figure 39). The maximal mean increase in fetal heartrate was 4O beats per minute and occurred between 30 and 60 minutesafter the fetal drug administration. Thereafter fetal heart rate wassignificantly elevated above control values for approximately 4 hours.The observation of delayed increase in fetal heart rate is also incontrast to studies in fetal lambs, where fetal ritodrine infusion forvarying periods was employed but where no changes in fetal arterialpressure were noted (Siimes et al., 1978;Bassett et al., 1989; van derWeyde 1990). Bassett et al. (1989) observed that fetal tachycardiapersisted for 12 -24 hours but that the fetal heart rate returned tocontrol values by 48 hours despite continuous ritodrine infusion.Desensitization of the fetal myocardium to ritodrine has beendemonstrated to account for this tachyphylaxis (Bassett et al., 1990).The previously mentioned studies (Siimes et al., 1978; Bassett et al.,1989; van der Weyde 1990) employed ritodrine infusion rates between 2.6and 5 zg/minute for time periods varying between 30 minutes and 80hours. Only in the study of van der Weyde (1990), however, were fetalarterial ritodrine concentrations measured, as reported here in Section3.2.. The fetal arterial ritodrine concentrations occurring during thefetal infusion studies were, however, much lower than those in theinitial period of the fetal bolus studies. In contrast to the fetal176infusion studies, the administration of the 2 mg fetal bolus doseresulted in a mean decrease in fetal arterial pressure of 13 mm Hg.Although it is clear that fl-adrenoreceptors exist in the myocardiumof the fetal lamb, their role in the control of fetal cardiovascularfunction has been the focus of much investigation. In a series ofstudies, Assali and coworkers have described the development of theneurohunioral control of cardiovascular function in the fetal lamb andhave also described this control in comparison to neonatal lambs andadult sheep (Assali et al., 1977; Nuwayhid et al., 1975a; Nuwayhid etal., 1975b; Woods et al., 1977). The studies conducted by theseinvestigators have demonstrated that although the sympathetic nervoussystem is well developed in the late gestation fetal lamb (Lebowitz etal., 1972), the parasympathetic system is less well developed and thatthere is little to no vagal control of the resting fetal heart rate(Assali et al., 1977; Nuwayhid et al., 1975). Paraysmpathetic influencecan, however, be stimulated through the Bezold-Jarisch reflex (Nuwayhidet al., 1975), via atrial stretching (Nuwayhid et al., 1975), or inresponse to hypoxia (Parer, 1983). It would appear that the autonomicnervous system exerts controlling influences during stress or whenactivated by specific reflexes (Assali et al., 1977). The systemiccirculation of the fetus excluding the placental circulation, however,is under a high degree of neurohumoral tone and resembles an adultanimal or human with neurogenic hypertension (Assali et al., 1977;Rankin and McLaughlin, 1979). Further, due to the high degree ofneurohumoral control of the peripheral vascular system existing in thefetus, fetal blood pressure is very sensitive to vasoactive substances(Assali et al., 1977).177A further experimental observation of the studies by Assali andcoworkers was that, although $-adrenergic responsiveness of the fetallamb increases as gestation progresses, the central cardiovasculareffects noted in the fetus were much less than those observed in theneonatal lamb or adult sheep (Woods et al., 1977; Assali et al., 1977).In fact, the studies in the fetus required the use of a relatively 10fold greater dose than those in the neonate or adult (Assail et al.,1977). A likely hypothesis is that the presence of the fetal shunts(e.g. ductus arteriosus, ductus venosus, and foramen ovale) and theumbilical circulation tend to dampen the circulatory effects ofvasomotor agents (Assaii et al., 1977).Arterial blood pressure is the product of cardiac output and totalperipheral resistance (Sheppard and Vanhoutte, 1981). Thus, the fall inarterial pressure seen in the fetal bolus studies may result from adecrease in one or both of cardiac output or total peripheralresistance. There is evidence from the acute inicrosphere studies thatblood flow to the placenta, kidneys, gastrointestinal tract, skeletalmuscle, bone, and fetal lower body is decreased by ritodrine (Table 17).Since cardiac output to these regions accounts for 80 % of the fetalbiventricular output it is likely inferred that fetal cardiac output hasdecreased. This possibility is strengthened by the rapid development offetal hypoxemia following the ritodrine bolus, since reduction inumbilical blood flow results in fetal hypoxemia (Itskovitz et al..1983). In addition we have recently made a ritodrine 2 mg bolusadministration to a chronically instrumented fetus in which anelectromagnetic flowmeter had been placed on the external iliac artery.Both the heart rate and blood pressure effects noted in the fetal bolus178studies were reproduced, and it was also observed that external iliacblood flow was decreased 5O % following the ritodrine bolus, which isconsistent with the fall in hind-limb muscle and bone flows observed inthe acute studies. This again suggests that fetal cardiac output hasfallen.An explanation for the lack of immediate tachycardia observed inthese fetal bolus experiments may be more complicated. From theobservation of the large fall in fetal arterial pressure occurring afterthe ritodrine administration it would be somewhat intuitive to expect atleast a reflex tachycardia with or without direct myocardial stimulationby ritodrine. A fall in fetal arterial pressure of < 15 %, induced bydecreasing venous return via a cuff implanted around the inferior venacava, result in a reflex tachycardia which is blocked by theadministration of propranolol (Walker et al., 1983). Larger falls inarterial pressure, > 30 % induced by the same method, result in aninitial bradycardia (Walker et al., 1983). The response in the fetallambs to hypotension was different to that observed in newborns wherereflex tachycardia was observed regardless of the magnitude of theinduced hypotension (Walker et al., 1983). The explanation of thebradycardia observed in the > 30 % fall in fetal arterial pressure hasbeen suggested to be an increased activity of vagal efferents to theheart which slow fetal heart rate (Walker et al., 1983). Such responses(i.e. bradycardia following large drops in fetal arterial pressure) havealso