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

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ANALYSIS, PHARMACOKINETICS, METABOLISM AND PHARMACODYNAMICS OF LABETALOL IN PREGNANT AND NONPREGNANT SHEEP By KRISHNASWAMY YELESWARAM B. Pharm., University of Madras, India, 1984. M. Pharm., Banaras Hindu University, India, 1987. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences) (Division of Biopharmaceutics and Pharmaceutics) We accept this thesis as conforming to the st. o • . d required.  THE UNIVERSITY OF BRITISH COLUMBIA September, 1992 © KRISHNASWAMY YELESWARAM  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  FACULTY OF PHARMACEUTICAL SCIENCES  Anorminix of ^  The University of British Columbia Vancouver, Canada  Date  ^September,  DE-6 (2/88)  1992  ii  ABSTRACT Labetalol, a combined a l and p-adrenoceptor blocker, is used as an antihypertensive, especially in the management of hypertensive disorders in pregnancy. While the antihypertensive efficacy of labetalol in pregnancy has been assessed by a number of clinical trials, there is little information in the literature about the disposition or pharmacodynamics of labetalol in the in utero fetus. Hence, a detailed investigation into the maternal-fetal pharmacokinetics and pharmacodynamics of labetalol was undertaken. Studies in adult nonpregnant sheep were also conducted to complement the data obtained from pregnant sheep. The maternal-fetal pharmacokinetics and pharmacodynamics of labetalol were studied in chronically instrumented pregnant sheep following a 100 mg i.v bolus. Labetalol was measured by a sensitive microbore high-performance liquid chromatographic assay with fluorescence detection, developed in this laboratory. The maternal total body clearance was 30.8 ± 3.83 inUmin/kg (mean ± SEM), elimination half-life was 2.79 ± 0.66 h and the volume of distribution at steadystate was 3.02 ± 0.18 LAT. Labetalol persisted in the amniotic and tracheal fluids up to 24 h. The fetal exposure to labetalol, calculated as the ratio of fetal to maternal area under the arterial plasma concentration-time curve, was equal to 14.4 ± 1.54%. Significant metabolic effects including maternal and fetal hyperglycemia and lactic acidosis and a decrease in fetal oxygen content were observed, suggesting potent r3 2 -agonist activity. The lactic acidosis was more pronounced in the fetus than in the ewe. In adult nonpregnant sheep, a 100 mg i.v. bolus administration of labetalol caused hyperglycemia, lactic acidosis, a significant increase in femoral blood flow, hypotension and tachycardia. The net output of lactate from the hind limb was found to be 6.25 ± 1.35 g (0.07 ± 0.015 mol). The metabolism of labetalol was  iii  studied using the urine and bile samples obtained in this study. The cumulative urinary excretion of labetalol as unchanged drug, glucuronide and sulfate were found to be 1.61 ± 0.38, 11.46 ± 2.83 and 1.47 ± 0.74% of the dose, respectively. A sensitive and specific gas chromatographic assay with mass selective detection was developed to identify and quantitate 3-amino-l-phenylbutane, an oxidative metabolite of labetalol and a close structural analog of amphetamine. Using this assay, the metabolite was identified in the bile and urine samples. The cumulative urinary excretion of the oxidative metabolite was equivalent to 0.044 ± 0.016% of labetalol dose. The exact role of this metabolite in the mediation of labetalol pharmacodynamics remains to be investigated. The vasodilation caused by labetalol in adult sheep, appears to be a direct effect of this drug, with little or no involvement of a-blockade or P-agonism. In the fetal hind limb, however, labetalol did not produce any consistent vasodilation. The pharmacokinetics and pharmacodynamics of dilevalol, the RR-isomer of labetalol, were studied in two adult nonpregnant sheep following a 25 mg intravenous bolus and the results suggest that most of the pharmacodynamics of labetalol in sheep could be attributed to dilevalol. The pharmacokinetics, metabolism and pharmacodynamics of labetalol in the fetus following exposure to clinically relevant labetalol concentrations were studied after a 4 mg direct fetal i.v. bolus. The total body clearance (50.45 ± 1.37 mL/min/kg), elimination half-life (4.35 ± 0.33 h) and apparent volume of distribution at steady-state (14.28 ± 0.95 L/kg) of labetalol in the fetal lamb were significantly higher than the corresponding values in the ewe following a 100 mg bolus. The calculated fetal transplacental clearance was 7.27 ± 2.11 mL/min/kg while the fetal nonplacental clearance was 43.18 ± 3.72 mL/min/kg. Glucuronide conjugates were present in the amniotic fluid samples, but sulphate conjugates and 3-amino-l-phenylbutane, could not be identified in fetal plasma or amniotic fluid  iv  samples. Significant hyperglycemia and lactic acidosis were observed, but the magnitude of fetal lactic acidosis, calculated as the incremental increase in arterial lactate AUC (61.11 ± 15.59 mMh), was not significantly different from that observed following maternal administration (67.8 ± 7.26 mMh), despite a roughly four-fold higher fetal exposure to labetalol. The exact reasons for this discrepancy are not clear.  V  TABLE OF CONTENTS Abstract^ Table of Contents^ List of Illustrations^ List of Tables^ List of Abbreviations^ Acknowledgements^ Dedication^  ii v xi xx xxi xxiv xxv  INTRODUCTION^  1  1.1.^Labetalol^ 1.1.1.^Pharmacology^ 1.1.2.^Clinical Use^ 1.1.2.1. General^ 1.1.2.2. Hypertension in Pregnancy^ 1.1.3.^Fetal Effects^ 1.1.4. Methods of Analysis^ 1.1.5.^Pharmacokinetics^ 1.1.6.^Metabolism^ 1.1.7.^Placental Transfer^  1 1 2 3 3 4 6 7 9 9  1.  1.2.^Dilevalol^  10  1.3.^Objectives and Rationale^ 1.3.1.^Specific Aims^  11 12  2.  EXPERIMENTAL^  ^ 2.1.^Materials ^ 2.1.1. Preparation of Standard Solutions and Buffers  13 13 15  2.2.^Equipment and Instrumentation^ 16 2.2.1. High-performance Liquid Chromatography ^ 16 2.2.3. Gas Chromatography with Mass Selective Detection^17 2.2.3. Physiological Monitoring^ 17 2.2.4. General Experimental^ 18 2.3.^Development of a HPLC Assay with Fluorescence Detection for the Quantitation of Labetalol in ^ Biological Fluids of Sheep  18  vi  18 2.3.1. Optimization of Mobile Phase ^ 19 2.3.2. Optimization of Detection ^ 20 2.3.3.^Optimization of Extraction^ 2.3.4. Procedures for the Analysis of Labetalol in Biological Fluids^ 20 2.3.4.1. HPLC Operating Conditions^ 20 2.3.4.2. Extraction from Biological Fluids^ 21 2.3.4.3. Preparation of Calibration Curve^ 22 2.3.4.4. Quantitation of Labetalol in Biological Fluids ^22 2.3.5. Labetalol Assay Validation^ 22 2.3.5.1. Precision of Quantitation^ 22 2.3.5.2. Extraction Recovery Studies^ 23 2.3.5.3. Determination of Minimum Quantitation Limit^23 2.3.6. Analysis of Glucuronide and Sulphate Conjugates of Labetalol^ 23 2.4.^Development of a GC-MSD Assay for the Identification and Quantitation of 3-APB in Biological Fluids of Sheep^24 2.4.1. Optimization of GC Conditions^ 24 2.4.2. Optimization of Extraction and Derivatization^25 2.4.3. Optimization of Sensitivity^ 25 2.4.4. Procedures for the Analysis of 3-APB in Biological Fluids^ 26 2.4.4.1. GC Operating Conditions^ 26 2.4.4.2. Extraction and Derivatization^ 26 2.4.4.3. Preparation of Calibration Curve ^ 27 2.4.4.4. Quantitation of 3-APB in Biological Fluid Samples ^28 2.4.5. Validation of 3-APB Assay^ 28 2.4.5.1. Precision of Quantitation ^ 28 2.4.5.2. Extraction Recovery Studies ^ 28 2.4.5.3. Determination of Minimum Quantitation Limit ^29 2.4.6. Analysis of Glucuronide and Sulphate Conjugates of 3-APB^ 29 2.5.^Standard Procedures for Sheep Experiments ^ 2.5.1. Recording of Hemodynamic Parameters ^ 2.5.2. Blood Gas Analysis^ 2.5.3. Glucose Measurement^ 2.5.4. Lactate Measurement^  29 29 30 30 31  2.6.^Animal Preparation^ 2.6.1. General Maintenance^ 2.6.2. Pregnant Sheep Studies ^  31 31 32  vii  2.6.3. Nonpregnant Sheep Studies^  36  2.7.^Experimental Protocols^ 2.7.1.^Maternal Bolus Studies^ 2.7.2. Bolus Studies in Nonpregnant Sheep^ 2.7.2.1. Labetalol Bolus^ 2.7.2.2. Dilevalol (RR-isomer of labetalol) Bolus ^ 2.7.3. Infusion Studies in Nonpregnant Sheep^ 2.7.3.1. Norepinephrine (NE) Challenge ^ 2.7.4. Intra-arterial Administration of Labetalol ^ 2.7.4.1. Studies in Adult Nonpregnant Sheep ^ 2.7.4.2. Studies in Fetal Sheep^ 2.7.5.^Fetal Bolus Studies^  36 36 38 38 39 39 40 40 40 41 42  2.8.^Data Analysis^ 43 2.8.1. Pharmacokinetic Analysis^ 43 2.8.1.1. Selection of Weighting Factor^ 43 2.8.1.2. Pharmacokinetic Fitting^ 43 2.8.1.3. General Calculation of Pharmacokinetic Parameters ^44 2.8.1.4. Calculation of Transplacental and Nonplacental Clearances^ 46 2.8.2. Analysis of Hemodynamic and Metabolic Data ^47 2.8.3.^Statistical Analysis^ 47 ^ 3.^RESULTS 48 3.1.^Development of a Microbore HPLC Assay with Fluorescence Detection for the Quantitation of Labetalol in Biological Fluids^ 48 3.1.1. Optimization of Mobile Phase^ 48 3.1.2. Optimization of Detection of Labetalol ^ 48 3.1.3. Optimization of Extraction of Labetalol ^ 50 3.1.4. Extraction of Labetalol from Biological Fluids ^50 3.1.5. Validation of Labetalol Assay^ 55 3.1.5.1. Precision of Quantitation^ 55 3.1.5.2. Extraction Recovery Studies^ 55 3.1.5.3. Minimum Quantitation Limit of Labetalol Assay^55 3.2.^Development of a GC-MSD Assay for the Identification and Quantitation of 3-APB, an Oxidative Metabolite of Labetalol in the Biological Fluids of Sheep ^59 3.2.1. Optimization of GC Conditions^ 59 3.2.2. Optimization of Extraction and Derivatization of 3-APB^59  viii  3.2.3. Derivatization and Extraction of 3-APB from Biological Fluids^ 3.2.4. Validation of 3-APB Assay^ 3.2.4.1. Precision of Quantitation^ 3.2.4.2. Extraction Recovery Studies^ 3.2.4.3. Minimum Quantitation Limit of 3-APB Assay ^  61 66 66 66 66  3.3.^Maternal Bolus Studies^ 3.3.1. Experimental Details^ 3.3.2. Pharmacokinetics^ 3.3.3. Hemodynamic Effects^ 3.3.3. Metabolic Effects^ 3.3.3.1. Changes in Blood Gas Parameters^ 3.3.3.2. Blood Glucose and Lactate Levels^  66 68 68 74 74 74 78  3.4.^Labetalol Bolus Studies in Nonpregnant Sheep^ 3.4.1. Pharmacokinetics^ 3.4.2. Metabolism^ 3.4.2.1. Conjugative Metabolism^ 3.4.2.2. Oxidative Metabolism^ 3.4.3. Hemodynamic Effects^ 3.4.4. Metabolic Effects^  80 80 86 86 86 92 92  3.5.^Dilevalol Bolus Studies in Nonpregnant Sheep^ 101 3.5.1.^Pharmacokinetics^ 101 3.5.2. Metabolism^ 101 3.5.3. Hemodynamic Effects^ 106 3.5.4. Metabolic Effects^ 106 ^ 3.6.^Labetalol Infusion in Nonpregnant Sheep ^ 3.6.1.^Disposition of Labetalol ^ 3.6.2. Hemodynamic Effects ^ 3.6.3. Metabolic Effects ^ 3.6.4. Intra-arterial Norepinephrine Studies  113 113 113 116 116  3.7.^Intra-arterial Labetalol Studies ^ 3.7.1. Studies in Adult Nonpregnant Sheep^ 3.7.2.^Studies in Fetal Sheep^  122 122 122  3.8.^Fetal Labetalol Bolus Studies^ 3.8.1.^Experimental Details^ 3.8.2.^Pharmacokinetics^ 3.8.3.^Metabolism^  122 122 126 130  ix  3.8.4. 3.8.5.  Hemodynamic Effects Metabolic Effects  130 130  4.  DISCUSSION  142  4.1.  Development of a HPLC Assay with Fluorescence Detection for the Quantitation of Labetalol in Biological Fluids  142  Development of a GC-MSD Assay for the Identification and Quantitation of 3-APB, an Oxidative Metabolite of Labetalol in Biological Fluids of Sheep  144  4.3. 4.3.1. 4.3.2. 4.3.3.  Maternal Bolus Studies Pharmacokinetics Hemodynamic Effects Metabolic Effects  146 146 149 150  4.4. 4.4.1. 4.4.2. 4.4.3. 4.4.4.  Labetalol Bolus Studies in Nonpregnant Sheep Pharmacokinetics Metabolism Hemodynamic Effects Metabolic Effects  157 158 158 160 161  4.5. 4.5.1. 4.5.2. 4.5.3. 4.5.4.  Dilevalol Bolus Studies in Nonpregnant Sheep Pharmacokinetics Metabolism Hemodynamic Effects Metabolic Effects  163 164 164 165 166  4.6. 4.6.1. 4.6.2. 4.6.3. 4.6.4.  Labetalol Infusion in Nonpregnant Sheep Disposition Hemodynamic Effects Metabolic Effects Intra-arterial Norepinephrine Studies  167 167 168 169 169  4.7. 4.7.1. 4.7.2.  Intra-arterial Labetalol Studies Studies in Adult Nonpregnant Sheep Studies in Fetal Sheep  170 170 171  4.8. 4.8.1. 4.8.2.  Fetal Labetalol Bolus Studies Pharmacokinetics Metabolism  172 173 176  4.2.  4.8.3. 4.8.4.  Hemodynamic Effects Metabolic Effects  177 177  5.  SUMMARY AND CONCLUSIONS  181  5.1. 5.2.  181  5.7. 5.8.  Quantitation of Labetalol in Biological Fluids Analysis of 3-amino-l-phenylbutane in Biological Fluids Maternal Bolus Administration of Labetalol Labetalol Bolus in Adult Nonpregnant Sheep Dilevalol Bolus in Adult Nonpregnant Sheep Labetalol Infusion to Steady-state in Adult Nonpregnant Sheep Intra-arterial Administration of Labetalol Labetalol Bolus Administration in the Fetal Lamb  184 184 185  6.  REFERENCES  187  5.3. 5.4. 5.5. 5.6.  181 182 183 184  xi  LIST OF ILLUSTRATIONS FIG 1: Chemical structure of labetalol (* denotes chiral centre).^1 FIG 2: Schematic diagram of ovine fetal hind limb to illustrate the position of the hind limb catheters and flowmeter. ^35 FIG 3: Effect of molarity of phosphate buffer in the mobile phase on the peak width and retention time of labetalol on a C-18 column.^  49  FIG 4: Absolute extraction recovery of labetalol with different solvents. A: Dichloromethane, B: Ethyl acetate, C: Diethyl ether and D: Toluene. ^  51  FIG 5: HPLC Chromatograms obtained from blank sheep plasma following a two-step (A) and one-step (B) extraction.^  52  FIG 6: Optimized procedure used for the extraction of labetalol from biological fluids. ^  53  FIG 7: Superimposed HPLC chromatograms of blank and spiked biological fluids (1 - Labetalol and 2 - Internal standard): A: Pregnant sheep plasma, B: Amniotic fluid, C: Fetal plasma and D: Fetal tracheal fluid. ^54 FIG 8: A typical calibration curve employed in the quantitation of labetalol (5 - 120 ng) in plasma (mean ± SD) (n=3). ^56 FIG 9: Extraction recovery of labetalol from sheep plasma as a function of amount added (mean ± SD) (n=3). ^58 FIG 10: HPLC chromatogram obtained from blank sheep plasma spiked with 0.5 ng labetalol (the lowest calibration point). (1 - Labetalol and 2 - Internal standard). Approximately 40 pg of labetalol was actually injected. 60 FIG 11: Effect of various reagents used for sample pH adjustment (prior to solvent extraction) on the absolute recovery of 3-APB in urine. 62  xii  FIG 12: Optimized procedure used for the extraction of 3-APB from biological fluids.^  63  FIG 13: EI GC-MS following HFBA derivatization of standard 3-APB and MPE (internal standard). A: Total Ion Chromatogram; B: Mass Spectrum of 3-APB Derivative and C: Mass Spectrum of the MPE Derivative.^  64  FIG 14: 3-APB Derivative: Suggested m/z assignments for the mass spectrum.^  65  FIG 15: Typical calibration curve following extraction of 3-APB (0.5 - 1000 ng) from urine (mean ± SD; n=3).^67  FIG 16: Representative plots of labetalol concentrations in two experiments following a 100 mg maternal intravenous bolus administration. (A - E# 109 and B - E# 201). (MA: Maternal arterial plasma, FA: Fetal arterial plasma, UT: Uterine venous plasma, UV: Umbilical venous plasma, AM: Amniotic fluid and TR: Tracheal fluid). ^71 FIG 17: Effect of a 100 mg i.v. labetalol bolus on maternal heart rate and arterial pressure (mean ± SEM) (n---6).^ 75 -  FIG 18:  Effect of a 100 mg i.v. labetalol bolus on fetal heart rate and arterial pressure (mean ± SEM) (n=7). ^ 76  FIG 19: Fetal and maternal arterial blood gas parameters (mean ± SEM) before and after a 100 mg maternal intravenous bolus administration of labetalol. (MA: Maternal arterial blood and FA: Fetal arterial blood). Asterisks (*) denote significant difference from control values.^ 77 FIG 20: Glucose and lactic acid concentrations (mean ± SEM) before and after a 100 mg maternal intravenous bolus administration of labetalol. (MA: Maternal arterial blood, FA: Fetal arterial blood and AM: Amniotic fluid). ^79 FIG 21: Glucose and lactic acid concentrations (mean ± SEM) before and after a 20 mL control saline administration. (MA: Maternal arterial blood, FA: Fetal arterial blood and AM: Amniotic fluid).  FIG 22: Fetal and maternal arterial blood gas parameters (mean  81  ± SEM) following a 20 mL saline administration (control experiment). (MA: Maternal arterial blood and FA: Fetal arterial blood).^  82  FIG 23: Disposition of labetalol in adult sheep plasma following a 100 mg i.v. bolus (mean ± SEM). ^  83  FIG 24: Cumulative urinary excretion of labetalol and its conjugates (mean ± SEM). A: Cumulative amount excreted and B: Percentage of labetalol dose recovered in urine as free drug and conjugates. ^  87  FIG 25: Concentrations of labetalol and its conjugates in adult sheep bile following a 100 mg i.v. bolus (E# 617). ^88 FIG 26: Total ion chromatogram (top panel) of urine sample obtained from E#105 (nonpregnant) following a 100 mg labetalol bolus. Asterisk denotes the 3-APB peak and the EI mass spectrum corresponding to that peak (bottom panel). ^  89  FIG 27: Excretion of 3-APB in adult non-pregnant sheep urine following a 100 mg labetalol administration. (mean ± SEM; n=5). A: Cumulative excretion over 24 h; B: Excretion rate plot. ^  90  FIG 28: Excretion of 3-APB conjugates following a 100 mg labetalol administration. A: Urine (amount) (E#105) and B: Bile (concentration) (E#248).^  93  FIG 29: Oxidative metabolism of labetalol. (Not exhaustive; dotted lines indicate potential pathways; structure of amphetamine is included for the sake of comparison; partly from Gal et al., 1988).^94 FIG 30: Hemodynamic changes following labetalol bolus in adult nonpregnant sheep (mean ± SEM) (n=5). (Asterisks denote significant difference from control values). ^95 FIG 31: Femoral arterial and venous blood gas changes following labetalol bolus in adult nonpregnant sheep. (mean ± SEM) (n=5). (Asterisks denote significant difference from control values). ^  97  xiv  FIG 32: Effect of labetalol on oxygen homeostasis in adult nonpregnant sheep (mean ± SEM) (n=5). (Asterisks denote significant difference from control values). ^98 FIG 33: Labetalol bolus in adult nonpregnant sheep: Femoral arterial and venous blood glucose and lactate concentrations (mean ± SEM; n=5). Asterisks denote significant difference from control values. ^  99  FIG 34: Changes in arterio-venous fluxes following labetalol administration in adult nonpregnant sheep (mean ± SEM; n=5). Positive values indicate net uptake and negative values indicate net release from the hind limb. Asterisks denote significant difference from control values.^  100  FIG 35: Disposition of dilevalol in adult nonpregnant sheep arterial plasma following a 25 mg i.v. bolus. ^  102  FIG 36: Cumulative urinary excretion of dilevalol as unconjugated, glucuronide and sulphate in an adult nonpregnant sheep (E#105). ^  104  FIG 37: Concentrations of unconjugated (free) and conjugated dilevalol in bile of adult nonpregnant sheep following a 25 mg i.v. bolus (E#105). 105 FIG 38: Excretion of 3-APB and its conjugates following a 25 mg dilevalol administration in an adult nonpregnant sheep (E#105). A: Urine (amount) and B: Bile (concentration). ^  107  FIG 39: Hemodynamic changes in an adult nonpregnant sheep (E#105) following a 25 mg dilevalol bolus. ^  108  FIG 40: Femoral arterial and venous blood gas changes in an adult nonpregnant sheep (E#105) following a 25 mg dilevalol bolus. ^  109  FIG 41: Femoral arterial and venous blood glucose and lactate concentrations in an adult nonpregnant sheep (E#105) following a 25 mg dilevalol bolus. 110  xv  FIG 42: Effect of dilevalol on oxygen homeostasis in an adult nonpregnant sheep (E#105).^  111  FIG 43: Changes in arterio-venous fluxes following dilevalol administration in an adult nonpregnant sheep (E# 105). Positive values indicate net uptake and negative values indicate net release from the hind limb. ^  112  FIG 44: Arterial plasma labetalol concentrations following a combined bolus (100 mg) and infusion (0.5 mg/min for 6 h) in adult nonpregnant sheep (mean ± SEM). (Solid line indicates infusion period).^  114  FIG 45: Hemodynamic changes during and after continuous infusion of labetalol in adult nonpregnant sheep (mean ± SEM; n=5). Solid line denotes infusion period and asterisks denote significant difference from respective control values.^  115  FIG 46: Changes in femoral arterial and venous blood gas parameters during and after continuous infusion of labetalol in adult nonpregnant sheep (mean ± SEM). Asterisks denote significant difference from control values; solid line indicates infusion period. ^ 117 FIG 47: Oxygen homeostasis in adult nonpregnant sheep before, during and after continuous infusion of labetalol (mean ± SEM). Asterisks denote significant difference from control values; solid line indicates infusion period.^  118  FIG 48: Effect of labetalol on arterial and venous blood glucose and lactate concentrations (mean ± SEM). (solid line indicates infusion period; arterial and venous blood lactate concentrations were significantly different from control between 30 min and 2 h post-infusion; arterial and venous glucose concentrations were significant between 3 h and 2 h post-infusion.). ^  119  FIG 49: Hind limb arterio-venous glucose and lactate flux before, during and after labetalol infusion in adult  xvi  nonpregnant sheep (mean ± SEM). (Asterisks denote significant difference from control values; dashed line indicates infusion period). ^  120  FIG 50: Intra-arterial norepinephrine dose-response curve before and during labetalol infusion (mean ± SEM; n=4).^121 FIG 51: A.: Intra-arterial labetalol dose-response relationship in adult nonpregnant sheep hind limb before and after phentolamine administration (E# 543). B.: Intra-arterial labetalol dose-response relationship in adult nonpregnant sheep hind limb before and after propranolol administration (E# 105). ^  123  FIG 52: Polygraph tracings showing the time course of hemodynamic changes corresponding to intra-arterial administration of A.: labetalol, B.: norepinephrine and C.: control saline. Vertical arrow on the time scale corresponds to the time of injection. [1: Femoral Blood Flow (mL/min), 2: Mean Arterial Pressure (mm Hg) and 3: Heart Rate (bpm)]. ^124 FIG 53: Intra-arterial (HLA) injection of labetalol in the fetal lamb (E#1154).^  125  FIG 54: Disposition of labetalol in pregnant sheep following a 4 mg fetal intravenous bolus administration (mean ± SEM; n=5). (FA: Fetal Arterial Plasma; AM: Amniotic Fluid; TR: Tracheal Fluid and MA: Maternal Arterial Plasma).^  128  FIG 55: Concentrations of labetalol and its glucuronide conjugate in the amniotic fluid following a 4 mg fetal intravenous bolus administration (mean ± SEM; n=5). ^131 FIG 56: Effect of a 4 mg fetal intravenous bolus on mean fetal heart rate and arterial pressure (mean ± SEM). Asterisk denotes significant difference. ^  132  FIG 57: Effect of a 4 mg fetal intravenous bolus on mean fetal hind limb blood flow and vascular resistance (mean ± SEM; n=5).^  133  xvii  FIG 58: Effect of labetalol and control (saline) fetal bolus administration on fetal arterial blood gas parameters (mean ± SEM; n=5). Asterisks denote significant difference.^  135  FIG 59: Effect of a 4 mg fetal intravenous labetalol bolus on glucose concentrations (mean ± SEM; n=5). (FA: Fetal arterial blood; HV: Hind limb venous blood and AM: Amniotic fluid).^  136  FIG 60: Effect of a 4 mg fetal intravenous labetalol bolus on lactic acid concentrations (mean ± SEM; n=5). (FA: Fetal arterial blood; HV: Hind limb venous blood and AM:Amniotic fluid). FA and HV concentrations were statistically significant between 90 min and 12 h and AMN concentrations between 2-24 h. 137 FIG 61: Glucose and lactate concentrations in maternal arterial blood following a 4 mg fetal intravenous bolus of labetalol (mean ± SEM; n=5). ^  138  FIG 62: Changes in glucose and lactate concentrations following control (saline) fetal intravenous bolus administration (mean ± SEM; n=5). (FA: Fetal arterial blood; HV: Hind limb venous blood; MA: Maternal arterial blood and AM: Amniotic fluid). ^  139  FIG 63: Effect of a 4 mg fetal intravenous bolus of labetalol on hind limb arterio-venous labetalol, glucose and lactate fluxes (mean ± SEM). Asterisks denote significant difference from control values.^  140  xviii  LIST OF TABLES Table 1^Intra-sample variability in labetalol assay. ^57 Table 2 Experimental details for maternal bolus experiments. ^69 Table 3 Pre-experimental maternal and fetal blood gas parameters (mean ± SEM).^  70  Table 4 Labetalol pharmacokinetics following maternal bolus.^72 Table 5 Comparison of the pharmacokinetics of labetalol in pregnant sheep with reported values in pregnant women.^  84  Table 6 Comparative pharmacokinetics of labetalol in pregnant and nonpregnant sheep.^  85  Table 7 Urinary excretion of 3-amino-l-phenylbutane.^91 Table 8 Pharmacokinetics of dilevalol (RR-isomer of labetalol) in adult nonpregnant sheep following a 25 mg i.v. bolus administration.^  103  Table 9^Fetal bolus studies: experimental details. ^ 127 Table 10 Pharmacokinetics of labetalol in the fetal lamb and ewe following a 4 mg fetal i.v. bolus (mean ± SEM).^129  xix  LIST OF ABBREVIATIONS 3-APB:^3 -amino- 1 -phenylbutane A:  Exponential equation constant  a:^Alpha, an exponential rate constant AUC:^Area under the concentration vs time curve AUMC:^Area under the first moment curve (i.e. time vs concentration*time) AM:^Amniotic fluid AUTOAN:^Computer program for pharmacokinetic modelling A-V:^Arterio-venous B:  Exponential equation constant  BE:^Blood base excess B:  Beta, an exponential rate constant  bpm:^Beats per minute °C:^Degree celsius C:  Arterial plasma concentration of labetalol  Css:^Arterial plasma concentration of labetalol at steadystate Ct-last^Arterial plasma concentration of labetalol in the last sample obtained or the last sample in which labetalol could be quantitated cAMP:^Cyclic adenosine monophosphate CI:^Chemical ionization CL:^Total body clearance CLf:^Fetal total body clearance CL f.:^Fetal nonplacental clearance  XX  CL fp :^Fetal transplacental clearance CLm :^Maternal total body clearance CLmn :^Maternal nonplacental clearance CLmp :^Maternal transplacental clearance CO 2 :^Carbon di-oxide C t :^Concentration of labetalol at time t CV:^Coefficient of variation EC 50 :^Concentration that corresponds to 50% of maximal response EI:^Electron impact ionization EM:^Emission wavelength Ems :^Maximum pharmacological effect eV:^Electron volts EX:^Excitation wavelength FA:^Fetal arterial g:^gram g:  acceleration due to gravity  GC:^Gas chromatography h:  hour(s)  Hb:^Hemoglobin HFBA:^Heptafluorobutyric anhydride Hg:^Mercury HLA:^Hind limb artery (pudendo-epigastric artery) HLV:^Hind limb vein (pudendo-epigastric vein) HPLC:^High performance liquid chromatography Hz:^Hertz i.d.:^Internal diameter  xxi  I.U.:^International units i.v.:^Intravenous JANA:^Computer program for pharmacokinetic modelling ko:^Infusion rate kg:^kilogram L:  Liter  [Lc]:^Concentration of lactic acid [Lc] t :^Concentration of lactic acid at time t M:  Molar (as a concentration term)  MA:^Maternal arterial MANOVA:^Multivariate analysis of variance meq:^Milliequivalents 14:^Microgram pL:^Microliter gm:^Micrometer mg:^milligram min:^Minute(s) mL:^milliliter mm:^ millimeters  mmol:^millimole(s) mol:^Mole(s) MPE:^1-methyl-2-phenoxyethylamine MS:^Mass Spectrometry MSD:^Mass selective detection mh:^Mass to charge ratio n:^Number of samples N:^Normal (as a concentration term)  NAD:^Nicotinamide adenine dinucleotide NADH:^Reduced form of nicotinamide adenine dinucleotide ND:^Not detectable NE:^Norepinephrine ng:^nanogram nm:^nanometer NONLIN:^Computer program for pharmacokinetic modelling NS:^No sample 0 2 :^Oxygen p:^Probability factor P:^Exponential equation constant pCO 2 :^Partial pressure of carbon dioxide pg:^Picogram pH:^Negative logarithm (base 10) of the hydrogen ion concentration Tr:^Pi, an exponential rate constant p0 2 :^Partial pressure of oxygen PFTBA:^Perfluorotributylamine PTFE:^polytetrafluoroethylene r:^ Correlation coefficient r2 :^Coefficient of determination rpm:^revolutions per minute sec:^seconds SCH:^Schering Corporation's compound, 5-124444-methyl phenyl)-2-butylamino)-1-hydroxyethyl) salicylamide hydrochloride hemihydrate SEM:^Standard error of mean  SIM:^Selected ion monitoring a 2..^Sigma squared, notation for variance ss:^(as a suffix) steady-state t-test:^Student's t-test for statistical analysis LA :^Half-life  -  TEA:^Triethylamine t-last:^Time at which the last sample was collected tmax :^Time corresponding to the maximum concentration TR:^Tracheal fluid TRIS:^Tris(hydroxymethyl)aminomethane UT:^Uterine vein UV:^Umbilical vein VD:^Apparent volume of distribution *^ Notation for product, i.e., multiplication  xxiv  Acknowledgements I would like to express my sincere appreciation and thanks to my research supervisors Drs. James E. Axelson and Dan W. Rurak, for their guidance, encouragement and support throughout my graduate training. Thanks to Drs. Frank S. Abbott, Graham H. McMorland and Joanne Douglas for their keen interest in the project and for the several valuable suggestions. Special thanks to Dr. Wayne Riggs for his support and friendship, which helped me tide over the testing times. My sincere thanks to Mr. Eddie Kwan and Ms. Caroline Hall for their exceptional technical assistance in sheep studies, which was often above and beyond the call of duty. I would like to thank Dr. Timothy Stratton and Dr. Johnathan Berkowitz for their assistance with the statistical analysis. Many thanks are due to Mr. Ahmad Doroudian for his help in sample analysis and his friendship. I would like to thank Mr. George Tonn and Ms. Meike Tonn for their valuable friendship and for providing a home away from home. I was fortunate to enjoy the company of my colleagues in the laboratory, Dr. Sun Dong Yoo, Dr. Grace Chan, Dr. Matthew Wright, Dr. Andras Szeitz, Ms. Jing Wang, Ms. Judit Orbay, and Mr. John Kim. Special thanks to Dr. Matthew Wright for his help in conducting experiments and helpful criticism during the preparation of manuscripts. This work would not have been possible without the love, affection and moral support of my family. Financial support in the form of studentship from the B.C. & Yukon Heart and University Graduate Fellowship are gratefully appreciated. This project was supported by the Medical Research Council of Canada.  XXV  Dedication  This thesis is dedicated to my beloved mother, Ms. Parvatham Yeleswaram and to the memory of my dad, Mr. Rajaram Yeleswaram.  1. INTRODUCTION  1.1. Labetalol  Labetalol { 2-hydroxy-5- [1-hydroxy-2-(1-methy1-3-phenylpropylamino) ethyl] benzamide } hydrochloride (TrandateR) is a compound with two chiral centres (Fig 1) and is marketed as a racemic mixture of all four isomers in roughly equal proportions (Gold et al., 1982). Labetalol is a combined post-synaptic a l and non-selective 0-adrenoceptor blocking agent (Farmer et al., 1972) and is used as an antihypertensive.  H 2 N OC  HO  FIG 1: Chemical Structure of Labetalol (* denotes chiral centre). 1.1.1. Pharmacology  The pharmacological effects of labetalol have been studied in vitro and in vivo. In rabbit and rat aortic strips, rat vas deferens and guinea-pig isolated left  atrium, labetalol competitively inhibits both a and 0-adrenoceptors (Brittain and Levy, 1976). The in vitro a-blockade was 6-10 times less potent than phentolamine; the 0-blockade was 1.5-3 times less potent than propranolol and in itself, labetalol was 4-8 times more potent at p- than at a-adrenoceptors (Farmer et  2  al., 1972; Brittain and Levy, 1976). The competitive a and (3-adrenergic antagonism of labetalol was also demonstrated in barbitone-anesthetized dogs, pithed rats as well as in conscious, normotensive and hypertensive dogs and hypertensive rats (Farmer et al., 1972; Kennedy and Levy, 1975; Brittain and Levy, 1976). In anesthetized dogs following intravenous administration, labetalol was about 7 times less potent than phentolamine in its a-blockade, about 4 times less potent than propranolol in blocking cardiac P i -receptors, 11-17 times less potent than propranolol in blocking vascular and bronchial 13 2 -receptors and in itself, 16 times more potent at cardiac P i than at vascular a-adrenoceptors (Kennedy and Levy, 1975; Brittain and Levy, 1976). Labetalol has also been shown to possess partial P 2 -agonist activity in isolated uterus of the rat (Carey and Whalley, 1979), isolated trachea of the guinea-pig (Carpenter, 1981) and innervated and denervated femoral vascular beds of dogs (Baum et al., 1981). However, labetalol is devoid of any agonist activity at cardiac P i -receptors (Brittain and Levy, 1976). Brittain et al., 1981, studied the pharmacology of the individual isomers of labetalol and found that the adrenergic activities of labetalol were not equally distributed amongst its four stereoisomers. These authors and Gold et al., 1982, found that the RR-stereoisomer contributes to most of the P-adrenoceptor blocking activity of labetalol and that this isomer was virtually devoid of any a-blocking activity. The SR-stereoisomer, on the other hand, has most of the a-blocking activity and shows insignificant 13-antagonism. The other two isomers, SS and RS, were found to have no significant a or P-adrenoceptor blocking activities (Brittain et al., 1981; Gold et al., 1982). 1.1.2. Clinical Use  3  The results from the various clinical trials that were conducted to assess the antihypertensive efficacy of labetalol are summarized in this section. The outcome of clinical studies of labetalol in pregnancy is presented separately. 