been observed when a decrease of z30 % or greater in fetal arterialpressure has been caused by haemmorrhage (Kwan 1989; Meyers et al.,1991), hypoxemia (Boddy et al., 1974;Bocking et al., 1988 and 1989), andby pilocarpine administration (Szeto and Hinman, 1990). It is179interesting to note that in all of these studies that the initial periodof bradycardia was followed by the progressive development of fetaltachycardia as in the fetal ritodrine bolus studies. Further, in asingle chronically instrumented fetus graded doses of 0.01, 0.04, 0.2and 2 mg of ritodrine were recently administered. In all but the 2 mgdose immediate tachycardia without significant decreases in fetalarterial pressure were observed. Following the 2 mg dose a fall infetal arterial pressure of > 30 % was seen accompanied by a slowlydeveloping tachycardia over a period of 1 hour that was sustained for atleast 12 hours following the dose. These observations, although only ina single animal, suggest that the observed heart rate effect is aproduct of the marked fetal hypotension brought about by the 2 mgritodrine bolus.The progressive development of tachycardia in the fetus followingthe hypotension may be the result of increased circulating levels ofcatecholamines and angiotensin. Studies of the tachycardia associatedwith haemmorrhage have used this explanation of the delayed progressivenature of the fetal tachycardia (Brace and Cheung, 1986; Iwamoto andRudolph, 1981). This may also be the case in the fetal bolus studiessince, during the period of fetal tachycardia, fetal arterial ritodrineconcentrations are continuously falling. The fall in fetal ritodrineconcentration over this time period makes a direct relationship betweenfetal ritodrine concentration and fetal heart rate unlikely.Furthermore, as noted above fetal hypotension induced by widelydiffering experimental techniques results in a delayed tachycardiaregardless of the presence or absence of exogenously administeredsympathomimetics.1804.4.4. Metabolic EffectsThe changes in fetal blood gas parameters during the fetal bolusstudies are qualitatively similar to those observed in the ewe duringthe maternal bolus studies (Section 3.3.). In addition, the effectsnoted are similar to those observed in the fetus during fetal infusionof ritodrine (Silmes et al., 1978; Bassett et al., 1989; van der Weyde,1990). Following the fetal bolus administration, the fetus develops aprogressive metabolic acidemia (Figure 41) which is apparent at thefirst sampling point (5 minutes) and persists for 6-8 hours. The fallin fetal arterial pH during the fetal bolus studies is greater thanthose observed by Bassett et al. (1989) or van der Weyde et al. (1991)during constant rate infusion to the fetus. In addition, fetal arterialp02 falls progressively from a control value of 21.1 ± 2.0 mm Hg (mean ±s.e.m.) to a minimum value of 12.0 ± 1.0 mm Hg at 30 minutes post-infusion. The fall in fetal arterial P02 seen during the fetal bolusstudies is greater in magnitude than those noted by Bassett et al.(1989) and van der Weyde et al. (1991) during constant rate infusions (5g/min and 2.6 ig/min). In the fetal bolus studies then, the fetus isexposed not only to acidemia but also to a period of fairly markedhypoxemia. There is also a marked fall in fetal arterial oxygensaturation to approximately half of the control value (Figure 41) whichpersists for up to 24 hours. Although the fetal arterial pH returns tocontrol values after approximately 6 hours, both fetal arterial [HC03]and whole blood base excess remain below control values much longer,suggesting compensation by some other buffering system. The lack ofchanges in blood gas parameters on the maternal side reflects the lack181of placental transfer of ritodrine demonstrated in the present work(Sections 3.2., 3.3., 3.4.).In common with the studies of fetal ritodrine infusion (van derWeyde et a?., 1991; Bassett et a?., 1989) elevations of both fetalarterial lactate and glucose were seen after the fetal bolus ritodrineadministration. The concentration of lactate in fetal arterial plasmaincreases approximately 5 fold and remains above control for 24 hoursfollowing the fetal bolus administration (Figure 44). There is fairlyclose parallel behavior between the lactate concentration in theumbilical vein and the fetal femoral artery. The increase in umbilicalvenous lactate concentration and increase in the umbilical venoarteriallactate difference would suggest that some of the lactate is derivedfrom placental lactate production assuming that umbilical blood flow isunchanged following the fetal ritodrine bolus. Similar to the maternalbolus studies the incremental increase in fetal arterial lactate hasbeen calculated (904.3 ± 59.6 mg h/lOU mL) and from the study of Sparkset a?. (1982) a fetal lactate clearance of 13 mL/kg/min can beestimated. The product of these values would suggest that 7.1 g oflactate is produced per kilogram of fetal weight. In contrast, usingthe metabolic clearance rate for lactate determined by Kitts andKrishnamurti (1982) of 37.4 mL/kg/min the fetus would produce 20.3 g oflactate per kilogram fetal weight. Again this calculation assumes thatthe clearance of lactate is independent of the arterial lactateconcentration and that ritodrine would only affect the production ratherthan the elimination of lactate.Similar to the studies employing fetal ritodrine infusion (Siimeset al., 1978; Warburton et al., 1988; Bassett et al., 1989; van der182Weyde et al.., 1991) there was an approximate doubling of the fetalarterial glucose concentration in response to the fetal ritodrine bolus.Since the umbilical venous glucose concentration remained essentiallyconstant, it is likely that the increase in glucose is caused bybreakdown of hepatic glycogen stores as has been demonstrated byWarburton et al. (1988).4.4.5. Uterine ContracturesIn the analysis of the uterine contractures following fetal bolusadministration the data have been compared to the maternal salinecontrol group. This was done since the fetal bolus control studies werenot adequate to give a statistically representative estimation ofuterine contractures. Since there is little reason to suspect thatfetal saline administration would alter maternal uterine contractures,comparison between the fetal bolus and maternal control groups was made.The lack of effect of ritodrine on the number, duration or temporaldistribution of uterine contractures following fetal administration wasnot unexpected. Since the effect