1.1.2.1. General  Labetalol has been used in the management of systemic hypertension of various etiologies, either alone or in combination with diuretics (Goa et al., 1989). Labetalol has been found effective in achieving target blood pressures in about 65% of all hypertensive patients (Arinsoy and Oram, 1986). In a placebo controlled trial, labetalol was found to cause significant decreases in both standing and supine systolic and diastolic blood pressures (Davidov et al., 1983). The efficacy of labetalol has also been compared with that of other antihypertensive agents in several clinical studies. The overall efficacy of labetalol in mild to severe hypertension was found to be better than that of propranolol (Flamenbaum et al., 1985) and acebutolol (Thibonnier et al., 1980) and comparable to that of  methyldopa (Frishman et al., 1983), atenolol (Nilsson et al., 1982), pindolol (Romo et al., 1984), prazosin (Gray et al., 1988), verapamil (Anavekar et al., 1982) and clonidine (Lilja et al., 1982). 1.1.2.2. Hypertension in Pregnancy  A number of clinical studies have been conducted to determine the efficacy  of labetalol in lowering hypertension in pregnancy without adversely affecting the fetus. Michael, 1979 and 1982, reported that orally administered labetalol (3001200 mg p.o. daily for 1-19 wks) caused effective and sustained reduction in systolic and diastolic blood pressures and no maternal or fetal side effects. Pickles  4  et al., 1989, found that the fetal outcome in hypertensive patients treated with  labetalol was not different from that in the placebo treatment group. However, Mabie et al., 1987, who compared the efficacy and neonatal outcome of labetalol plus hospitalization with hospitalization alone, observed that labetalol treatment was associated with a higher frequency of fetal growth retardation. The antihypertensive efficacy and perinatal safety of treatment with labetalol was found to be similar to that of methyldopa (Lamming and Symonds, 1979; Lamming et al., 1980; Plouin et al., 1988). In a comparative trial involving labetalol and hydralazine, the incidence of fetal distress was found to be less in the labetalol group (Sibai et al., 1987). Lardoux et al., 1983, observed decreased incidences of fetal growth retardation with labetalol treatment than with atenolol treatment. In summary, most of the clinical studies have found labetalol to be a safe and effective drug in the management of hypertension in pregnancy. Labetalol has also been used in the management of hypertensive crises in pregnancy. Satisfactory results with intravenous labetalol administration were reported by Garden et al., 1982; Michael, 1986; Ashe et al., 1987; and Mabie et al., 1987. Ashe et al., 1987, however, noted that the efficacy of labetalol in  emergencies was less than that achieved with dihydralazine. 1.1.3. Fetal Effects  Many of the conclusions about the effects of labetalol on the human fetus were based on uteroplacental hemodynamics and/or neonatal follow-ups. Lunell et al., 1982 and Nylund et al., 1984, observed no significant changes in  uteroplacental blood flow calculated using placental scintigraphy. Using combined real time and Doppler ultrasound techniques, Joupilla et al., 1986, found that maternal labetalol caused no changes in the blood flows in the intervillous  5  space, umbilical vein and fetal descending aorta. Umbilical artery flow velocity waveforms and pulsatility index were studied by Harper and Murnaghan, 1991, using pulsed Doppler ultrasound. They found that labetalol increased the umbilical artery pulsatility index and attributed the increase to vasoconstriction in the fetoplacental unit due to beta-blockade in the fetus. But, Pirnohen  et al.,  1991,  found no significant changes in uterine artery, umbilical artery or fetal middle cerebral artery flow velocity waveforms. In a neonatal follow-up study, MacPherson et a/., 1986, monitored the systolic blood pressure, heart rate, palmar sweating, response to cold stress and blood glucose for 72 h postnatal in newborns whose mothers had received labetalol and found that the only significant difference in comparison to the control group was a transient hypotension at 2h. It was concluded that maternal labetalol does not cause significant sympathetic blockade in the newborn. Pickles et al., 1989, studied the fetal outcome following maternal labetalol in terms of birth weight, incidence of neonatal respiratory distress syndrome, hypoglycemia and bradycardia and inferred that labetalol is apparently safe to the fetus. However, Sibai et al., 1987, found that labetalol was associated with a higher incidence of fetal growth retardation than untreated controls and that the perinatal outcome was not improved despite reductions in the maternal blood pressure. Information on the fetal effects of labetalol from animal studies is also quite limited. Nicholas et al., 1978, observed that labetalol increased the amount of alveolar surfactant in fetal rabbit lungs, possibly through its partial f3 2 -agonism. Ahokas et a/., 1989, found that while labetalol effectively lowered blood pressure, it was not associated with any decrease in placental perfusion, in the hypertensive term-pregnant rat. Mohan  et al.,  1990, studied the hemodynamics of varying  doses of labetalol in normotensive and hypertensive pregnant ewes and found that the fetal mean arterial pressure and heart rate did not change significantly.  6  Eisenach et al., 1991, investigated the effect of labetalol in pregnant sheep during acute hypertension produced by norepinephrine as well as the degree of alpha and beta-adrenergic blockade in the mother and fetus, determined by isoproterenol and phenylephrine challenges. Those authors found that labetalol effectively decreases the elevated arterial pressure, overcomes the decrease in uterine blood flow as well as improves the fetal acidemia and hypoxemia induced by noradrenaline. Also, the adrenergic blockade was found to be significantly less in the fetus than in the mother (Eisenach et al., 1991). 1.1.4. Methods of Analysis  A number of HPLC assay procedures have been reported for the  determination of labetalol in human plasma. Methods involving ultra-violet detection (Dusci and Hackett, 1979; Woodman and Johnson, 1981; Hidalgo and Muir, 1984) in general, were limited in application due to lack of sufficient sensitivity to detect concentrations below 20 ng/mL. Hence, electrochemical and fluorescence detection systems were used in an attempt to improve the lower limits of detection. Wang et al., 1985, and Abernethy et al., 1986, employed electrochemical detection using the inherent redox behaviour of labetalol and improved the sensitivity to about 5 ng/mL. HPLC assays with fluorescence detection were reported by Meredith et al., 1981, Oosterhuis et al, 1981, and Ostrovska et al., 1988, with minimum detection limit in the range of 2-4 ng (amount injected). Alton et al., 1984, used a macroporous co-polymer (PRP-1), which withstands alkaline pH conditions as the stationary phase instead of the conventional reverse-phases (C-18 or C-8) which have a pH tolerance range of 2.5-7.5. They used pH 9.5 carbonate buffer and acetonitrile as the mobile phase since it has been shown that the optimum pH for the fluorescence detection of  7  labetalol is between 9-10 (Oosterhuis et al., 1981). However, the limit of detection was only marginally improved (1.5 ng injected). Luke et al., 1987 reported an improved method, which employs PRP-1 column as well as postcolumn addition of ammonium hydroxide to adjust the pH of the eluent to 11.0 and obtained a minimum detection limit of about 0.45 ng. A thermospray mass spectrometric method for the determination of labetalol was also reported (Lant et al., 1987), but the authors noted deterioration in sensitivity with repeated injections, thus limiting the applications of that method. It should be mentioned that the assay procedures discussed above are achiral and measure total, i.e., racemic labetalol. There has been no validated report to date on the quantitation of the individual isomers of labetalol. Lalonde et al., 1990, attempted to study the stereoselectivity in the disposition of labetalol in humans, using a HPLC assay with oc i -acid glycoprotein chiral stationary phase. However, the details of the assay conditions provided were sketchy and there was no mention of any validation studies.  1.1.5. Pharmacokinetics  Labetalol undergoes rapid absorption following oral administration, with peak plasma concentrations reached within 1 to 2 h after drug administration (Abernethy et al., 1985; McNeil et al., 1979). Like other P-blockers, labetalol undergoes significant first-pass metabolism (McNeil and Louis, 1984). Further, considerable inter-patient variability in the extent of first-pass metabolism and hence, the oral bioavailability (11 to 86%) has been observed (McNeil et al., 1979). Daneshmand and Roberts (1982), have shown that food increases the bioavailability of labetalol by about 11%.  8  The protein binding of labetalol in human plasma was reported to be about 50% (Martin et al., 1976). Studies in rats, rabbits and dogs have shown that labetalol undergoes extensive distribution into the lung, liver and kidney, with little being present in brain tissue (Martin et al., 1976). The reported estimates of apparent volume of distribution of labetalol in humans are in the range of 2.5-15.5 L/kg (Goa et al., 1989). The plasma clearance of labetalol following intravenous administration in hypertensive patients was found to be in the range of 10 to 20 mL/min./kg (Abernethy et al., 1987; McNeil et al., 1982; Nyberg et al., 1982). The reported estimates of elimination half-life following oral or intravenous administration in normotensive or hypertensive subjects have ranged from 3 to 8 h (Goa et al., 1989). The phannacokinetics of labetalol is essentially unchanged by pregnancy (Rubin et al., 1983). A decrease in clearance and increases in elimination halflife, bioavailability and peak plasma concentrations were observed in elderly subjects as compared to young subjects (Abernethy et al., 1985). Decreased clearance was also observed in patients with chronic liver diseases (Homeida et al., 1978; Daneshmand et al., 1982), but the pharmacokinetics of labetalol was unchanged in patients with chronic renal diseases (Wood et al., 1982). Evidence of stereoselectivity in the disposition of labetalol in humans was reported by Lalonde et al., 1990, who used a stereoselective assay to measure the proportions of the individual stereoisomers in steady-state plasma samples, obtained following labetalol administration. They found that the percentage of RR-isomer at steady-state was significantly lower than that of the other three isomers. Further, co-administration of cimetidine, a hepatic enzyme inhibitor, was found to cause a significant increase in the steady-state concentrations of all the isomers except the RR-isomer. These results, although based on an assay whose  9  validity is not clearly established, may suggest stereoselectivity in the disposition and hepatic elimination of labetalol.  1.1.6. Metabolism  Mass balance studies with 3 H and 14 C labelled labetalol were conducted by Martin et al., 1976 in rats, rabbits, guinea-pigs and humans and they found that less than 5% of an oral dose of labetalol was excreted unchanged in the urine. About 20% of the dose was recovered as glucuronide and in humans, the major metabolite was an unidentified conjugate. No sulfate conjugates were found in any of the species studied (Martin et al., 1976). Recently, Gal et al., 1988, reported that labetalol undergoes oxidative metabolism via N-dealkylation to form 3-amino-l-phenylbutane and 3-amino-1-(4-hydroxyphenyl) butane. These compounds, which are structural analogs of amphetamine, were identified in the urine samples of three patients on labetalol therapy. The fraction of dose that undergoes N-dealkylation, however, was not known.  1.1.7. Placental Transfer  There has been no report in the literature that has attempted to quantitate placental transfer and fetal exposure to labetalol following maternal administration in any species. Martin et al., 1976, used whole body autoradiography following administration of 14 C-labelled labetalol in pregnant rats and rabbits and found that the total radioactivity in the fetus was less than 1% of the maternal levels. There are no published data in any animal species regarding disposition and pharmacokinetics of labetalol in the in utero fetus following maternal or fetal administration. In humans, the cord blood/ maternal venous labetalol  I0  concentration ratio at the time of delivery averages 0.5 (range: 0.2 to 1.0) (Michael, 1979; Nylund et al., 1984; Rogers et al., 1990), suggesting significant fetal exposure to the drug. However, a single point estimate obtained at the time of delivery is of limited value and could even be misleading since it changes constantly with respect to the time of drug administration (Anderson et al., 1980b). The concentrations of labetalol in amniotic fluid and breast milk were less than that in maternal plasma (Michael 1979; Lunell et al., 1985). 1.2. Dilevalol  Dilevalol is the RR-stereoisomer of labetalol and has 3 to 6 times the activity of labetalol in blocking p-adrenoceptors, but only about 10% of a l blocking activity of labetalol (Sybertz et al., 1981). The relative selectivity of dilevalol for p i - vs oc r adrenoceptors was around 500:1 in rats (Gold et al., 1982) and 400:1 in anesthetized dogs (Brittain et al., 1982). The partial agonist activity of dilevalol is mostly at the 0 2 -receptor subtype and is about 7 times more potent than labetalol (Sybertz et al., 1981). The antihypertensive effect of dilevalol is believed to be caused primarily through a reduction in peripheral vascular resistance (Clifton et al., 1988). The hypotension caused by dilevalol may be accompanied by tachycardia (Baum et al., 1981; Baum and Sybertz, 1983). Intravenous dilevalol administration results in an increase in norepinephrine and epinephrine levels (Grossman et al., 1989). Dilevalol is rapidly and completely absorbed after oral administration but the bioavailability is only about 11 to 30% (Kramer et al., 1988). The apparent volume of distribution of dilevalol of about 25 L/kg is higher than that of labetalol (Chrisp and Goa, 1990). About 73-77% of the drug was bound to plasma proteins (Sugeno et al., 1987a). The reported total body clearance of dilevalol in humans  11  was 23 mUmin/kg and the elimination half-life was around 8 to 12 h (Kramer et al., 1988). While about 1.25% of the dose was excreted unchanged in urine, about 80% of the dose was recovered as glucuronide conjugates (Sugeno et al., 1987a). Only about 0.03% of the maternal dose was transferred to the fetus in studies conducted in pregnant rats (Sugeno et al., 1987b). Dilevalol was withdrawn from the North American market by its manufacturers (Allen Hanburys) in 1991 because of suspected hepatotoxicity.  1.3. Objectives and Rationale  Although a number of clinical studies have been conducted to assess the efficacy of labetalol in pregnancy, there is no information in the literature regarding the in utero fetal exposure to maternal labetalol as well as the fetal effects of labetalol. The primary objectives of this study were to characterize the maternal-fetal pharmacokinetics, metabolism and pharmacodynamics of labetalol in the chronically instrumented pregnant sheep. The rationale for these objectives was that labetalol may cause adrenergic blockade in the fetus if it undergoes substantial placental transfer. Fetal exposure to drugs and pharmacological/toxicological effects of these agents in the fetus cannot be accurately determined in humans due to technical and ethical constraints. The chronically instrumented pregnant sheep has been extensively used as a model to understand developmental physiology and pharmacology since the fetal physiology and biochemistry in sheep are similar to that of the human fetus (Comline and Silver, 1974; Van Petten et al., 1978). Further, the rigorous maternal and fetal blood sampling that can be accomplished in this model cannot be reproduced in small animal models like guinea-pig or rats due to the physical size and limited blood volumes in those animals.  12  1.3.1. Specific Aims  The specific aims of the project at its outset were: 1. To develop a sensitive and selective assay method for the quantitation of labetalol in various biological fluids. The assay should be sensitive enough to quantitate a few nanograms of labetalol present in a few hundred microliters of biological fluid. 2.  To determine the maternal pharmacokinetics, placental transfer and extent of fetal exposure and maternal-fetal pharmacodynamics following maternal intravenous bolus administration.  3.  To determine the fetal total body clearance and placental and nonplacental contributions to fetal total body clearance of labetalol.  4.  To study the oxidative and conjugative metabolism of labetalol in both fetus and ewe.  13  2. EXPERIMENTAL  2.1. Materials  The reference standards, chemicals, reagents and other materials used are listed below, along with information on purity (where applicable) and the source. Unless otherwise specified, the materials were used without further purification or modification. Labetalol hydrochloride (>99% purity) (lot # 85F-0253) (Sigma Chemical Co., St. Louis, MO); injectable labetalol hydrochloride (TrandateR) (5 mg/mL); the internal standard for labetalol assay, 5424444  -  methyl phenyl) 2 Initylaminol 1 hydroxyethyl) salicylamide hydrochloride -  -  -  -  hemihydrate (SCH) (>99% pure) and dilevalol hydrochloride (RR-isomer of labetalol) (>99% pure) (gifts from Schering Corporation, Bloomfield, NJ); 3APB (>98% pure) (lot # 243006 584) (Fluka AG, Federal Republic of Germany); the internal standard for 3-APB assay, 1 methyl 2 phenoxyethylamine -  -  -  (MPE), (90% pure) (lot # 00128HL) (Aldrich Chemical Co., Milwaukee, WI); injectable norepinephrine bitartrate (LevophedR); injectable phentolamine mesylate (RogitineR); injectable propranolol hydrochloride (InderalR); injectable thiopental sodium (1g/vial) (PentothalR); injectable ampicillin (250 mg/vial) (PenbritinR); injectable gentamicin sulphate (40 mg/vial) (GaramycinR); injectable Atropine sulphate (0.6 mg/mL); halothane (FluothaneR); injectable heparin (HepaleanR); sodium chloride for injection USP (Abbott Laboratories, Montreal, Quebec). All the injectable drug formulations were obtained from the Pharmacy Department, Grace Hospital, Vancouver, B.C. Heptafluorobutyric anhydride and triethylamine (Sequanal Grade) (Pierce Chemical Co., Rockford, IL); ACS reagent grade potassium dihydrogen orthophosphate (monobasic), potassium carbonate, sodium acetate, sodium  14  bicarbonate, and tris(hydroxymethyl)aminomethane (TRIS free base) (BDH Chemicals, Toronto, Ontario); phosphoric acid, glacial acetic acid and ammonium hydroxide (all USP grades) (Fisher Scientific Co., Fair Lawn, NJ); 13 -glucuronidase, (GlucuraseR, from bovine liver, Sigma G-4882), aryl-sulfatase from Aerobacter aerogenes (Sigma S-1629), glucose standard solution (1 mg/mL), zinc sulphate (0.3 N), barium hydroxide (0.3 N), o-dianisidine dihydrochloride, PGO capsules (500 I.U. glucose oxidase, 100 Purpurogallin units peroxidase and buffer salts), lactate dehydrogenase, glycine buffer, and nicotinamide adenine dinucleotide (NAD) (Sigma Chemical Co., St. Louis Co., MO), and ACS reagent grade sodium hydroxide pellets (Fisher Scientific Co., Fair Lawn, NJ). Diethyl ether, ethyl acetate, and toluene (all glass distilled, pesticide grade), and acetonitrile (Caledon Laboratories, Georgetown, Ontario); methanol (OmnisolvR), BDH Chemicals (Toronto, Ontario). Distilled water of high purity (referred to in the text as "distilled water") was produced on-site by reverse osmosis, followed by filtration through ion-exchange cartridges and 0.45 p. membrane filter (Milli-QR) (Millipore, Mississauga, Ontario). Nitrogen USP (Union Carbide Canada Ltd., Toronto, Ontario); prepurified helium (for degassing HPLC mobile phase) and ultra-pure grade helium (for GC-MSD) (Matheson Gas Products Canada Ltd., Edmonton, Alberta). Needles and plastic disposable Luer-LokR syringes for drug administration and sample collection (Beckton-Dickinson Canada, Mississauga, Ontario); membrane filters (0.45p.) (Millipore, Mississauga, Ontario); disposable plastic pipet tips (National Scientific, San Rafael, CA); borosilicate glass pasteur pipets (John Scientific, Toronto, Ontario); heparinized blood gas syringes (Marquest Medical Products Inc., Englewood, CO); heparinized VacutainerR tubes (Vacutainer Systems, Rutherford, NJ); 15 mL PyrexR disposable culture  15  tubes (Corning Glass Works, Corning, NY); polytetrafluoroethylene (PTFE) lined screw caps (Canlab, Vancouver, B.C.); polystyrene tubes (Evergreen Scientific International Inc., Los Angeles, CA) and silicone rubber tubing for catheter preparation (Dow Corning, Midland, MI). 2.1.1. Preparation of Standard Solutions and Buffers  Standard stock solutions (1 mg/mL) of labetalol hydrochloride, SCH, 3APB, and MPE were prepared by dissolving accurately weighed quantities of the respective analyte in methanol. The stock solutions were stored at 4°C. The labetalol and SCH stock solutions were used within three months of preparation while 3-APB and MPE stock solutions were used within one month of preparation. No evidence of degradation was found within these time periods. Dilutions of the stock solutions were made with distilled water to yield 1 pg/mL, 100 ng/mL and 10 ng/mL concentrations. Standard solutions of labetalol and SCH in 0.05 M phosphoric acid were used in labetalol extraction recovery studies whereas standard solutions of 3-APB and MPE in toluene were used in 3APB extraction recovery studies. Phosphate buffer (0.015 M, pH 3.1) was prepared by dissolving potassium dihydrogen orthophosphate (monobasic) in distilled water. The pH of the solution was adjusted to 3.1 (range: 2.95-3.20) with phosphoric acid. A solution of triethylamine in toluene (0.0125 M) was prepared by diluting triethylamine with toluene. Four or five pellets of sodium hydroxide were added to the solution. Carbonate buffer (1M, pH 9.5) was prepared by dissolving potassium carbonate and sodium bicarbonate in distilled water. The pH was adjusted to 9.5  16  (range: 9.40-9.65) using either concentrated hydrochloric acid or 5 N sodium hydroxide. The pH of the buffer was determined daily prior to use. Ammonium hydroxide solution (4%) was prepared by diluting ammonium hydroxide USP with distilled water. Sodium acetate solution (0.2 M) was prepared by dissolving sodium acetate in distilled water and adjusting the final pH to 5.0 using glacial acetic acid. Tris(hydroxymethyl)aminomethane (TRIS) (0.05 M) was prepared by dissolving TRIS free base in distilled water at room temperature (---.22 °C) and adjusting to a final pH of 7.5 using 1 N hydrochloric acid. 0-dianisidine dihydrochloride (50 mg/vial) was reconstituted in 20 mL distilled water. A 1.6 mL aliquot of this solution was mixed with 100 mL distilled water and a PGO capsule (see section 2.1.) was added and dissolved. The final solution was stable for up to one month at 4°C. Nicotinamide adenine dinucleotide (NAD) (50 mg) was dissolved in 20 mL distilled water. To this solution, 10 mL of glycine buffer and 0.5 mL lactate dehydrogenase were added. The final solution was stored at 4°C for not more than two days. 2.2. Equipment and Instrumentation  2.2.1. High-Performance Liquid Chromatography  An integrated HP 1090 liquid chromatograph system (Hewlett Packard Ltd., Palo Alto, CA) comprising a microbore solvent delivery system, a sample loop injector of 250 AL capacity, autoinjector, autosampler, a Model 310 HP (9000 series) workstation for acquiring, integrating and storing data files, a HypersilODS reverse phase microbore column (200 x 2.1 mm, 5/2 particle size), a  17  Hypersil-ODS reverse phase microbore guard column (20 x 2.1 mm, 5/A), an online solvent filter assembly, and flexible microbore stainless steel tubings (0.12 mm i.d.) (Hewlett Packard Ltd., Palo Alto, CA). A Model 1046A programmable fluorescence detector with replaceable excitation and emission slits and cut-off filters and interfaced to the HP 1090 liquid chromatograph through an analog-to digital converter (Hewlett Packard Ltd., Palo Alto, CA). 2.2.2. Gas Chromatography with Mass Selective Detection  A Model 5890 (Series II) gas chromatograph equipped with a splitsplitless capillary inlet system, a Model 7673 autoinjector, Model G1030A workstation (DOS Series) and connected to a Model 5971A quadrupole mass selective detector (all from Hewlett Packard Ltd., Avondale, PA); cross-linked fused silica capillary column (25m x 0.31 mm i.d., film thickness 0.25, 5% phenylmethylsilicone) (Ultra 2) and 4 mm borosilicate glass inlet liner (Hewlett Packard Ltd., Avondale, PA); silicone rubber septa (ThermogreenR LB-2) (Supelco, Bellafonte, CA). 2.2.3. Physiological Monitoring  A Beckman R-711 Dynograph Recorder (Sensormedics, Anaheim, CA); strain-gauge transducers (Statham model P23Db, Gould Inc., Oxnard, CA); Gould DTX disposable pressure transducers (Spectramed Inc., Oxnard, CA); cardiotachometers (Model 9857, Sensormedics, Anaheim, CA); transit-time blood flow transducers (Transonic Systems Inc., Ithaca, NY); Apple Ile computer and computer data acquisition system consisting of Interactive Systems (Daisy Electronics, Newton Square, PA), analog to digital converter and clock  18  card (Mountain Software, Scott's Valley, CA). A IL 1306 pH/blood gas analyzer (Allied Instrumentation Laboratory, Milan, Italy) and HemoximeterR (Radiometer, Copenhagen, Denmark). 2.2.4. General Experimental  An automatic Speed VacR concentrator (Model AS 290, Savant, Farmingdale, NY); a vortex-type mixer (Vortex-GenieR) and incubation oven (IsotempR model 350) (Fisher Scientific Industries, Springfield, MA); IEC model 2K centrifuge (Damon/IEC division, Needham Hts., MA); rotating type mixer (LabquakeR model 415-110, Lab Industries, Berkeley, CA); infusion pump (Harvard model 944) (Harvard Apparatus, Millis, MA); Pye Unicam SP8400 spectrophotometer (Pye Unicam Ltd., Cambridge, UK). 2.3. Development of a HPLC Assay with Fluorescence Detection for the Quantitation of Labetalol in Biological Fluids of Sheep 2.3.1. Optimization of Mobile Phase  The following parameters were studied in an attempt to determine the optimum composition and flow rate of the mobile phase to be used to obtain satisfactory chromatography of labetalol and SCH on a C-18 microbore column: 1) pH of the phosphate buffer (from 2.5 to 7.5). 2) molarity of the phosphate buffer (1 to 20 mM). 3) selection of organic modifier (methanol and acetonitrile) 4) proportion of organic modifier (0-70%). 5) mobile phase flow rate (0.3 to 0.8 mL/min).  19  The sample for injection was prepared by mixing 1 mL aliquots of aqueous solutions of labetalol and SCH (lmg/mL each) and 10 ttL of the mixture was injected into the HPLC under varying mobile phase conditions. UV detection at 254 nm was used in these initial experiments. The mobile phase variables were evaluated against the following: separation of drug and internal standard, their peak shapes and symmetry quantitated by the assymmetry factor. Separation was evaluated by the resolution factor (R s), which is calculated as [R, = 2 * (difference in retention times) ÷ (sum of peak widths at base)], peak shape by the peak width at base and peak symmetry by the assymmetry factor, which is obtained by dividing the peak at its apex into two halves and taking the ratio of the rear half to front half areas.  2.3.2. Optimization of Detection  The excitation and emission wavelengths that provide the best signal-to-noise ratio were determined as follows. A 10 R1_, aliquot of a 1 i.tg/mL aqueous solution of labetalol was injected into the HPLC with the fluorescence detector on line. The labetalol peak (from a knowledge of retention time obtained using UV detection) was "trapped" in the detector cell by turning the pump off when the peak was beginning to elute. With the excitation wavelength set at zero, the emission wavelength was scanned from 190 to 600 nm, in increments of 10 nm. Emission peaks were chosen from a fluorescence intensity vs emission wavelength plot. The excitation wavelength was then scanned against each of the emission peaks. The process was repeated with the scanning done over narrow ranges at increments of 1 nm to obtain the optimum wavelength pair.  20  Sensitivity of detection was further optimized by using excitation and emission slits of varying widths (1, 2 and 4 mm) and emission filters of different cut-off values (210 to 400 nm). 2.3.3. Optimization of Extraction  A sample of an aqueous solution of labetalol (1 lig/mL) was subjected to extraction and the following parameters were studied: (i) pH of the buffer, (ii) extraction efficiency of solvents (dichloromethane, ethyl acetate and toluene), (iii) volume of the organic solvent, (iv) volume of aqueous phase, and (v) duration of mixing. The organic phase was separated, dried and reconstituted in 500 µL of 0.05 M phosphoric acid and 50^was injected into the HPLC. In addition, a two-step extraction, in which the separated organic layer was reextracted with 0.05 M phosphoric acid, was also attempted. In the latter case, an aliquot of aqueous layer at the end of the second step of extraction was directly injected into the HPLC. Following preliminary optimization, the extractions were repeated with 100, 250, 500 and 1000 !IL of biological matrices (maternal and fetal plasma, amniotic and tracheal fluid) spiked with labetalol. 2.3.4. Procedures for the Analysis of Labetalol in Biological Fluids 2.3.4.1. HPLC Operating Conditions  Chromatography was achieved on a 200 x 2.1 mm analytical column, which was connected in series with a guard column (20 x 2.1 mm) and an online solvent filter. The connections between the solvent delivery system, the columns and the detector were made with 0.12 mm i.d. flexible stainless steel  21  tubing. The mobile phase used was pH 3.1 phosphate buffer (0.015 M) and acetonitrile in a ratio of 56:44. The phosphate buffer was first filtered through a membrane filter of 0.45 A pore size aided by suction. The buffer and acetonitrile, which were pumped separately in the binary HP 1090 liquid chromatograph system, were degassed with helium. The flow rate used was 0.5 mL/min. The fluorescence intensity of labetalol was monitored at an excitational wavelength of 196 nm and an emission wavelength of 412 nm with a 370 nm emission cut-off filter. The "Photo Multiplier Tube Gain" (i.e., signal attenuation) used was 17 or 16 in a scale of 1-18. The optical system of the detector included a 2 mm wide excitational slit and two 4 mm wide emission slits. 2.3.4.2. Extraction of Labetalol from Biological Fluids  The procedure used for the extraction of labetalol from plasma, amniotic and tracheal fluid samples is as follows. To 0.25 mL of the biological fluid in a glass culture tube, 100 !IL of internal standard solution (1 tg/mL) and 0.5 mL of pH 9.5 carbonate buffer (1 M) were added and the total volume adjusted to 0.75 mL with distilled water. The samples were then extracted with 6 mL of ethyl acetate by mixing for 20 min on a rotary shaker. The tubes were then refrigerated for 15 min to break any emulsion that might have formed during mixing. This was followed by centrifugation at 1000 g for 6 min. The organic layer was transferred to a clean, dry screw-capped glass tube and mixed with 0.6 mL of 0.05 M phosphoric acid for 20 min and then centrifuged. The organic layer was discarded and the aqueous layer transferred to 300 AL microvials and 60 ILL was injected into the HPLC.  22  2.3.4.3. Preparation of Calibration Curve  Standard solutions of labetalol and the internal standard were prepared from methanolic stock solutions (1 mg/mL) by dilution with HPLC grade water to produce concentrations ranging from 10 to 1000 ng/mL. Blank biological fluid samples (0.25 mL) were spiked with varying amounts of labetalol and identical amounts of the internal standard (100 ng). The spiked samples were extracted as described previously. Duplicate samples were prepared and analyzed for each of the labetalol concentrations in the standard curve. The calibration curves were constructed by plotting the mean labetalol/internal standard peak area ratio against the amount of labetalol added to the sample. 2.3.4.4. Quantitation of Labetalol in Biological Fluid Samples  Duplicate aliquots of the biological fluid samples were extracted simultaneously with the calibration curve samples, prepared from the respective biological fluid. Labetalol concentration in the biological fluid sample was determined from the peak area ratio corresponding to the sample and the linear regression equation of the standard curve. Samples with peak area ratios lower than that of the lowest concentration or higher than that of the highest concentration of the calibration curve were not quantitated. 2.3.5. Labetalol Assay Validation  2.3.5.1. Precision of Quantitation  23  Intra-sample variation was studied using replicate samples (n=4) at each of the concentrations of the calibration curve in each of the biological fluids. The percent coefficients of variation were determined. Variability due to the injector was studied by multiple injections of standard solution of labetalol (1 p. g/mL).  2.3.5.2. Extraction Recovery Studies The efficiency of extraction of labetalol from each of the biological fluids was determined over the entire concentration range of the calibration curve by comparing the peak area ratio of the extracted samples with that of direct injection of a corresponding amount of labetalol.  2.3.5.3. Determination of Minimum Quantitation Limit The minimum limit of quantitation was defined as the amount that yields a signal-to-noise ratio of at least 3 and an intra-sample coefficient of variation of less than 10% .  