of ritodrine on uterine contractureswould likely require the interaction of ritodrine with myonietrialadrenoreceptors, ritodrine would have to reach the maternal bloodcirculation to exert this effect. As ritodrine could not be determinedin either uterine venous or maternal femoral arterial plasma it isunlikely that any significant amount of the fetal bolus was transferredto the mother.1834.4.6. Microsphere StudiesSiimes et al. (1978) examined the effects of fetal ritodrineinfusion (30 minutes at a mean rate of 1.2 g/kg/min) on fetal cardiacoutput and the distribution of cardiac output in the fetal lamb usingboth microsphere and electromagnetic flow probes in chronically preparedanimals. These short term infusions demonstrated minimal change infetal cardiac output and no significant alterations in the distributionof cardiac output other than an increase in flow to the adrenals.In the present study an acute preparation was used to examine theeffects of the fetal ritodrine bolus on fetal organ blood flow. Inthese studies the fetal arterial ritodrine concentrations observed werevery similar to those observed in the chronically instrumented fetuses,but, of course in the initial period after drug injection, were muchhigher than was the case in the fetal infusion studies (Silmes et al.,1978). Further, it was felt that the use of microspheres may aid inproviding an explanation for the fetal hypotension and lack of immediatetachycardia following the ritodrine bolus. Since a possible explanationfor the fetal hypotension could be a reduction in cardiac output,microspheres were used to attempt to delineate changes in fetal regionalblood flows resulting from the ritodrine bolus. From the fetal bolusstudies, the fall in fetal p02 suggests a decrease in umbilical bloodflow. This was confirmed in the microsphere studies, where a 20 %decrease in flow was observed 5 minutes following ritodrineadministration. This was accompanied by a 74 % decrease in flow tohind limb muscle and bone as well as reduced flow to various otherorgans (e.g. gastrintestina1 tract, kidney). Together these tissues184(including the placenta) account for 8O % of fetal cardiac output.Hence the overall reduction of their flow is most readily explainable bya decrease in fetal cardiac output caused by the high drug levelspresent in the initial period after drug administration. These changeswere not apparent with lower fetal ritodrine concentrations (Silmes eta!., 1978). The mechanisms of the decrease in cardiac output remain tobe elucidated but would most likely involve a decrease in stroke volume.In human fetuses exposed in utero to ritodrine there are reports offetal arrhythniias, cardiac failure, myocardial ischemia and infarctionthat may be due to ritodrine induced increases in myocardialintracellular calcium leading to overexcitation and cell necrosis (Katzand Seeds, 1989).Various fetal tissues exhibited an increase in blood flow followingritodrine, although some of these are unlikely to be the direct resultof the drug. Thus the increases in brain blood flow more likelyresulted from the acidemia, while the increase in myocardial blood flowlikely is due to the tachycardia. However, in the case of the increasedflow to brown adipose tissue and skin, a direct effect of the drug seemspossible. The increase in blood flow to the perirenal brown fat isconsistent with the effects of other adrenergic agonists (e.g.norepinephrine) (Schroeder et a!., 1987). A possible interpretation ofthe increase in blood flow to the perirenal brown fat and the increasein blood flow to the skin may be a metabolic mobilization of the brownadipose tissue accompanied by an attempt to dissipate the energyliberated from the brown adipose tissue. There is considerableevidence, however, that the perirenal brown fat cannot be activated inutero before 140 days of gestation despite increases in perirenal brown185fat blood flow induced by hypoxia, cold, norepinephrine, or 3,5,3’-triiodothyronine (Rurak et al., 1990; Schimmel and McCarthy, 1986; Poweret a!., 1987; Gunn et al., 1986). Possible explanations for themetabolic non-responsiveness of fetal perirenal brown fat are the lowoxygen delivery to the brown fat (Power et al., 1987) and the placentalproduction of an inhibitory factor, possible adenosine, of brown fatrespiration (Schinimel and McCarthy, 1986). It may be possible that inthe present study that the acute nature of the experiment and the highritodrine concentrations may have overridden the normal inhibition ofbrown fat mobilization.In the acutely prepared fetuses, however, tachycardia lasting 26and 55 minutes and a large fall in arterial pressure (24.5 mm Hg) whichpersisted for the duration of the study (60 minutes) occurred. Theobservation of the tachycardia is different from the chronic fetal bolusstudies where no significant increase in fetal heart rate was seen(Section 3.4.). In addition, although fetal arterial pressure fell inthe chronic fetal bolus studies the fall in fetal arterial pressure inthe acute microsphere studies is 2 fold greater in magnitude andpersists for at least 4 times as long as in the chronic studies.There may be several reasons for the differing effects notedbetween the chronic and acutely prepared fetuses. Acute surgical stressmay cause a number of alterations on the fetal cardiovascular systemthat may make comparison to the in utero or chronically prepared fetusdifficult (Rudolph and Heymann, 1980). In acute preparationsmanipulation of the fetus may cause autonomic nervous systemstimulation, release of fetal ACTH, vasopressin and other hormones whichmay affect the circulation and/or responses of the cardiovascular system186(Rudolph and Heymann, 1980). In the acute preparation used in thisstudy the fetus is also under halothane anaesthesia. In general,anaesthetics can abolish either partial or totally maternal or fetalneural reflexes control 1 i ng vascul ar refl exes and/or alter vascul arresponses to vasoactive substances (Assali et al.. 1974). Halothane inparticular can cause alteration in the baroreceptor reflex, hypotension,and bradycardia (Marshall and Woilman, 1980). In addition, halothanecan increase the automaticity of the heart, an effect which isexaggerated by adrenergic agonists (Marshall and Woliman, 1980). Thediffering observations between the acute and chronic experiments maythen be reflections of the acute nature of the experiment and theeffects of anaesthesia on the fetus. However, a definitive explanationfor the