2.3.6. Analysis of Glucuronide and Sulphate Conjugates of Labetalol Urine and bile samples obtained from adult non-pregnant sheep following bolus administration of labetalol and amniotic fluid samples obtained following direct fetal administration of labetalol were also analyzed for the presence of conjugated metabolites of labetalol using the general procedure described by Brashear  et al., 1988. Aliquots of urine (50 pt each), bile and amniotic fluid  (250 [IL each) samples were treated with 0.5 mL pH 5.0 sodium acetate buffer  24  (0.2 M) and 0.5 mL GlucuraseR for the determination of glucuronide and 0.5 mL pH 7.5 Tris buffer (0.05 M) and 30 "IL of aryl-sulfatase preparation for sulphate determination. The enzyme treated samples were incubated overnight at 37°C in a water bath with gentle shaking. Following incubation, the samples were cooled to room temperature and the liberated labetalol was quantitated as previously described. Glucuronide or sulphate conjugate concentrations were expressed as the difference between the post-incubation and pre-incubation labetalol concentrations. 2.4. Development of a GC-MSD Assay for the Identification and Quantitation of 3-APB, an Oxidative Metabolite of Labetalol, in the Biological Fluids of Sheep 2.4.1. Optimization of GC Conditions Samples of 1 mL each of methanolic solutions of 3-APB and MPE (1 pi g/mL) were mixed and dried under a stream of nitrogen at 30°C, reconstituted in 1 mL toluene and derivatized with HFBA at 55°C for 1 h. The excess derivatizing reagent was neutralized by vortex mixing with 2 mL 0.067 M phosphate buffer (pH 6). The organic layer was removed to autosampler vials and 1 !IL was injected onto the GC. The default derivatization conditions used in the preliminary experiments were from an assay method for ritodrine, developed in this laboratory (Wright et al., 1991 a). The following parameters were studied with respect to separation of the drug and internal standard, peak shape and symmetry. 1) Injector temperature: 160, 180, 200, 220 and 250°C. 2) Initial column temperature: 90, 100, 125 and 150°C.  25  3) Column temperature ramping: 5, 10 and 15°C/min. 2.4.2. Optimization of Derivatization and Extraction  The following factors were studied: 1) Duration of HFBA derivatization (15 min to 2 h). 2) Neutralization of HFBA (water, pH 6.0 phosphate buffer (0.067 M), water + 4% ammonium hydroxide). 3) Adjustment of sample pH (7.0-14.0) using carbonate buffers and sodium hydroxide. 4) Extraction efficiency of solvents (ethyl acetate, ether, hexane and toluene). 5) Volume of solvent and duration of mixing. 6) Drying conditions (nitrogen vs vacuum, temperature, incomplete vs complete drying). 7) Reconstitution volume (0.1 to 1.0 mL). 2.4.3. Optimization of Sensitivity and Selectivity  The following steps were used in the optimization of selectivity and sensitivity: 1) Selection of two ions from the mass spectrum of 3-APB derivative obtained in the scan mode that are diagnostic and with at least one of them having a relative abundance of 50% or more. 2) Calculation of the dwell time required to obtain a minimum number of 10 scans to define each peak. 3) Selection of the appropriate tuning method (reagent and target ions).  26  4) Determination of the exact fractional mass (correct to two decimal places) for selected ion monitoring that produces the best signal-tonoise ratio.  2.4.4. Procedures for the Analysis of 3-APB in Biological Fluids 2.4.4.1. GC-MSD Operating Conditions  A cross-linked fused-silica capillary column (25m x 0.31 mm i.d. , film thickness 0.25 , 5% phenylmethylsilicone) and a wide bore (4 mm i.d.) borosilicate glass inlet liner with silanized glass wool plug were used for chromatography. The splitless mode of injection was employed in all cases. The GC injection port temperature was kept at 200°C. The oven temperature was initially set at 100°C and following injection was raised to 200°C at the rate of 15°C/min and then to 280°C at the rate of 30°C/min. The final temperature (280°C) was maintained for 1 min to give a run time of 11.33 min. Helium was used as the carrier gas at a flow rate of 30 cm/sec. The electron energy used for  EI was 70 eV. Tuning of the mass selective detector was accomplished using PFTBA as the tuning reagent. Two groups of ions were monitored in the SIM mode: m/z 347.4 and 134.0 in group I (internal standard) and 345.4 and 132.0 (3-APB) and the switch (from group I to II) was made at 8.15 min. A dwell time of 200 ms was used to obtain a detection frequency of 2.25 cycles/sec.  2.4.4.2. Extraction and Derivatization of 3-APB from Biological Fluids  The biological fluids studied were urine, bile, and plasma obtained from adult non-pregnant sheep and fetal plasma and amniotic fluid obtained from  27  pregnant sheep. One mL samples of the biological fluid were added to clean 10 mL borosilicate glass tubes with PTFE lined screw caps (Corning Laboratory, Corning, NY). Subsequently, 100 juL of aqueous internal standard solution (1 tt g/mL) and 300 pL of 5N NaOH were added and this mixture was extracted with 6 mL of n-hexane by tumble mixing for 5 min. The tubes were then chilled (at 20°C for 15 min) to facilitate breakage of any emulsion formed during mixing. Following centrifugation for 3 min at 1000 g, the organic layer was separated with borosilicate Pasteur pipets and transferred to clean tubes. The drying of the solvent was accomplished using the automatic Speed VacR concentrator at ambient temperature, using a target vaccuum of 2000 mtorr. The samples were reconstituted with 300 (IL of 0.0125 M solution of triethylamine in toluene and treated with 20 jut of HFBA at 55°C for 1 h for derivatization. The samples were then cooled to room temperature and the derivatizing reagent was neutralized by treating first with 0.5 mL distilled water and then with 0.5 mL 4% ammonium hydroxide solution with a 30 sec vortexing after each addition. The aqueous layer was discarded while the organic layer was transferred to autosampler vial inserts and 1 [iL was injected into the GC. 2.4.4.3. Preparation of Calibration Curve Blank biological fluid (1 mL) samples were spiked with varying amounts of 3-APB (range: 0.5-1000 ng), 100 ng of internal standard and 300 !IL of 5N NaOH. After adjusting the total volume of the aqueous phase to 1.5 mL with distilled water, the samples were extracted and derivatized as described above. The peak area ratio (3-APB/internal standard) was plotted against the amount added to yield the calibration curve. Calibration curves with six points, prepared in duplicate, were used for routine sample analyis. A calibration curve  28  in the appropriate biological fluid was prepared each time the samples were analyzed.  2.4.4.4. Quantitation of 3-APB in Biological Fluid Samples Concentrations of 3-APB in biological fluids was determined as described for labetalol (section 2.3.4.4.).  2.4.5. Validation of 3-APB Assay 2.4.5.1. Precision of Quantitation Intra-sample variation was studied using replicate samples (n=3) at each of the concentrations of the calibration curve in each of the biological fluids. The percent coefficient of variations were determined. Variability due to the injector was studied by multiple injections (1 ilL) of standard solution of 3-APB (1 µg/mL).  2.4.5.2. Extraction Recovery Studies  Specified amounts of 3-APB (in aqueous solution) were added to blank biological fluid (1 mL) samples and the spiked samples were extracted without the internal standard. The organic layer was separated and dried, following which, 100 ng of internal standard (in toluene) was added and the samples were dried again and derivatized as described previously. The amount of 3-APB recovered by the extraction procedure was determined using a calibration curve  29  constructed by derivatizing corresponding amounts of standard 3-APB (in toluene) with 100 ng of internal standard (in toluene) (i.e. , without extraction). 2.4.5.3. Determination of Minimum Quantitation Limit  The minimum quantitation limit for 3-APB was determined as explained previously (section 2.3.5.3.). 2.4.6. Analysis of Glucuronide and Sulphate Conjugates of 3-APB  The conjugates of 3-APB were analyzed in identical manner as described for labetalol (section 2.3.6.). 2.5. Standard Procedures for Sheep Experiments 2.5.1. Recording of Hemodynamic Parameters  The various parameters that were continuously monitored (varies with the type of experiment; see section 2.7.) include fetal and maternal arterial pressures, fetal hind limb venous pressure, tracheal and amniotic pressures, which were measured with strain-gauge manometers (Statham Model P23Db, Gould Inc., Oxnard, CA) or disposable DTX transducers (Spectramed, Oxnard, CA). Fetal and maternal heart rates were measured from the arterial pulse by means of cardiotachometers (Model 9857, Sensormedics, Anaheim, CA). Femoral and hind limb blood flows were measured with transit-time flow transducers (Transonic Corporations Inc., Ithaca, NY). All of these variables were continuously recorded using a Beckman R-711 polygraph recorder. The  30  analog signals were converted simultaneously to digital form through an analog to digital conversion board (Daisi Electronics, Newton Square, PA) and an online Apple IIe computer (Apple Computers Inc., Cupertino, CA) equipped with a clock board (Mountain Computers Inc., Scotts Valley, CA) (Kwan, 1989). The sampling rate was 2.5 Hz. At the end of each minute, the measurements were averaged, fetal arterial pressure corrected for amniotic pressure and the variable values displayed on the monitor. Every 30 minutes, the minute average measurements were automatically transferred to floppy diskettes for subsequent analysis. 2.5.2. Blood Gas Analysis Blood pH, p0 2 , and pCO 2 were measured by an IL 1306 pH/blood gas analyzer following injection of -200 [IL whole blood. The instrument also calculates base excess and bicarbonate. Blood oxygen saturation and hemoglobin content were measured in duplicate using a HemoximeterR. Oxygen content was calculated as hemoglobin concentration (g/100 mL) * oxygen saturation (%) * 0.0139 2.5.3. Glucose Measurement Glucose concentrations were measured in whole blood and amniotic fluid (where applicable) samples. Freshly drawn samples were transferred to heparinized tubes and 0.2 mL was added (within 30 min of collection) to polystyrene tubes containing 0.9 mL distilled water. To this mixture was added 0.55 mL of zinc sulphate (0.3 N), vortex mixed and the mixture was allowed to stand for 10 minutes. Barium hydroxide (0.3 N), 0.55 mL, was then added,  31  followed by vortex mixing and then the solution was allowed to stand for a further 10 minutes prior to centrifugation at 4000 rpm for 15 min. The supernatant was transferred through cotton-tipped pasteur pipets to clean polystyrene tubes, covered with ParafilmR and refrigerated until analysis. All samples were analyzed in duplicate within two weeks using glucose assay kits. The assay method involves enzymatic oxidation of glucose to form gluconic acid and hydrogen peroxide. The hydrogen peroxide then oxidizes o-dianisidine (in the presence of peroxidase) to a product which can be quantitated colorimetrically. 2.5.4. Lactate Measurement  For the analysis of lactate, 0.3 mL of the sample (whole blood or amniotic fluid) was added to polystyrene tubes containing 0.6 mL perchloric acid (8%) followed by centrifugation at 4000 rpm for 15 min. The supernatant was removed to clean polystyrene tubes, covered with ParafilmR and refrigerated until analysis. The samples were analyzed in duplicate within 2 weeks using lactate assay kits, which employ enzymatic conversion of lactate in the presence of nicotinamide adenine dinucleotide (NAD) to pyruvate and the reduced form of NAD (NADH). The NADH concentration, which is equivalent to the concentration of lactate in the sample, was then determined spectrophotometrically. 2.6. Animal Preparation  2.6.1. General Maintenance  32  Both pregnant and non-pregnant ewes of Suffolk, Finn and Dorset mixed breed were used in these studies. The animals were brought into the animal unit at the Children's Variety Research Center, at least 1 week prior to surgery, and kept in groups of 2 or more in large pens in full view of one another. The animals received a standard diet and free access to water. Ethical approval for the studies was obtained from the Animal Care Committee of the University of British Columbia and the procedures used were in accordance to the guidelines of the Canadian Council on Animal Care.  2.6.2. Pregnant Sheep Studies  Eleven time dated pregnant sheep were operated on between 115 and 125 days of gestation (term 145 days). Food was withheld for about 18 h prior to surgery. Aseptic techniques were employed throughout the surgical procedure. Following intravenous atropine (3 mg) administration to control salivation, anesthesia was induced with intravenous sodium pentothal (1g). The animals were intubated with an endotracheal tube and anesthesia was maintained with a mixture of halothane (1-2%), nitrous oxide (60%) and oxygen. An intravenous bolus injection of 500 mg ampicillin and 5% dextrose solution, at a rate of 1.0-1.5 mL/min, via an intravenous drip, were administered to the ewe. The ewe's abdomen was shaved, and the surgical area was sterilized with 10% povidone-iodine topical solution, while other areas were covered with sterile sheets and drapes. A midline abdominal incision was made in the ewe and the uterus identified. Access to the head of the fetus was gained through an incision of the uterine wall in an area devoid of placental cotyledons and major blood vessels. With a small incision, the trachea was exposed. The tracheal catheter (2.2 mm o.d.), was inserted through a small incision 1-2 cm below the larynx  33  between two rings of cartilage and advanced 4-5 cm into the trachea. The tracheal catheter did not obstruct lung fluid efflux from the airway into the pharynx. The catheter was anchored onto the skin overlying the incision with the 3-0 silk attached to the catheter. A drop of KrazyglueR (Feature Products Inc., Mississauga, Ontario) was applied on the catheter's point of entry to the trachea. The tracheal incision was then closed. A catheter was placed in the amniotic fluid and anchored to the neck. After this, a second small uterine incision was made to expose the fetal hindlimbs. The hindquarters of the fetus were withdrawn from the uterus. An incision of about 3 cm in length was made above the femoral arterial pulse in the groin. The femoral artery was exposed and 3 pieces of 3-0 silk sutures were passed underneath the vessel. After the vessel was tied off with the distal silk and temporarily constricted with the proximal one, a partial cut was made on the vessel between the middle and the distal sutures. Approximately 5 to 6 cm of the catheter was threaded through the vessel into the descending aorta. Then the catheter was secured to the vessel with all three silk sutures and a drop of KrazyglueR was applied on the site of entry. The catheter was anchored to the adjacent muscle on either side of the incision with the sutures. Both the right and left femoral arteries were catheterized in this manner. In a similar manner, silicone rubber catheter was also placed in the right lateral tarsal vein and a small branch of the uterine vein draining the uterine horn containing the operated fetus. In the case of tarsal vein catheter, approximately 11 to 12 cm of the silastic catheter was inserted into the vessel to reach the inferior vena cava. To catheterize the common umbilical vein, a small incision was made in the umbilicus overlying one of the two umbilical veins. Two sutures were placed, in parallel, through the vessel wall at right angles to the long axis. Using an 18-gauge needle, a hole was made in the vessel wall between the sutures and a silicone catheter inserted for — 2 cm, so  34  that the tip lay in the intra-abdominal common umbilical vein. The sutures were tied in a "figure 8" fashion around the catheters and a drop of KrazyglueR was applied. For fetal bolus studies, catheters were placed in the pudendo-epigastric artery (HLA) and vein (HLV) of right hind limb and a 4R or 4S series transittime blood flow transducer (Transonic Corporations Inc., Ithaca, NY) was placed around the right external iliac artery distal to the origin of the circumflex iliac artery (Fig 2). The fetus was then gently returned to the uterus and the uterine incision closed with a continuous 2-0 gut chromic suture and then oversewn. Amniotic fluid lost during surgery was replaced with irrigation saline (Travenol Canada Inc., Mississauga, Ontario). The catheters were filled with heparinized normal saline (1.2 U heparin/mL), tunnelled subcutaneously and exited through a small incision on the maternal right flank. The ewe's midline abdominal incision was closed in layers and the right flank incision was sewn up as well. Medical adhesive spray was applied onto the incisions. Finally, the maternal femoral artery and vein were catheterized. All the catheters were capped and stored in a denim pouch which was secured in place with two adhesive bandages on the right flank. The ewe's abdomen was then wrapped around with elastic crepe bandages. All vascular catheters were flushed daily with 2 mL of heparinized normal saline. In the case of umbilical venous catheter, an additional 0.5 mL heparin was used to prevent clot formation. Ampicillin (500 mg) and gentamicin (40 mg) were administered prophylactically to the ewe on the day of surgery and for the first four days following surgery while ampicillin (500 mg) and gentamicin (10 mg) were administered to the fetus at the time of surgery. Ampicillin (500 mg) and gentamicin (20 mg) were also administered into the amniotic cavity on a daily basis until delivery. The ewes were moved to holding pens in the company of other sheep and were allowed to  35  Internal Mix t Arteries Umbilical Arterkis  Femoral catheter Deep Femora11 Arte ry  Hindlimb Artery (& Vein)^ Catheter  Femoral Artery  Pudendoepigastric Artery  Lateral Tarsal Vein . Tarsal Vein Caiheter  FIG 2: Schematic diagram of ovine fetal hind limb to illustrate the position of the hind limb catheters and flowmeter.  36  recover for at least 5 days following surgery. The catheters were flushed daily with 2 mL hepaiinized normal saline (1.2 U heparin/mL). 2.6.3. Nonpregnant Sheep Studies  Nonpregnant Dorset or Suffolk breed ewes aged 5-11 years were used in these studies. The ewes were sedated with i.v. sodium pentothal (1-1.5 g) and anesthetised with a mixture of halothane (1-2%), nitrous oxide (60%) and oxygen. Silicone rubber catheters were implanted in the femoral artery and vein in one limb while a 6R-series transit-time blood flow transducer (Transonic Systems Inc., Ithaca, NY) was placed on the femoral artery of the other hind limb. A catheter was also placed in the jugular vein for drug administration. An abdominal incision to the right of the umbilicus was made to gain access to the gall bladder, where a catheter was placed to allow bile collection. The catheters were exteriorized, tunnelled subcutaneously through an incision on the flank and secured in a denim pouch. The catheters were filled with heparinised saline and capped when not in use. Ampicillin (500 mg) and gentamicin (40 mg) were administered prophylactically on the day of surgery and the following four days. The ewes were allowed to recover for at least five days before they were used in the experiments. Just before the beginning of each experiment, a Foley catheter was inserted for total urine drainage. 2.7. Experimental Protocols 2.7.1. Maternal Bolus Studies  37  A total of fourteen experiments were performed on eleven ewes. Nine experiments involved administration of a 100 mg dose of labetalol (20 mL) (TrandateR), administered as an intravenous bolus through the maternal femoral vein. The catheter was flushed immediately with heparinized saline (10 mL). The other five experiments were control studies involving a bolus of normal saline (20 mL) instead of labetalol. In both types of experiments, samples for labetalol analysis were obtained from maternal artery, amniotic and fetal tracheal fluid (3 mL each), fetal artery, umbilical and uterine veins (1.5 mL each) at -30, -15, 3, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 360 min and then every 2 h till 12 h, as well as at 24 h and 48 h following drug administration. Simultaneously, maternal and fetal arterial samples (0.5 mL) were taken for blood gas analysis. The fetal blood taken for each sample was replaced with an equal volume of drug-free maternal blood, collected at the beginning of the experiment. The blood samples were immediately transferred to heparinized tubes and centrifuged at 3000 g for 15 min to harvest the plasma. The plasma, as well as the amniotic and tracheal fluid samples, were transferred to clean PTFE lined screw capped glass tubes and stored at -20 ° C until analysis. In six of the experiments (four labetalol and two control experiments), fetal and maternal blood and amniotic fluid samples were also analyzed for glucose and lactic acid. In those cases, an aliquot was removed from the samples for the analysis of glucose and lactate before they were centrifuged. For at least 24 h before and 48 h after labetalol or saline administration, fetal and maternal arterial pressures, tracheal and amniotic pressures, fetal and maternal heart rates were continuously monitored on a polygraph recorder as well as processed by an on-line computerized data acquisition system as described previously.  38  2.7.2. Bolus Studies in Nonpregnant Sheep  2.7.2.1. Labetalol Bolus  Five animals were used in this study. A 100 mg dose of labetalol was administered as an intravenous bolus through the jugular vein. Blood samples (3 mL) for the quantitation of labetalol, glucose and lactate were obtained from femoral artery and vein at -30, -15, 3, 10, 15, 20, 30, 45 and 60 min, every 30 min until 4 h and then at 5, 6, 8, 10, 12 and 24 h following drug administration. The blood samples were immediately transferred to heparinized tubes, aliquots removed for glucose and lactate determinations and centrifuged at 3000 x g for 15 min to harvest the plasma. Samples (0.5 mL) for blood gas analysis (pH, base excess, p0 2 , pCO 2 , hemoglobin content and percent oxygen saturation) were also obtained simultaneously from femoral artery and vein. Urine was collected continuously and aliquots (10 mL) corresponding to fixed intervals (030 min, 30-60 min and then every hour until 6 h, and then at 6-8, 8-10, 10-12 and 12-24 h following drug administration) were retained. Bile samples were obtained from only two of the animals (E#617 and 248). Since continuous draining of bile will deplete the physiologically essential components (e.g. bile salts), samples were obtained over discrete half-hour intervals by gravity assisted draining at 30 min, 2, 4 and 10 h following labetalol administration. The plasma, as well as the urine and bile samples were stored at -20°C until analysis. The urine and bile samples and some of the arterial plasma samples were also used for the identification and quantitation of 3-APB, an oxidative metabolite of labetalol.  39  The femoral arterial and venous pressures, heart rate and femoral blood flow were continuously monitored on a polygraph recorder as well as processed by an on-line computerized data acquisition system as described previously. 2.7.2.2. Dilevalol (RR isomer of Labetalol) Bolus -  This study was conducted only in two animals (E#105 and E#248). A 25 mg dose of dilevalol (in 10 mL normal saline) was administered through the jugular vein. The sampling and recording protocol used was identical to that used for labetalol (section 2.7.2.1.). Femoral blood flow data was obtained from only one animal (E# 105). 2.7.3. Infusion Studies in Nonpregnant Sheep Five animals were used in these studies. Injectable labetalol was infused through the jugular vein at a constant rate of 0.5 mg/min immediately following a 100 mg bolus and the infusion was continued for 6 h. Blood samples (3 mL) for the quantitation of labetalol, glucose and lactate were obtained from femoral artery and vein at -30, -15, 15, 30, 45, 60, 90, and 120 min, and every hour until 6 h during infusion and at 15, 30, 45, 60, 90, and 120 min, 3, 4, 6 and 18 h post-infusion. The blood samples were immediately transferred to heparinized tubes, aliquots removed for glucose and lactate determinations and centrifuged at 3000 g for 15 min to harvest the plasma. Samples (0.5 mL) for blood gas analysis (pH, base excess, p0 2 , pCO 2 , hemoglobin content and oxygen saturation) were also obtained simultaneously from femoral artery and vein.  40  The femoral arterial and venous pressures, heart rate and femoral blood flow were continuously monitored on a polygraph recorder as well as processed by an on-line computerized data acquisition system as described previously.  2.7.3.1. Norepinephrine (NE) Challenge The degree of x-adrenergic blockade caused by labetalol infusion was assessed by intra-arterial injections of NE. Five different doses of NE (0.025, 0.05, 0.1, 0.2 and 0.4 Kg/kg; q. s. to 3 mL with normal saline) and a control dose (3 mL saline) were administered through the femoral artery in a randomised order. The injections were followed by a 5 mL saline flush. The doses were separated by 6 min. The graded NE challenge was repeated three times - at 30 min before the start of labetalol infusion, at 2 and 5 h after the start of infusion. The response was calculated as the magnitude of change in femoral blood flow from baseline values, i.e., E max -E zero , where E max represents the maximum change in flow following the injection and E uro , the mean flow corresponding to the minute preceeding the injection.  2.7.4. Intra-arterial Administration of Labetalol  The direct vascular effects of labetalol were studied with experiments involving intra-arterial injections of labetalol, which were conducted in naive animals (i.e., no drug was administered for at least 72 h before the experiment).  2.7.4.1. Studies in Adult Nonpregnant Sheep  41  In five adult nonpregnant sheep, doses of labetalol ranging from 0.001 to 1 mg in saline were injected intra-arterially, i.e., via the femoral arterial catheter, starting with the lowest dose and separated by a 10 min interval. Control injections (5 mL normal saline) were administered at the beginning and end of the experiment. The changes in femoral blood flow over 10 sec intervals were recorded. A dose-response plot was constructed in each case. In two animals, the intra-arterial injections (0.1 and 1.0 mg labetalol) were repeated following administration of phentolamine (30 mg i.v. bolus followed by continuous infusion at the rate of 1 mg/min for 90 min) to produce oc-blockade and in one animal, following administration of propranolol (20 mg infused over 10 min), to produce 0-blockade. Alpha blockade was indicated by the lack of any vasoconstictory response to intra-arterial injection of 0.8 i.tg/kg of NE, which produces complete vasoconstriction (flow momentarily reduced to 0 mL/min) in the control sheep. Lack of pressor response to 1 pg/kg of isoprenaline (increase in heart rate to 200 bpm) was taken as an indication of f3blockade. The change in flow was calculated as E.-E zra , as explained previously under NE challenge (section 2.7.3.1.). For at least one hour before, during and for at least one hour after the experiments, the femoral arterial and venous pressures, heart rate and femoral blood flow were continuously monitored on a polygraph recorder as well as processed by an on-line computerized data acquisition system as described previously.  2.7.4.2. Studies in Fetal Sheep  The fetal hind limb preparation was used for these studies (Fig 2), which were conducted in a total of six animals. The following doses of labetalol were  42  injected through the external pudendal epigastric artery (HLA): 0 (control), 0.1, 1.0, 10, 25, 50, 100, 250 and 500 lug. The volume of the injection in all cases was 3 mL, which was followed by a 5 mL saline flush. The doses were separated by a 6 min interval. The changes in hind limb blood flow, arterial pressure and hind limb venous pressure over 10 sec intervals were recorded. 2.7.5. Fetal Bolus Studies  A total of nine experiments were performed on seven chronically instrumented fetal lambs. In five of the experiments, a 4 mg dose of labetalol was administered as an intravenous bolus through the fetal tarsal vein while in the other four, 2 mL of saline was injected, to serve as controls. Samples for the determination of labetalol, glucose and lactic acid were obtained from fetal hind limb artery and vein (HLA and HLV respectively) (1.5 mL each), amniotic fluid, tracheal fluid (only for labetalol analysis) and maternal femoral artery (3 mL each) at -30, -15, 3, 10, 15, 20, 30, 45 and 60 min, every 30 min until 4 h and then at 5, 6, 8, 10, 12 and 24 h following drug administration. Samples (0.5 mL) for blood gas analysis (pH, base excess, p0 2 , pCO 2 , hemoglobin content and percent oxygen saturation) were also obtained simultaneously from the fetal and maternal arteries. The fetal blood withdrawn at each sample was replaced via the tarsal vein with an equal volume of drug-free maternal blood, collected at the beginning of each experiment. The blood samples were immediately transferred to heparinized tubes on ice and aliquots removed for the determination of glucose and lactate. The remainder of the blood samples were centrifuged at 3000 g for 15 min to harvest the plasma. The plasma, as well as the amniotic and tracheal fluid samples, were transferred to clean PTFE lined screw capped glass tubes and stored at -20°C until analysis.  43  Fetal and maternal arterial, fetal hind limb venous, amniotic and tracheal pressures, fetal and maternal heart rates and fetal external iliac artery blood flow were monitored continuously for at least 24 h before and after the experiment.  2.8. Data Analysis 2.8.1. Pharmacokinetic Analysis 2.8.1.1. Selection of Weighting Factor  Plasma concentration data were weighted before they were subjected to curve-fitting. An appropriate weighting factor was chosen by the method of Albert et al., 1974. The steps involved in this method are as follows. First, concentration-time data from the individual subjects were pooled and estimates of mean concentrations at each time point and the associated variances were calculated. The variance of the concentrations is related to the mean concentration values by the following equation:  ln G 2 = ln a + n ln C  where In refers to natural logarithm, 6 2 is the variance, C is the mean concentration and a and n are constants. The appropriate weighting factor is n, -  i.e., any concentration C t is weighted as (Cd -n. 2.8.1.2. Pharmacokinetic Fitting  44  The weighted plasma drug concentration-time data were fitted by the computer program AUTOANR (Sedman and Wagner, 1976) or JANAR (Statistical Consultants Inc., Lexington, KY) to choose the most appropriate equation to describe the decline in plasma concentrations and to obtain initial estimates of exponents and constants (e.g. P, A, B, 7E, a, and 0). The initial estimates were used in iterative fitting to obtain final estimates using PCNONLINR (version 3.0) (Statistical Consultants Inc. Lexington, KY). 2.8.1.3. General Calculation of Pharmacokinetic Parameters  With the exception of p, the terminal elimination rate constant, which was obtained through model specific fitting, the rest of the pharmacokinetic parameters were calculated in a model independent manner. The formulae used in the estimation of pharmacokinetic parameters were obtained from Gibaldi and Perrier, 1982, unless otherwise specified. The area under the plasma concentration-time curve (AUC) and area under the first moment curve (AUMC) were calculated as follows: AUC o c° = AUC o t last + AUC t-Iastc° -  AUMC0 0° AUMC0t -last AUMC t _i ast c"  where t-last represents the time of the last sample. The terms AUC o t last and -  AUMC o t last were determined by trapezoidal approximations. An estimate of -  AUC t _ last °° was made according to the equation AUC t-last °°^Ct-lastiO  45  and an estimate of AUMC t-last c° was made by the equation (Gouyette, 1983) AUMC t_ iast °° = t-last * Cr-last/0 + Ct-last/1 32 Terminal elimination half-life (t 1/213) was calculated as t w = 0.693/0 The mean residence time (MRT) was estimated by the equation MRT = AUMC o c"/AUCe° Total body clearance (CL) following an intravenous bolus as CL = Dose/AUCe° while steady-state clearance (CL„), i.e. , during constant rate infusion to steadystate as cLss = Ico/c ss  where lco is the infusion rate and C ss is the steady-state concentration of labetalol. The apparent volumes of distribution were calculated as VD a„a = Dose/(AUCe/(3) VD ss = Dose * AUMC/(AUC) 2  46  2.8.1.4. Calculation of Transplacental and Nonplacental Clearances  Maternal and fetal transplacental clearances were calculated by a modification of the equation derived by Anderson et al. 1980a, for steady-state infusion (Eqn. 1): [CL fp] ss = [CLf]SS * (FssiMs)^Eqn. 1 where [CL fp] ss is fetal transplacental clearance, [C1. 4] ss is fetal total body clearance, F and M are the concentrations in the fetus and mother, respectively, following maternal infusion to steady-state. Integrating the above equation from time zero to infinity yields CL 4, = CL f * (-FAuc/MAuc)^Eqn. 2 where CL fi, and CL f represent the time averaged fetal transplacental and total body clearances respectively, while  FAUC  and MAUL represent the fetal and  maternal AUC from time zero to infinity, respectively. An estimate of CL f was obtained following direct fetal intravenous labetalol administration (see section 2.7.5.) while F^/M AUC- -AUC was obtained in the studies following maternal drug  administration (see section 2.7.1.). The maternal transplacental clearance (CL.p) could be calculated in a similar manner from the maternal total body clearance (CL m) (Eqn. 3). 