different fetal heart rate responses in the chronicallymaintained and acutely prepared fetuses is not obvious at this time.Clearly further studies of this phenomenon are warranted.It must be emphasized that these microsphere studies must beregarded as very preliminary, because of their acute nature and the lownumbers involved. However, some of the effects, particularly thedecrease in umbilical, muscle, bone, renal, and gastrointestinal bloodflows (and, hence, probably cardiac output) and the opposite change inblood flow to brown adipose tissue and skin, are of considerableinterest, and warrant further investigation. Although the initial fetalarterial ritodrine concentrations (400 ng/mL) in the fetal bolusexperiments are much higher than those achieved in the infusion studies(20 ng/mL), they are not much greater than levels in human umbilicalcord blood (up to 282 ng/mL (Kuhnert et al., 1986; Fujimoto et al.,1871990; Gross et al., 1985; Fujimoto et a!., 1986)(at delivery) associatedwith ritodrine tocolysis.4.5. Comparative Maternal and Fetal Kinetics and Effects4.5.1. KineticsPrior to these studies there have been no pharmacokineticdescriptions of the kinetics of ritodrine in the fetus of any species.In the present study it has been observed that ritodrine has extremelylimited placental transfer across the sheep placenta. Thus, the fetusand mother are pharmacokinetically independent of one another. In thefetal and maternal bolus studies an attempt was made to administersimilar mg/kg doses to ewe and fetus in order to compare thepharmacokinetic behavior between mother and fetus.In assessing the relative exposure of the ewe and fetus in thebolus studies there was no statistically significant difference(unpaired t-test) between the doses given to the mother (50 mg) or fetus(2 mg) when normalized to body weight (in utero fetal weight). Further,there is no significant difference between the maternal and fetal AUC(unpaired t-test) in the maternal and fetal bolus studies, respectively.Also, the concentration ranges observed in the mother and fetus duringthe maternal and fetal bolus studies were not different. Therefore itwould seem likely that the fetus and mother were exposed to ritodrine torelatively the same extents between the fetal and maternal bolusadmini strations.188The only statistically significant difference observed between thematernal and fetal kinetic parameters was the difference between(t1,213 maternal = 8.7 ± 1.8 h; fetal = 2.9 ± 0.7 h) (unpaired ttest). The interpretation of this difference is uncertain, however, itshould be noted that the terminal elimination half-life is a dependentparameter that is determined by the relatively independent parameters,clearance and volume of distribution (Vdss) (Jusko, 1987). In spite ofthis difference there are no significant differences (unpaired t-tests)between the maternal and fetal clearance, Vc, Vdp or Vd55 parametersestimated during these studies.In the maternal and fetal bolus experiments total body clearancesof 24.3 ± 5.0 and 31.7 ± 10.0 mL/min/kg were estimated in the ewe andfetus, respectively. In the fetal infusion studies a fetal total bodyclearance of 62 ± 8.1 mL/min/kg was determined in the fetus. Theapparent discrepancy between the two estimates of fetal total bodyclearance have been discussed in Section 4.2. Thus, the total bodyclearances in the bolus studies are not significantly different betweenmother and fetus (unpaired t-test). Given that the placental clearanceof ritodrine is very low, the calculated total body clearances representprimarily non-placental elimination. In examining the literature itwould appear that a similar relationship between fetal and maternal nonplacental clearance exists for morphine (maternal Clmo = 40 ± 4, fetalClf0 = 42 ± 5 mL/min/kg), methadone (maternal Clmo = 108 ± 12, fetalClf0 = 127 ± 30 mL/min/kg), acetaminophen (maternal Clmo = 15 ± 1,fetal Clf0 = 11 ± 1 mL/min/kg), and metoclopramide (maternal Clmo = ±3, fetal Clf0 = 28 ± 7 mL/min/kg), but not for diphenhydramine (maternalClmo = 43 ± 3, fetal Clf0 = 100 ± 13 mL/min/kg) (Rurak et al., 1991).189It would appear from these studies that with the possible exception ofdiphenhydramine that fetal and maternal non-placental clearances aregenerally of similar orders of magnitude.Another interesting observation that can be made is that in thematernal bolus studies the AUCf/AUCm was 0.03 ± 0.01 (i.e. fetalexposure is 3 % that of the mother) (Table 5) giving a mean fetal AUCfof 19.7 ng h/mL. If this AUCf value is multiplied by the fetal totalbody clearances (62 and 31.7 mL/min/kg), the fetus can be estimated tohave received between 37.5 - 73 ig or 0.08 - 0.15 % of the 50 nigmaternal bolus. Thus the apparent amount of drug received by the fetusis far smaller when compared to the maternal dose than is the fetal drugexposure as expressed by the AUCf/AUCm ratio.4.5.2. Pharmacological EffectsIn spite of the observation of essentially equivalent drug exposureand similar pharmacokinetic behavior between the ewe and fetus it isevident that the pharmacological responses in the ewe and fetus due toritodrine are qualitatively and quantitatively different.In the ewe, following maternal bolus administration, the mostevident cardiovascular response was an approximate doubling of heartrate with little change in the mean maternal arterial pressure.Following fetal bolus administration no such tachycardia was evident butthere was a significant fall ( 13 mm Hg) in the mean fetal arterialpressure. The cardiovascular effects in the fetus have been discussedin Section 4.4.3..190Metabolic acidosis was created in both ewe and fetus followingritodrine administration. In the fetus (Section 4.3.3.), however, theacidosis was of much greater intensity (approximately double as shown bythe fall in arterial pH or base excess), persisted for almost double theduration than that in the ewe (Section 4.4.4.), and was also associatedwith significant hypoxemia as demonstrated by the fall in fetal arterialP°2. Further, due to the ability of the ewe to alter respiration inresponse to changing acid-base status, changes in maternal pCO2 werealso evident in the ewe. Changes in respiratory depth and rate areobviously not possible in the fetus since the placenta acts as the organof fetal gas exchange.In both ewe and fetus the metabolic acidemia was in large part theresult of lactic acidosis (Sections 4.3.3. and 4.4.4.). The increase inarterial lactate concentrations was greater in the fetus as demonstratedby the incremental AUC for lactate. Further the lactic acidemiapersisted for a much longer period in the fetus (24 h) than in the ewe(8 h). As estimated by the amount of lactate produced (AUC xCliactate), the fetus produced 7-20 g of lactate per kg fetal weight(Section 4.4.4.) while the ewe produced O.5 g per kg maternal weight(Section 4.3.3.) in response to ritodrine administration.Thus, there are significant qualitative (e.g. cardiovascularresponses) as well as quantitative differences (e.g. metabolic effects)between the ewe and fetus in response to equivalent pharmacokineticexposure to ritodrine. These differences are likely to result fromdifferent pharmacological sensitivities between mother and fetus as wellas differing abilities of the ewe and fetus to maintain homeostasis.1914.5.3. Indices of Fetal Drug ExposureMany experimental approaches have been used to examine theplacental transfer and fetal exposure of drugs. The factors involved inthe movement of drugs across the placenta have been thoroughly reviewed(Rurak et al., 1991; Reynolds and Knott 1989; Mitani et al., 1987;Mucklow, 1986; Mihaly and Morgan, 1984; Yurth, 1982; Levy, 1981;Waddell and Marlowe, 1981;). Studies of the fetal exposure of drugs arelimited in humans by both technical and ethical restraints. As a resultthe largest proportion of information regarding the exposure of humanfetuses following maternal drug administration (either chronic or acute)has come from measurement of maternal and umbilical cord blood andoccasionally amniotic fluid at delivery (Rurak et a!., 1991). Althoughstudies conducted in this manner are of clinical relevance, the fetalexposure, as estimated by the fetal to maternal concentration ratio, ishighly dependent on the time of sampling when the drug is not at steady-state (Anderson et al.,1980c). Some studies have attempted to overcomethis by creating a composite of fetal/maternal concentration ratios atdifferent sampling times from different subjects (e.g. Bray et a!.,1966). A possible complication of measuring maternal and fetal drugconcentrations at delivery is that alterations in uterine and umbilicalblood flow rates which normally occur during labor could alter thematernal/fetal concentration ratio (Hamshaw-Thomas et a!., 1984).Recently in utero sampling procedures have allowed fetal/maternalconcentration ratios to be determined in the antepartum period (Daffoset a!., 1984). Serial sampling of fetal blood following acute orchronic maternal drug administration has not been accomplished.192As a result of the technical and ethical restraints on serial bloodsampling from human fetuses, both chronically-catheterized pregnantanimals (especially sheep) (e.g. Riggs et al., 1987; Wang et al., 1986)and isolated perfused placentas, both in situ and in vitro, of humansand animals (e.g. Hanishaw-Thomas et al., 1984) have been employed.Analysis of fetal drug exposure using these preparations has usuallybeen made using the fetal/maternal concentration ratio (Reynolds andKnott, 1989) or on the basis of the fetal/maternal AUC ratio proposed byLevy and Hayton (1973).Both the fetal/maternal concentration and AUC ratios compare fetaldrug concentrations with maternal concentrations. Values approaching orgreater than 1 suggest that the fetus is exposed to a similar extent asis the mother. The implications of fetal exposure as measured by theseindices to fetal pharmacological effects is less clear. In the presentstudy both fetal and maternal bolus administrations have been carriedout. It has been established both in this (Sections 3.2., 3.3., 3.4.)and other studies (Fujimoto et al., 1984) that ritodrine has extremelylimited placental transfer in the sheep. Further the administration ofritodrine on one side of the ovine placenta does not result in thedevelopment of significant physiological changes on the opposite side ofthe placenta (van der Weyde, 1990; van der Weyde et al.; Sections 3.2.,3.3., 3.4.). Thus for ritodrine, in the pregnant sheep model, the eweand fetus are essentially in pharmacokinetic and pharmacologicalisolation from one another. In the maternal and fetal experiments,equivalent doses on a mg/kg basis were administered. The administrationof these doses resulted in equivalent AUC in both mother and fetus andthe concentrations achieved in both mother and fetus were of similar193orders of magnitude. Therefore by all conventional estimates of drugexposure the fetus and the mother have been equally exposed toritodrine. The pharmacological responses that have been measured,however, are both qualitatively (e.g. heart rate effect, blood pressureeffect) and quantitatively (e.g. extent and duration of acidosis,lactate production, hypoxemia) different. This would obviously suggestthat the late gestation fetus has a different pharmacologicalsensitivity to ritodrine than the mother. Further the apparentsensitivity of the fetus also depends on the effect in question (e.g.heart rate vs. acidosis).The implication of these results is that, for ritodrine at least,one unit of fetal concentration or AUC does not have the same endphysiological result as one unit of concentration or AUC in the mother.These observations have previously been suggested by the pharmacologicalstudies of Assail et al. (1977) which were however, made in the absenceof either fetal or maternal drug concentration measurements. Furtherthe geometry of the fetal circulation may have profound influences(Section 4.4.3.) on both the distribution of drug and the effectsobserved. For this reason even similar doses of drugs given by similarroutes (e.g. intravenous via the inferior vena cava) may result indiffering distributions and effects between mother and fetus. Anadditional complication is that the fetus is maturing throughoutgestation both in terms of organ function and neurohumoral influences(Assali et al., 1977) which may suggest that both the pharmacokineticand pharmacodynamic relationships may change throughout pregnancy.1944.5.4. In viva Pharmacokinetic/Pharmacodynamic ModellingIn the present work there are a number of observations ofphysiological changes induced in both the ewe and fetus through theadministration of ritodrine. Although it has been possible to measureboth the effect and plasma ritodrine concentration, in most casesattempting to establish a concentration-effect relationship has beendifficult. This section seeks to discuss the difficulties of developingconcentration-effect relationships from in viva data of the typegathered during