1 CL„,p = CLm * (M^ Eqn. 3 -AUC - AUC,^  The value of CL m was obtained from maternal i.v. bolus administration of labetalol (see section 2.7.1.), while MAUC/FAUC was determined following direct fetal administration (see section 2.7.5.). The maternal nonplacental and fetal nonplacental clearances^and CL f,.,, respectively) were calculated as the difference between the total body clearance and transplacental clearance (Eqns. 4 & 5). CL fi, = CL f - CL f,^Eqn. 4  47  CL nin = CL m - CL mp^Eqn. 5  2.8.2. Analysis of Hemodynamic and Metabolic Data  The arterial pressure, blood flow and heart rate values were averaged over 30 min periods. The mean values obtained from all the animals along with the SEM were plotted against time. Glucose and lactate concentrations were expressed as mM. The fluxes of glucose, lactate and labetalol across the hind limb were calculated as [(arterial concentration - venous concentration) * hind limb blood flow]. Fetal hind limb vascular resistance was calculated as [(arterial pressure - hind limb venous pressure) ÷ (hind limb blood flow)]. 2.8.2. Statistical Analysis  The blood gas parameters, concentrations of glucose, lactate, hind limb fluxes of glucose and lactate following labetalol administration were analyzed for statistical significance against changes in control experiments (in the case of maternal bolus and feta bolus experiments) by Multivariate Analysis of Variance (MANOVA) or against pre-experiment values (in the case of experiments in nonpregnant animals) by Fisher's Least Square Difference using SPSS/PC+ program (SPSS Inc., Chicago, IL). Changes in arterial pressure, heart rate and flow values following labetalol administration were tested for significant difference from control values by Fisher's Least Square Difference. Values are expressed as mean ± standard error of the mean, unless otherwise specified. The level of significance used in all cases was equal to 0.05.  48  3. RESULTS 3.1. Development of a Microbore HPLC Assay with Fluorescence Detection for the Quantitation of Labetalol in Biological Fluids.  3.1.1. Optimization of Mobile Phase.  The pH of the phosphate buffer used in the mobile phase had significant effect on the retention time, peak shape and resolution. pH values greater than 4.0 provided broad peaks (peak width >2 min). The optimum pH range was found to be 2.8-3.2. Increasing the molarity of the phosphate buffer (from 1 mM to 20 mM in steps of 5 mM) decreased the peak width and the retention time (Fig 3). The ideal concentration was between 15-20 mM. Acetonitrile and methanol were studied as organic modifiers in combination with pH 3.1 phosphate buffer (0.015 M). Less tailing of labetalol and SCH peaks was observed with acetonitrile than with methanol at similar concentrations. Best results in teens of retention time (4.1 min for labetalol and 6.0 min for SCH), peak symmetry (1.05-1.15) and resolution between labetalol and SCH (>1.5) were obtained with a flow rate of 0.5 mL/min with 44% acetonitrile. 3.1.2. Optimization of Detection of Labetalol  The optimum excitation and emission wavelengths, determined from scanning the trapped labetalol peak, were found to be 196 and 412 nm, respectively. The signal-to-noise ratio was significantly improved by using a 2 mm excitation slit and two 4 mm emission slits. Use of emission cut-off filters  49  FIG 3: Effect of molarity of phosphate buffer in the mobile phase on the peak width and retention time of labetalol on a C-18 column.  50  resulted in reduction in the background noise and the 370 nm filter provided the optimum sensitivity. 3.1.3. Optimization of Extraction of Labetalol  Of the four solvents studied (dichloromethane, ether, ethyl acetate and toluene), only ether and ethyl acetate provided an extraction efficiency of 50% (Fig 4). Further optimizations were done using ethyl acetate. The extraction efficiency improved with an increase in sample pH from 6.0, but no appreciable increase was seen beyond 9.0. The optimum volume of ethyl acetate was 6 mL, which provides a phase volume ratio (organic to aqueous) of 5 and the duration of mixing was optimized at 20 min. Under these conditions, the absolute recovery of labetalol was about 81%. Two-step extractions were also attempted. Extraction with 2 x 3mL ethyl acetate followed by drying of the pooled solvent and reconstitution provided about 85% recovery. Extraction with 6 mL ethyl acetate followed by re-extraction of the ethyl acetate layer with dilute phosphoric acid (0.01, 0.05 or 0.1 M) provided a cleaner chromatogram as compared to single extraction (Fig 5), but the absolute recovery decreased to about 75%. 3.1.4. Extraction of Labetalol from Biological Fluids  The procedure used in the extraction of labetalol from biological fluids is shown in Fig 6. Representative chromatograms obtained from spiked biological fluids following a two-step extraction using 6 mL ethyl acetate and 0.6 mL 0.01 M phosphoric acid are shown in Fig 7.  51  100 -  81.1%  A  84.2%  C  FIG 4: Absolute extraction recovery of labetalol with different solvents. A: Dichloromethane, B: Ethyl acetate, C: Diethyl ether and D: Toluene.  52  A  711-  498-  i98-  255-  255-  LL  0  12  12 1^2^3^i^5^6^  1^2^3^i^5  FIG 5: HPLC Chromatograms obtained from blank sheep plasma following a two-step (A) and one-step (B) extraction. An interfering peak was seen at around 3.5 min following one-step extraction (B).  Plasma (or other fluid) 250 uL + Int. Std.(100 ng) + 500 uL pH 9.5 (1M) buffer + 6 mL ethyl acetate  Discard aqueous layer  20 min extraction 20 min chilling 5 min spin  To the organic layer, add 600 uL of 0.01M phosphoric acid  Discard organic layer  20 min extraction 5 min spin Inject 60 uL  FIG 6: Optimized procedure used for the extraction of labetalol from biological fluids.  ^  B  A 1 000  600 : .  8007  >  E  1  7007.  -  600  -  400  -  200  -  5007 400  >  E  300 2007 100  =t)  -  ^A 0 0 8.0 ^0.0 Time (min.)  ^  .  >  E  Time (min.)  C  D  sea.]  700 :  500.  Sea:  8.0  -  500:  4007  >  E  300':  400 : -  300 :  ^I  -  2007 100  2007 1007  -  0.0  8  .^  1  0.0  Time (min.)^  Time (min.)  FIG 7: Superimposed HPLC chromatograms of blank and spiked (100 ng labetalol) biological fluids (1 Labetalol and 2 - Internal Standard): A: Pregnant sheep plasma, B: Amniotic fluid, C: Fetal plasma and D: Fetal tracheal fluid. -  8.o  55  3.1.5. Validation of Labetalol Assay  3.1.5.1. Precision of Quantitation A typical calibration curve following extraction from sheep plasma is shown in Fig 8. The coefficients of determination were between 0.996 and 1.000 following extraction from various biological fluids. The coefficients of variation over the calibration range are shown in Table 1. The mean intra-sample coefficient of variation over the concentration range in plasma was found to be 2.95 ± 2.76%, while in the case of amniotic and fetal tracheal fluids, the values were 3.85 ± 3.2% and 4.12 ± 2.7% respectively. The coefficient of intra-injection variability (a measure of variability due to the injector) was consistently less than 0.5%  3.1.5.2. Extraction Recovery Studies The mean extraction recovery of labetalol from plasma over the entire range studied (i.e. 0.5-120 ng) was 76.01 ± 2.8%. Extraction recovery, as a function of amount added, is shown in Fig 9. Mean extraction recoveries from amniotic and tracheal fluids over the calibration range (0.5-20 ng) were found to be 70.56 ± 3.76% and 75.12 ± 8.8%, respectively.  3.1.5.3. Minimum Quantitation Limit of Labetalol Assay  The minimum quantifiable limit in this assay was .-- 30 pg of labetalol injected (absolute sensitivity). In terms of amount added (apparent sensitivity), this was equivalent to about 0.4 ng and in concentration terms was equivalent to  2.0  Y = 0.0212 + 0.01558 X  /1  r 2 = 0.9996 -,:  1.5  1.0  0.5  0.0  0^25  50  75  100  125  AMOUNT OF LABETALOL ADDED (ng) FIG 8: A typical calibration curve employed in the quantitation of labetalol (5 120 ng) in plasma (mean ± SD) (n=3).  57  TABLE 1: INTRA-SAMPLE VARIABILITY IN LABETALOL ASSAY AMOUNT OF LABETALOL ADDED (g)  MEAN PEAK AREA RATIO  0.5  0.0931  9.11  2.5  0.1122  5.56  5.0  0.2724  0.83  15.0  0.2347  1.65  30.0  0.4939  1.09  50.0  0.8032  0.67  100.0  1.5845  3.29  120.0  1.8715  2.15  n=4  CV (%)@  125 -  ....--  10-  Y --= -0.12 + 0.7548 X r = 0.9998  0 Li^75  >  -  O  LIJ  50  z 0  25 -  25^50^75  ^  100  AMOUNT ADDED (ng) FIG 9: Extraction recovery of labetalol from sheep plasma as a function of amount added (mean ± SD) (n=3).  125  59  about 1.6 ng/mL (using 250 ptL volume of sample). Fig 10 shows a chromatogram obtained from plasma spiked with 0.5 ng labetalol.  3.2. Development of a GC-MSD Assay for the Identification and Quantitation of 3-APB, an Oxidative Metabolite of Labetalol in the Biological Fluids of Sheep  3.2.1. Optimization of GC Conditions  The splitless mode of injection was used in all cases to maximize sensitivity. The optimum injector temperature was 200°C, with lower temperatures (160 and 180°C) resulting in tailing peaks and higher temperatures (220-250°C) resulting in reduced peak areas. Significant "cold-trapping" effect was obtained with initial oven temperature of 100°C or lower, but initial temperature at 90°C increased the retention time by about 3 min Hence 100°C was chosen as the initial temperature. The oven temperature programming of 15° C/min up to 200°C, followed by 30°C/min to 280°C yielded satisfactory resolution, peak shape and total run time (-11 min).  3.2.2. Optimization of Derivatization and Extraction of 3-APB Treatment with HFBA at 55°C for lh (Wright et al., 1991) provided near complete derivatization. No appreciable increase in peak area was seen beyond lh. Neutralization with pH 6.0 phosphate buffer (0.065 M) yielded a complex chromatogram with a high baseline noise. Use of 0.5 mL distilled water followed by 0.5 mL ammonium hydroxide (4%) (Riggs et al., 1983) provided significant  400  300  200  -  -  -  1  CD . 0  Time (min.)  FIG 10: HPLC chromatogram obtained from blank sheep plasma spiked with 0.5 ng labetalol (the lowest calibration point) and internal standard. (1 - Labetalol and 2 - Internal standard). Approximately 40 pg of labetalol was actually injected.  61  improvement in the form of a cleaner chromatogram even with 2 11,1. injection volume (optimized injection volume = 1p.L). Extraction of 3-APB at different pH conditions of the aqueous phase was studied using 1M carbonate buffer (pH 9.5), 1N, 2N, and 5N sodium hydroxide solutions (200-500 pL volume). Extraction efficiency improved by about 25% with 2N and 5N sodium hydroxide solutions as compared to pH 9.5 buffer or 1N sodium hydroxide (Fig 11). Of the four solvents studied (ethyl acetate, ether, hexane and toluene), ether and n-hexane provided extraction recoveries > 80%. Hexane was used in subsequent optimization studies. Optimal extraction recovery was obtained with 6 mL hexane and mixing time of 5 min. Longer durations of mixing resulted in extensive emulsification, which prevented complete removal of the organic layer. No significant differences in recovery were found between drying the hexane layer under a stream of nitrogen at 30°C and drying under vacuum using SpeedVacR concentrator at ambient temperature. Also, complete drying of the solvent at ambient temperature did not result in any loss of the analyte due to volatilization. The reconstitution volume of 300 p,L, was chosen to increase the apparent sensitivity; volumes less than 300 iiL do not provide any significant increase in the signal-to-noise ratio. 3.2.3. Derivatization and Extraction of 3-APB from Biological Fluids  The optimized procedure for the derivatization and extraction of 3-APB from urine, bile or plasma is shown in Fig 12. The total ion chromatogram of the HFBA derivatives of 3-APB and the internal standard following extraction from control urine spiked with 100 ng each of 3-APB and MPE and their mass spectra are shown in Fig 13. The suggested m/z assignments for some of the fragments are shown in Fig 14.  62  ^ ^ ^ pH 9.5 NaOH NaOH ^ ^ ^NaOH (1M) (1N) (2N) (5N)  FIG 11: Effect of various reagents used for sample pH adjustment (prior to solvent extraction) on the absolute recovery of 3-APB in urine.  URINE, BILE OR PLASMA (1 mL) + 100 ng 1-METHOXY-2-PHENYLETHYLAMINE + 0.3 mL 5N NaOH + 6 mL n-HEXANE TUMBLE MIX (6 min) & REMOVE ORGANIC LAYER DRY HEXANE & RECONSTITUTE IN 0.3 mL TEA in TOLUENE  [DERIVATIZE WITH HFBA AT 55 deg C for 1 hr NEUTALIZE HFBA WITH WATER & AMM. HYDROXIDE (4%) INJECT 1 uL OF ORGANIC LAYER INTO GC FIG 12: Optimized procedure used for the extraction of 3-APB from biological fluids.  64  Fig 13: EI GC-MS following HFBA derivatization of standard 3-APB and MPE (internal standard). A: Total Ion Chromatogram; B: Mass Spectrum of 3-APB Derivative and C: Mass Spectrum of the MPE Derivative.  A  TIC:  Abundance  0100121.D  250000-  3-APE:  200000'STD 150000• 100000  CaFrOO-H  0  C,FrOC-H  -  CH,  CH.  50000-  • 1.,, 8.00 T^8.50  --------------------  B  1/2^->6 00^6.50  7.00  Abundance  Scan 308^(8.219 min): 1'7  91  18000  7.50  16000  -,_____ 9.00  9.50  10.00  0100121.D  132  14000 12000  241  10000 8000 600069  4000  t  0  C  Time ->  44^ ; I ll i  -( J  I  J. .^,^,^I^.  254  14  1  50^100^150^200 ^' -^250^300  Abundance  1  345  169 2000  Scan 293 (8.015 min): 0100121.D 2 4  40000 35000 30000 25000 20000 94 15000 10000  107^134  240 169  5000  226 192^ Time  -  >  50^100  150  200  250  347 300  65  Fig 14: 3-APB Derivative: Suggested m/z Assignments for the mass spectrum  MW=345 0 II C3 F7 C-FiN 169  OTHER IONS m/z 44^CH3-CH-NH2  69^CF3 117^CH=CH-CH2-C6H5 and/or CH 3 -C =CH-C 6 H 5 132^CH3-CH=CH-CH2-C6H5  66  3.2.4. Validation of 3-APB Assay  3.2.4.1. Precision of Quantitation  Fig 15 shows a typical calibration curve (peak area ratio expressed as mean ± SD) obtained following extraction and derivatization of spiked urine samples. Quantitation was found to be linear in the examined range of 0.5-1000 ng 3-APB (0.98 r2 1.00). The intra-sample coefficients of variation (n=5) were found to be less than 12% in all cases over the entire calibration range. Inter-day variability was overcome by running a fresh standard curve every time the samples were analyzed.  3.2.4.2. Extraction Recovery Studies  The efficiency of the extraction procedure was studied at five different concentrations (10, 100, 250, 500 and 1000 ng/mL of urine/bile) (n=3) and the mean extraction recovery was found to be 102.9 ± 4.9% from urine and 98.3 ± 1.45% from bile.  3.2.4.3. Minimum Quantitation Limit of 3-APB Assay  The minimum limit of quantitation was found to be equivalent to about 2 pg of 3-APB (amount injected) or 0.5 ng/mL (in concentration terms).  3.3. Maternal Bolus Studies  10.0 -  A = -2.300, B = 0.957 & r 2 = 0.999  1.0 -  0.1 -  1.0E-2 -  1.0E-3 1.0E 4 ^ 0.1  1.0^10.0^100.0  1000.0  AMOUNT 3-APB ADDED (ng) Fig 15: Typical calibration curve following extraction of 3-APB (0.5 - 1000 ng) from urine (mean ± SD; n=3).  68  3.3.1. Experimental Details Table 2 lists the particulars about the animals used in the study. The gestational age of the ewes in the labetalol group was 132.6 ± 1.6 days while in the control group it was 132.8 ± 1.4 days. The mean birth weight of the operated fetus was 3255 ± 207 g in the labetalol group and 2914 ± 154 g in the control group. The fetal and maternal cardiovascular and acid-base status were within the normal range at the beginning of each experiment and the mean values are shown in Table 3. 3.3.2. Pharmacokinetics Fig 16A shows a typical disposition profile of labetalol in the maternal and fetal femoral artery, amniotic and tracheal fluid compartments of the pregnant sheep (E# 109) following a 100 mg bolus. In the mother, the maximum concentration (at 3 min) was in the range of 1.1-2.3 gg/mL. The weighting factor for the maternal labetalol concentrations was determined from the plot of log variance vs mean concentration (Albert et al., 1974). The slope of the plot was found to be 1.65 and accordingly a weighting factor of [1/C 2 ] was chosen. The disposition in the maternal plasma is best described by a tri-exponential equation of the type Pe - nt + Acca + Be -Bt (except in one animal, where the data is bestfitted by a bi-exponential equation of the form Acat + Be -I3 t) with a very rapid distribution phase. The maternal pharmacokinetic parameters calculated for the individual subjects are shown in Table 4. The mean total body clearance was 135.86 ± 16.93 L/h (30.8 ± 3.83 mL/min/kg) while the mean terminal elimination half-life was 2.79 ± 0.66 h. The apparent volume of distribution calculated as VD area was 477.51 ± 52.98 L (6.48 ± 0.72 L/kg) while the nonparametric  TABLE 2: MATERNAL LABETALOL BOLUS: EXPERIMENTAL DETAILS:  EWE#  BODY^GEST. WT.