the present experiments.Considerable effort in the recent pharmacokinetic literature hasbeen expended attempting to relate drug concentration andpharmacological effect, particularly when there is a temporal lagbetween the measured drug concentrations and the observedpharmacological effects of the drug. In his pioneering descriptions ofthe kinetics of pharmacological effect, Levy (1964, 1965, 1966; Levy andNelson, 1965) provided elegant derivation and experimental evidence forthe relationships describing both the time course and magnitude ofpharmacologic response. It should be noted that this work occurredprior to the development of assays of sufficient capability to measureplasma/serum/blood levels of the drugs under investigation. Thesemethods of analysis made several assumptions regarding both thepharmacokinetics of the drug and the pharmacologic response beingmeasured. Essentially these methods require a single compartment modelgoverned by first-order processes with pharmacological effect resultingfrom the drug only (no active metabolites) and that the pharmacologicaleffect be related linearly to the logarithm of the amount of drug in the195body (Levy, 1965). As Levy himself admits (Levy, 1965), there are manyoccasions when these assumptions are not upheld; with the advent of moresophisticated analytical methods and the possibility of more widespreadpharmacological response measurement more complexpharmacokinetic/pharmacodynamic analysis methods have been required.Three general approaches have been developed to overcome thelimitations of the assumptions required by Levy (1965). Holford andSheiner (1981a, 1981b, 1982) have proposed a purely mathematicalapproach to account for temporal lags between measured drugconcentration and observed effect. This method involves the estimationof drug concentrations in a theoretical effect compartment using theparameters obtained via pharmacokinetic modelling (Holford and Sheiner,1981a). These effect compartment concentrations are then related to theeffect measurements using a selected pharmacological response model(e.g. linear, Emax, Smax) (Holford and Sheiner, 1981a). The effectcompartment is described as being of negligible volume and receiving anegligible portion of the administered dose such that it does not appearin the pharmacokinetic modelling’ (Holford and Sheiner, 1982).Modifications of the approaches of Holford and Sheiner (1981a) havebeen attempted such that either a nonparametric pharmacodynamic model(Fuseau and Sheiner, 1984) or nonparametric models of bothpharmacokinetics and pharmacodynamics can be employed (Unadkat et al.,1986; Verotta and Sheiner, 1987). These models still, however, retain aparametric link between pharmacokinetics and pharmacodynamics in theform of an effect compartment, the entry and exit from which are byfirst-order processes (Fuseau and Sheiner, 1984; Unadkat et al., 1986;Verotta and Sheiner, 1987).196Most recently, generalized systems approaches have been presentedto describe both pharmacokinetic and pharmacodynamic behavior (VengPedersen and Gillespie, 1988; Gillespie et al., 1988). In this approachplasma/serum/blood concentration is linked to pharmacological effectthrough the use of a series of operator functions (12) which representthe conduction, transduction, and propagation processes within ageneralized model (Veng-Pedersen and Gillespie, 1988). In general thismethod allows the mathematical description of general properties of agiven system without requiring description or modelling of theindividual processes contributing to the expression of a particularproperty (Veng-Pedersen and Gillespie, 1988). Although the theoreticalframework has been presented few experimental applications have beenmade.There has been widespread application of all of these methods, withthe possible exception of the systems approach, in the recentpharmacokinetic literature and the importance of simultaneouspharmacokinetic/pharmacodynamic modeling has been thoughtfully reviewedby Galeazzi (1986) and Gibaldi (1989). There would appear to be,however, several considerations that do not seem to have been addressedin the pharmacokinetic/pharmacodynamic literature that may influence theinterpretations of concentration-effect relationships determined bythese methods.In general, the previously discussed methods of analysis treat themeasured effect as the terminal event of a stochastic process of one ormore steps. That is, drug is viewed to interact with its site ofaction, the signal resulting from this interaction is transduced,propagated and finally an effect is observed (Veng-Pedersen and197Gillespie, 1988). This interpretation of the production of apharmacological response, however, cannot be universally applied. Anexample of a case where a response may not be able to be viewed as theterminal event of a stochastic process is the change in arterial pHfollowing ritodrine administration (Sections 3.3. and 3.4.). Althoughin both cases the fall and subsequent rise of arterial pH in eithermother or fetus could be related to the plasma concentration ofritodrine through the use of an effect compartment, such an analysiswould ignore well recognized concepts regarding the control of mammalianacid-base balance. In broad terms the acid-base balance of an adultmammal may be controlled by either respiratory and non-respiratoryroutes (Laiken and Fanestil, 1985). Respiratory routes would includecontrol over both the rate and depth of breathing which in turn wouldresult in either the retention or release of CO2. Non-respiratoryroutes would include renal retention or excretion of H+ or othersubstances (e.g. (P04)3, lactate) which may act as buffers. Thusarterial pH must be viewed not as the endpoint of a stochastic processbut as a centerpoint balanced between and interdependent upon bothrespiratory and non-respiratory control. Further, each control ofarterial pH has different subroutes. In the present study, it has beendemonstrated that at least a portion of the metabolic acidosis ismediated through lactic acid production but that in spite of continuedrelatively high concentrations of lactic acid, arterial pH returns tonormal. This suggests the presence of compensation by other bufferingsystems (Laiken and Fanestil, 1985). The contribution of these othersystems cannot, however, be determined. Therefore to set up arelationship between ritodrine concentration and arterial pH would198ignore the modulating effects of other buffer systems and would estimateparameters not entirely the product of the administered drug.The modulation of pharmacologic response by homeostatic mechanismsmay introduce a further complication in the interpretation ofpharmacodynamic data. If a response (e.g. arterial pH in the presentstudy) is viewed as being influenced not only by the drug administeredbut also by homeostatic reflexes, the observed response can becomedependent on the physiological state of the experimental subject priorto drug administration. This speculation may not alter theinterpretations within an experiment but rather would be important incomparisons of one or more studies.The use of an effect compartment has been suggested to account fortime lags between peak drug concentrations and peak drug effects(Holford and Sheiner, 1981b). Concentrations in the effect compartmentare estimated from the measured plasma/serum/blood concentrations,pharmacokinetic parameters, and effect observations (Holford and Sheiner1981b; Unadkat et a]., 1986). Although many studies have employed thesemethods (ex. Stanski et a]., 1979; Holford et al., 1981; Sheiner et al.,1979; Whiting et al., 1980; Fisher et al., 1990), there are severalfactors that must be considered in the interpretation of this approach.Since the effect compartment is outside the central compartment (Holfordand Sheiner 1981b) the concentrations of drug within the effectcompartment can never be measured. Further, the estimates of the effectcompartment drug concentrations are largely dependent on the adequacy ofthe pharmacokinetic model (Holford and Sheiner 1981b) or on assumedproperties and estimated parametric constants of the effect compartment(Verotta and Sheiner, 1987). Also in the methods of Holford and Sheiner199(1981b) the form of the pharmacologic model must also be assumed. Theeffect compartment models attempt to resolve time lags between peak drugconcentrations and peak drug effects by fitting the observed effect-timemeasurements to an assumed relationship with estimated concentration-time data. In doing this, other possible explanations for the lack ofcorrelation between plasma/serum blood drug concentration and effect(such as the production of an active metabolite, the production of anendogenous modulator, the modulation of effect by homeostaticmechanisms) may be obscured. In the present work such modelling wouldlimit the interpretation of these effects solely to an immeasurable drugconcentration and thereby exclude many reasonable alternativeinterpretations.Given the relatively large numbers of parameters that are requiredto be estimated even in the simplest of pharmacokinetic/pharmacodynaniicmodelling, the question of the dependence of the results on the adequacyof sampling (both pharmacokinetic and pharmacologic) and on thestatistical methods of data analysis should be addressed. It is wellrecognized in the literature describing generalized procedures forfitting data to mathematical models that, for any given level ofprecision, the larger the number of parameters to be estimated thelarger the number of observations required (Schwartz, 1978). Inaddition, not only is the total number of observations important but thedistribution of the observations within the experimental period also hasa significant impact on the mathematical description of the data(Colburn et al., 1988). Thus the transferability of interpretationsbetween studies is likely to be dependent on the manner in which thedata is gathered and the mathematical processes by which it is analyzed.200The objectives of modelling pharmacokinetic/pharmacodynamic datahave been variously described as simulation, parameter estimation, modelcomparison, extrapolation (prediction), and summarization (datareduction) (Colburn et al., 1988; Holford, 1982). When these objectivesare applied to solely pharmacokinetic data many of these objectives canbe faithfully fulfilled since the modeling is dependent only on thegathered concentration-time data. With the addition of pharmacodynamicdata the complexity of the modeling is of a much higher degree since notonly must concentration-time relationship be estimated but also theeffect-time and concentration-effect relationships determined. The keyassumption implicit in such an approach is that the observed effect istotally dependent on the concentration of drug in some area of the body.However, the processes of homeostatic adaptations to physiologicalstresses are well documented and the assumption that an observed effectis solely the result of drug may ignore physiological reality. Thus,the establishment of concentration-effect relationships by these meansmay unnecessarily restrict the appropriate interpretation of thepharmacokinetic/pharmacodynamic data obtained.2015. SUMMARY AND CONCLUSIONSAn electron capture detection capillary gas-chromatographic assaymethod (Wright et al., 1991a) has been developed and applied to thestudy of the maternal and fetal pharmacokinetics of ritodrine in thechronically instrumented pregnant sheep model. The analytical method ishighly sensitive and has proven to be suitable for the quantitation ofritodrine in single dose pharmacokinetic studies from a variety ofbiological fluids. The primary advantages of the developed method overpreviously published methods include:i) the smaller biological fluid volumes required.ii) the ability to perform multiple injections of eachsample.iii) improved sensitivity, in terms of amount injectedon column or reaching the detectoriv) improved reproducibilityThe assay method has been shown to be reproducible (inter- and intra-daycoefficients of variation < 10 %) and stable during the pharmacokineticstudies of ritodrine.The developed assay method has been applied to the study of theclearance and disposition of ritodrine in the fetal lamb during andafter constant rate fetal intravenous infusion (Wright et al., 1991b).Ritodrine appears to have only very limited clearance across the sheepplacenta (Cl 9.2 ± 2 mL/min/kg; mean ± s.e.m.). Furthermore, ritodrinecould not be detected in either maternal arterial or uterine venousplasma. There is, however, significant fetal non-placental clearance ofritodrine (Clf0 52.8 ± 8.0 mL/min/kg). At least part of this clearance202appears to be due to fetal drug metabolism, as evidenced by theaccumulation of glucuronide conjugates of ritodrine in the amnioticfluid. Ritodrine was also shown to accumulate in the amniotic and fetaltracheal fluids and persist after fetal arterial plasma concentrationsbecame undetectable. The accumulation of ritodrine in the trachealfluid may be of pharmacologic significance, given the well documentedand potent effects of2-agonists on fetal lung function anddevelopment.The pharmacokinetics and pharmacodynamics of ritodrine were studiedin 10 pregnant ewes following the administration of a 50 mg maternalintravenous bolus dose. In all but one animal, a biexponential declinein maternal arterial plasma concentrations was observed. Fetal arterialconcentrations were very low and peaked at 1O minutes followingmaternal drug administration. Fetal drug exposure as determined by thefetal to maternal AUC ratio was very low (O.O3). Maternal total bodyclearance averaged 24 mL/min/kg. The volumes of distribution determinedby various methods were relatively large: Vd, 1.6 ± 0.3; Vdarea, 14.3 ±3.5; Vd5, 10.0 ± 1.9 L/kg. The maternal distributional half-life (a)averaged 0.5 hours while the elimination half-life () averaged 8.7hours. The apparent fetal elimination half-life averaged 2.3 hours.Ritodrine accumulated in both the amniotic (peak concentrations z10ng/mL) and fetal tracheal (peak concentrations 20 ng/mL) fluids andpersisted in these fluids longer than in the fetal arterial plasma. Asin the fetal infusion studies, glucuronide conjugates of ritodrine weredetectable in the amniotic fluid.A number of physiological effects were observed in the motherfollowing the maternal bolus administration. Following drug203administration there was an approximate doubling of maternal heart ratewith no apparent change in maternal arterial blood pressure. Maternalheart rate declined to control levels over a period of 4 - 6 hours. Theewe developed a metabolic acidosis which was in part mediated by lacticacid. This acidosis was partially compensated by respiratory alkalosis.There was no apparent change in the maternal arterial concentration ofglucose during these studies. There was also a decrease in uterinecontractures during the first 6 hours following drug administration.There were no apparent changes in fetal cardiovascular or metabolicparameters during these studies.There was a direct relationship between the maternal arterialritodrine concentration and the maternal heart rate. This relationshipwas modeled using an Emax equation. Direct and, therefore, obviousrelationships could not be found between the maternal arterial plasmaritodrine concentrations and the maternal metabolic effects.The pharmacokinetic and pharmacodynamics of ritodrine were studiedin 5 fetuses and ewes. following a 2 mg fetal intravenous bolusadministration. In all animals a biexponential decline in the fetalarterial plasma concentration was observed. A mean fetal total bodyclearance of 31.7 ± 10.0 mL/min/kg (mean ± s.e.m.) was noted, which wasnot significantly different from the maternal total body clearancecalculated in the maternal bolus studies but significantly less than thefetal total body clearance calculated in the fetal infusion studies. Itis felt that the effects of the profound fetal acidemia and hypoxiacreated in the fetal bolus studies may account for this difference.Similar to the maternal bolus studies the fetal volumes of distributionwere very large (Vc, 1.6 ± 0.8; Vdarea, 8.7 ± 1.8; Vd55, 8.5 ± 2.6 L/kg)204but were not significantly different from the comparable maternalvolumes of distribution. The fetal distributional (a) half-life was 0.2± 0.1 hours and the fetal elimination ($) half-life was 2.9 ± 0.7 hours(mean ± s.e.m.). The fetal distributional half-life was notsignificantly different from that of the mother. The fetal elimination() half-life was significantly less than that of the mother but was notsignificantly different from the apparent fetal half-life determined inthe maternal bolus studies. Ritodrine could not be determined in eitherthe maternal arterial or uterine venous plasma further confirming thelack of placental transfer of ritodrine across the ovine placenta.There were no significant differences between either the dose (on anmg/kg basis), AUC, AUMC, or concentrations achieved in the fetus duringthe fetal bolus studies and the ewe in the maternal bolus studies.There were both qualitative and quantitative differences betweenthe fetal physiological effects observed in the fetal bolus studies andthe maternal effects seen during the maternal bolus studies. In thefetus the cardiovascular responses were characterized by a fall in thefetal mean arterial pressure (by 13 mm Hg) without the immediatetachycardia seen in the maternal bolus studies. A slow progressivetachycardia (40 beats per minute above control heart rate) was seen andthis persisted for approximately 4 hours following the fetal bolus.After this time fetal heart rate returned toward control values. Thefetus also developed metabolic acidosis in response to the ritodrinedose. Similar to the maternal bolus studies this acidosis was largelymediated by lactic acid. The acidosis in the fetus was much moreintense and persisted for approximately twice as long as the maternalmetabolic acidosis in the maternal bolus studies. There was also a205significant increase in the fetal arterial glucose concentration duringthese studies. There was no apparent effect of the fetal ritodrinebolus on the rate of uterine contractures. There were no apparentmaternal effects following the fetal bolus administration.There were no direct relationships between the fetal arterialplasma concentration of ritodrine and the fetal physiological effectsobserved. The fetal physiological effects were compared with thematernal effects using model independent measures.Two fetuses were acutely prepared for study using radioactivemicrospheres to examine fetal regional blood flows following fetal bolusadministration. Both tachycardia and hypotension were seen followingthe fetal bolus dose which may be the result of the acute nature of thepreparation, the halothane anaesthesia, and the hypercapneic/acidoticcondition of these fetuses. The fetal arterial ritodrine concentrationsdetermined in these studies were not different from those seen in thefetal bolus studies. The fetal ritodrine bolus resulted in a decreasein renal blood flow but increased flow to the adrenals and perirenalbrown fat. The increase in flow to the brown fat was mirrored by anincrease in blood flow to the hind limb skin.In conclusion, when viewed as a whole the results of these studieswould suggest the following:i) There is very little transfer of ritodrine across the ovineplacenta. 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