^AGE (Kg)^(days)  # of Fetus  TERM (days)  BIRTH WT. OF FETUS (grams)  SAMPLES OBTAINED^LACTATE  GLUCOSE  I. LABETALOL EXPERIMENTS: 145  76.4^139  1  144  2364*  MA, FA & TR  NO  NO  109  82.7^127  1  144  4082*  MA, FA, UTV, UV & AMN  NO  NO  091  73.2^142  1  145  3728*  MV, FTV, UV, UTV, AMN & TR  NO  NO  127  85.9^133  2  141  2933*, 3478  MA, FA, UV, AMN & TR  NO  NO  248  80.0^131  2  141  2550*, 2730  MA, FA, UV, UTV & AMN  NO  NO  NTG  40.5^130  1  142  3300*  MA, FA & AMN  YES  YES  105  69.1^131  2  138  3165*, 2535  MA, FA, AMN & TR  YES  NO  137  81.0^129  2  135  3063*, 2874  MA, FA, AMN, & TR  YES  YES  201  73.8^131  2  139  4110*, 3320  MA, FA, UV, UTV & AMN  YES  YES  MA, FA, UV, AMN & TR  NO  NO  MA, FA, UV, UTV, AMN & TR  NO  NO  H. CONTROL EXPERIMENTS: 127  85.9^133  2  141  2933*, 3478  141  75.0^138  2  140  @*, 3092  248  80.0^131  2  141  2550*, 2730  MA, FA, UV, UTV & AMN  NO  NO  NTG  40.5^130  1  142  3300*  MA, FA & AMN  YES  YES  617  82.3^132  2  138  2873*, 2547  MA, FA, UV, AMN & TR  YES  YES  * @  Operated fetus Weight not available; fetus trampled upon by ewe after unsupervised delivery.  70  TABLE 3: PRE-EXPERIMENTAL MATERNAL AND FETAL BLOOD GAS PARAMETERS (mean ± SEM).  MATERNAL ARTERIAL  PARAMETER  FETAL ARTERIAL  A. MATERNAL BOLUS EXPERIMENTS (n=8) pH^  7.481 ± 0.016  7.356 ± 0.017  p0 2 (mm Hg) ^  137.5 ± 6.9  22.3 ± 1.4  pCO 2 (mm Hg)^ 36.1 ± 1.0  50.2 ± 0.7  Base Excess (mEq/L)  4.3 ± 0.5  2.3 ± 0.9  Hemoglobin (g/100 mL)  9.1 ± 0.4  11.1 ± 0.4  02 Saturation (%)  100.7 ± 4.2  57.8 ± 2.2  0 2 Content (mL 0 2/100 mL)  12.7 ± 1.1  9.0 ± 0.9  B. FETAL BOLUS STUDIES (n=5)  pH  7.505 ± 0.011  7.351 ± 0.017  p0 2 (nun Hg)  131.1 ± 5.8  25.4 ± 1.9  pCO 2 (mm Hg)  33.3 ± 1.1  47.3 ± 1.4  Base Excess (mEq/L)  3.8 ± 0.5  1.6 ± 0.9  Hemoglobin (g/100 mL)  9.5 ± 0.6  10.8 ± 0.8  0 2 Saturation (%)  99.3 ± 3.1  65.7 ± 2.8  0 2 Content (mL 0 2/100 mL)  12.8 ± 1.7  10.2 ± 1.1  ^ ^  71  1000  ^— o MA • — • FA o —o AM  0 OD  A - A  13o-o_.  o 100^\  TR  ^ -^ N  ^0 ...6,6.-AAN.^.,-.A-- N N.^A 0^---..,,  A 0-A 0\2  1:1..  ti ^  ^6 i^-0-0 .-4" 14 3 4 • •^ 10^ • ^V / \O 0 A..^  A  O  12  TIME (h) FIG 16: Representative plots of labetalol concentrations in two experiments following a 100 mg maternal intravenous bolus administration. (A - E# 109 and B - E# 201). (MA: Maternal arterial plasma, FA: Fetal arterial plasma, UT: Uterine venous plasma, UV: Umbilical venous plasma, AM: Amniotic fluid and TR: Tracheal fluid).  72  TABLE 4: LABETALOL PHARMACOKINETICS IN PREGNANT SHEEP FOLLOWING MATERNAL BOLUS ADMINISTRATION. A. EWE EWE #^AUC (mg*h/L)  A^C (mg*h-/L)  (1/%1  tv  TBCL^Vdarea (L/hl^(L)  Vd (L)  1VIRT (h)  E145^0.581  0.964  0.322  2.15  172.12^534.50  285.58  1.66  E109^0.624  0.939  0.320  2.17  160.26^500.81  241.16  1.50  ENTG^0.518  0.591  0.347  2.00  193.05^556.34  220.26  1.14  E105^0.997  1.944  0.359  1.93  100.3^279.39  195.57  1.95  E201^0.977  2.073  0.196  3.54  102.35^522.19  217.18  2.12  E248^0.658  0.771  0.52  1.33  151.94^292.19  178.07  1.17  E127^1.409  4.336  0.108  6.42  70.97^657.15  218.41  3.08  MEAN^0.823  1.660  0.310  2.79  135.86^477.51  222.32  1.80  S.E.M.^0.121  0.496  0.049  0.655  16.930^52.975  12.979  0.25  B. FETUS EWE #^8 (1/h)  ty (hi  Cmax (ng/mL)  E145^0.265  2.62  15.4  0.058^0.123  9.91  E109^0.137  5.06  66.4  0.131  0. 5 51  19.23  ENTG^0.232  2.99  23.1  0.087  0.379  16.84  E105^0.238  2.91  42.8  0.157  0.930  15.70  E201^0.160  4.33  36.3  0.136  0.814  13.90  E248^0.130  5.33  24.0  0.117  0.837  17.74  E137^0.140  4.95  40.7  0.107  0.714  ----@  E127^0.212  1.42  21.2  0.102  0.404  7.25  MEAN^0.189  3.71  33.7  0.112  0.594  14.37  S.E.M.^0.019  0.50  5.8  0.011  0.098  1.54  @ Maternal sampling incomplete.  AUMC AUC (mg*h/L)^(mg*h-4/L)  AUC (F/M) %  73  estimate (VD ss ) was 222.32 ± 12.98 L (3.02 ± 0.18 L/kg). Labetalol showed a mono or bi-exponential decline in the fetal plasma, with the terminal elimination rate constant significantly smaller than that seen in the maternal plasma (0.189 ± 0.019 Ir 1 vs 0.31 ± 0.04911 -1 ) and a mean half-life of 3.71 ± 0.5 h, which was significantly higher than that in the mother (2.79 ± 0.66 h) (Student's t-test). The peak concentration in the fetal plasma (33.7 ± 5.8 ng/mL), which is roughly 10% of that in the maternal plasma, was observed at 3 min in all but one animal, where tmax was 10 min. The characteristic features of disposition of labetalol in the fetus are shown in Table 4. The extent of fetal exposure to labetalol following the maternal bolus, as expressed by the mean fetal to maternal plasma AUC ratio was 14.37 ± 1.54%. Labetalol accumulated in the fetal tracheal fluid with concentrations consistently higher than that in fetal plasma after 30 min. The tracheal fluid to fetal plasma labetalol concentration ratio beyond 2 h was — 2 to 4. The drug appeared in the amniotic fluid at a much slower rate as compared to the fetal tracheal fluid, but beyond 4 h, similar concentrations are seen in both the fluid compartments. The drug persisted in these fluid compartments for 24-48 h, while the concentrations in fetal arterial plasma fell below the limits of detection between 6-12 h. Fig 16B shows the uteroplacental and fetoplacental arteriovenous labetalol concentration profile (E# 201). The concentrations in the uterine vein were mostly identical to those in the maternal artery and there was no significant difference between the labetalol AUCs (0.873 ± 0.09 mgh/L in the uterine vein and 0.823 ± 0.12 mgh/L in the maternal artery; paired t-test). In the umbilical vein, the peak labetalol concentration (57.3 ± 14.3 ng/mL) tended to be higher than the peak concentration in the femoral artery (33.7 ± 5.8 ng/mL), but the difference in the peak concentrations as well as the AUCs (0.133 ± 0.02 mgh/L in the umbilical vein and 0.113 ± 0.033 mgh/L in the fetal artery) were not statistically significant (paired t-test).  74  3.3.3. Hemodynamic Effects  No consistent changes in the maternal cardiovascular parameters (mean arterial pressure and heart rate) were seen following labetalol administration. The mean values obtained from all the animals are shown in Fig 17. In two of the animals (ENTG and E105), an initial phase of hypotension (maximum change of about 10 mm Hg in the mean arterial pressure) lasting 1-3 h, with a corresponding increase in the heart rate (maximum change of about 20 bpm) was observed while the other animals did not show any change. There were no apparent changes in the fetal mean arterial pressure and heart rate values in any of the animals following maternal labetalol administration (Fig 18). 3.3.3. Metabolic Effects 3.3.3.1. Changes in Blood Gas Parameters  Significant changes were seen in the fetal and maternal arterial blood gas parameters following maternal labetalol (Fig 19). In the fetus, pH decreased gradually over 2 h and remained significantly below the control values until 12 h. An almost corresponding decrease in the base excess was also seen, with the mean value at 4 h (-4.7 ± 1.34 mEq/L) representing a maximum decrease of 7 mEq/L from the control value of 2.3 ± 0.63 mEq/L. Fetal p02 decreased significantly with the mean value at 3 min, which represents the maximum change, being 18.6 ± 1.53 mm Hg (control value = 22.0 ± 1.39 mm Hg). Fetal pCO2 increased significantly with the maximum change occurring at 30 min (52.8 ± 1.77 mm Hg; control = 50.1 ± 1.0 mm Hg). Fetal 02 saturation fell rapidly by 27 ± 6.5%  75  FIG 17: Effect of a 100 mg i.v. labetalol bolus on maternal heart rate and arterial pressure (mean ± SEM) (n=6). 150 -  cai " ,-0  Lo,' I 1/°1^T - TO i - 1 ,^i  125 -  E -4  ^1 T^j 1\'1 1.\(1)./C6-(I)-(1)-criv °^ ^10-0n., 11 () (3  )  E-4  100  ^  75  111  1\ 0 1 10 1 1  2^5  TIME (h)  110 TV)  100  1  I/ A  90 - 0 80 14 (1)  a.  70 -  60  11- 1-^I O. 1 ?\ 1\1 o I IY) I,o,f T/6kci poi I or?^1 T. /I 7 0] of^ 1. -j) - (1).  .  I  5^8^11  TIME (h)  76  FIG 18: Effect of a 100 mg i.v. labetalol bolus on fetal heart rate and arterial pressure  (mean ± SEM) (n=7). 180  160  I  i  .1 0/Tq l1,0 01 s ,01 /1\1101,i o , cf"\i-Jo_i 0,(1).1 rol  10(111^  E ^140 -1  120  2^5  111^TI  8^11  TIME (h)  60  50  o'N 0\1 1 1^I 1^ /Troi °I\,11/7\-[ I (I) 0.° 11°1\0/1 \10  40 Cf)  30  I^1 1.Y  T  7 011°1^  T-Ti  2^5^8^11  TIME (h)  77  FIG 19: Fetal and maternal arterial blood gas parameters (mean ± SEM) before and after a 100 mg maternal intravenous bolus administration .of labetalol. (MA: Maternal arterial blood and FA: Fetal arterial blood). Asterisks (*) denote. significant difference from control values.  • ••  7.800 -  • FA  180-  A 7.500 •  7.400 -  7.300 7.200  O  t;•.0 coo°  •  •  O  %Ise=  -•-- 11?  1  f. 100  11■••• ^  25 23  •  ° A^*Tr^ •144til^I  19^  15  110 -  0 too 80 •  Cf1  50  (5" 30  TIME (h)  •, .  ;0404111 . -6-  -^0^2^6^8^11^24^  ^  •  •L-^L •  Yr.^  T  •  •  -1 0^2^6^8 .^11  TIME (h)  ^  24  78  following drug administration and stayed significantly decreased for 4 h. Even at 24 h, the fetal 0 2 saturation was lower, although not significantly, than control. The calculated fetal 0 2 content decreased by 28.2 ± 8.1% following labetalol administration and remained significantly decreased until 5 h (data not shown). In contrast, the maternal pH and base excess showed only moderate decreases. The maximum decrease in pH was by 0.015 unit (cf 0.1 unit in the fetus) and base excess by 4.3 mEq/L (cf 7.0 mEq/L in the fetus) and the values were back to normal by 4h, unlike that in the fetus. A gradual but significant decrease in pCO 2 over 4h was observed (35.8 ± 0.8 to 30.2 ± 1.7 mm Hg) as well as a trend towards increase in p0 2 (not statistically significant) over 2-10 h. Maternal 0 2 saturation and content remained unchanged (data not shown). 3.3.3.2. Blood Glucose and Lactate Levels  Fig 20 shows the changes in lactic acid and glucose concentrations in fetal and maternal arterial blood and in the amniotic fluid. Significant lactic acidosis occurred in both the fetus and ewe following labetalol administration, but the effect was much more pronounced in the fetus and lasted longer (12-24 h). Accumulation of lactic acid, persisting beyond 24 h, was also seen in the amniotic fluid. The changes in the maternal lactate concentrations were characterized by a rapid increase in the first hour, with the peak concentration, which was attained at 60-90 min, remaining essentially unchanged till 5-6 h and then a return to baseline concentrations by 12 h. In the fetus, the lactate concentration continued to rise for about 4 h and then reached a plateau. The rate of decline in the fetus was much slower than that in the mother and baseline values were not reached before 24 h. The amniotic fluid lactate concentrations in the first 2 h show a distinct lag in comparison to the fetal blood lactate concentrations and peak concentrations were  79  o-o MA^• ^ • FA  A-A  AM  U  z O CD  U) O  -1 0  2  5^8^11  24  48  TIME (h) FIG 20: Glucose and lactic acid concentrations (mean ± SEM) before and after a 100 mg maternal intravenous bolus administration of labetalol. (MA: Maternal arterial blood, FA: Fetal arterial blood and AM: Amniotic fluid).  80  reached by 6 h. Beyond 10 h, the decline in the amniotic fluid lactate was much slower than in the fetus with the mean 24 h concentration being roughly twice the fetal arterial concentration. Blood glucose levels also rose in the ewe and fetus following labetalol dosing, but in this case, the effect was more pronounced in the ewe and the amniotic fluid glucose concentration showed only a marginal increase. In contrast to the situation with lactate levels, the changes in the fetal glucose concentrations paralleled those in the ewe. In all the animals studied, the fetal blood gas parameters and lactate concentrations returned to control values between 24-48 h. The control experiments did not show any change in fetal or maternal lactate and glucose concentrations (Fig 21) nor any consistent deviation in any of the blood gas parameters (Fig 22). 3.4. Labetalol Bolus Studies in Non-pregnant Sheep 3.4.1. Pharmacokinetics  The mean weight of the ewes (n=5) used in the study was 74.9 ± 4.0 kg. The disposition of labetalol in sheep arterial plasma following a 100 mg i.v. bolus administration is shown in Fig 23. The weighting factor chosen (Albert et al., 1974) for pharmacokinetic fitting of plasma labetalol concentrations was 1/C 2 . The decline of labetalol concentration was best described by a triexponential equation of the type Pert + Ae - at + Be - 13t. The estimates of the pharmacokinetic parameters obtained by nonparametric analysis of the arterial plasma concentration data are listed in Table 6. The total body clearance, elimination half-life and  81  4.0 -  E 3.0 -  0-0 FA  AA  z  O  u 2.0  rhAA  1  A-A  DAM  MA  _1 1  1 1 1  UTJ  O U  1.0 **^- 0 - 0 0  0.0  - 0- o D-0  -1  2  O  ^  8  5  3.0  1  2.0 0 1.0 -  0.0 ^ 1 -  - — A A  2  A  A  - ^  I  A^A  5^8^11  TIME (h) FIG 21 Glucose and lactic acid concentrations (mean ± SEM) before and after a 20 mL control saline administration. (MA: Maternal arterial blood, FA: Fetal arterial blood and AM: Amniotic fluid).  82  0 ^ o MATERNAL ARTERIAL  I  1  0\ 1(2430._____ 0,„„  7.500^  ^  • ^ • FETAL ARTERIAL  0  0^  11  25  cr) c/)  •  20 -  1^I^1  •—__  /1\  tn 0:1  11  5  TIME (h)  60  ▪  50  •  •  T^  •  1  ^  3^5^7  TIME (h)  •  40 C\2 30  20  c9.-^  9,c)  - 1^1^3^5  9  11  TIME (h)  FIG 22: Fetal and maternal arterial blood gas parameters (mean± SEM) following a 20 mL saline administration (control experiment).  ^  11  1000  100  io 0  6  10^12  TIME (h) FIG 23: Disposition of labetalol in adult sheep plasma following a 100 mg i.v. bolus (mean ± SEM).  84  TABLE 5: COMPARISON OF THE PHARMACOKINTETICS OF LABETALOL IN PREGNANT SHEEP WITH REPORTED VALUES IN PREGNANT WOMEN PARAMETER  ^  TOTAL BODY CL (L/h)  PREG. SHEEP^PREG. WOMENb  ^  ^  ^  (71.0-193.1)  ^  129.6  (92.4-188.4)  2.8  2.5  (1.9-6.4)  (1.9-3.3)  222c  274  (196-286)  (250-557)  0.14d  0.49e  (0.10-0.19)  (0.19-0.81)  TERMINAL ELIM ty2 (h)  VD ss (L)  135.9  FETAL DRUG EXPOSURE (ratio)  a Mean values; range is given in parentheses. b Pre-eclamptic women; from Rubin et al., 1983. c Non-compartmental determination. d Fetal/maternal plasma AUC ratio. e Cord blood/maternal plasma concentration ratio at delivery; from Michael, 1979.  85 TABLE 6: Pharmacokinetics of labetalol in pregnant and non-pregnant sheep.  (mean ± SEM; n=5)  PARAMETER  NONPREGNANT SHEEP  PREGNANT SHEEP  CL (ml/min/kg)  29.00 ± 2.67  30.80 ± 3.83  t y4 (hr)  2.41 ± 0.30  2.79 ± 0.66  MRT (hr)  1.85 ± 0.09  1.80 ± 0.25  Vd ss (liter/kg)  3.22 ± 0.31  3.02 ± 0.18  Vd area (liter/kg)  6.19 ± 1.13  6.49 ± 0.72  86  volume of distribution were not significantly different from the estimates obtained from pregnant sheep (two-tailed two sample t-test).  3.4.2. Metabolism  3.4.2.1. Conjugative Metabolism  Excretion of labetalol in urine and bile as free drug (unchanged labetalol) and as glucuronide and sulfate was studied to assess the contribution of these pathways in the overall elimination of labetalol. Cumulative urinary excretion of unchanged labetalol accounted for 1.61 ± 0.38% of labetalol dose, while the glucuronide and sulfate conjugates accounted for 11.46 ± 2.82 and 1.47 ± 0.74% of the dose respectively (Fig 24). Both glucuronides and sulphate conjugates of labetalol were also detected in the bile and their concentrations along with that of unconjugated (free) labetalol are shown in Fig 25.  3.4.2.2. Oxidative Metabolism  Plasma, urine and bile samples were also analyzed for the presence of 3APB, an oxidative metabolite of labetalol. While 3-APB could not be detected in the plasma samples, its presence in urine and bile samples was established by GCMSD using an authentic standard, as described previously. The total ion chromatogram and the mass spectrum corresponding to the peak at 8.22 min, which is identical to that of authentic standard (Fig 13B), are shown in Fig 26. The urinary excretion profile of 3-APB is shown in Fig 27 (A & B). The cumulative excretion of 3-APB (Table 7) over 24 h was 43.76 ± 16.4 lig, representing 0.044 ± 0.016% of the dose. The terminal slope of 3-APB urinary  87  A 15.00  I^1^1-/-/*  I^•^I  10.00 -  1^I  •  5.00 -  0.00  I  •  •  ITV  •/1  MIN FREE^0 GLUCURONIDE^[mil SULFATE  B  1E4tTi)  -r"  8000 6000  0 4000  T  T  2000  12  24  TIME (h) FIG 24: Cumulative urinary excretion of labetalol and its conjugates (mean ± SEM). A: Cumulative amount excreted and B: Percentage of labetalol dose recovered in urine as free drug and conjugates.  FREE,^INII FREE+ GLUCURONIDE & = FREE + SULPHATE 12.00 '1 :--■ W) 8.00 7 0 E:-: i-z:C 2  E-1^4.00  z  W 0  z  0^ 0  0.00  [  .75^2^5^9  TIME (h) FIG 25: Concentrations of labetalol and its conjugates in adult sheep bile following a 100 mg i.v. bolus (E# 617).  89  TIC:^0101006.D  Abundance  120000  -  100000-  *  80000-  60000-  VeN6  40000  20000-  ,^, 0 M/Z^->^6.00^7.00^8.00^9.00^10.00^11.00 Abundance  Scan 308  (8.222 min):  0101006.D  91 117  12000-  10000132  44 8000-  6000-  4000-  241  69  169  2000-  1 1  1  254  345  , 1 II^. 1^. i ^1 . ^-. I. ,^. .^ l„ , , ^, Time ->^50^100^150 200 250 300 FIG 26: Total ion chromatogram (top panel) of urine sample obtained from E#105 (nonpregnant) following a 100 mg labetalol bolus. Asterisk denotes the 3-APB peak and the EI mass spectrum corresponding to that peak (bottom panel).  ^0  90  A  "  • •  ---^  40  /".  •  •  •  •  •f/  30 -  r/'  20^•  1  10 -  /1  ^0 • 0^3  6^9  12  18  TIME (h)  B  1 \  01 0 T  O 1 0.1  0^3  6^9^12^18  TIME (h) FIG 27: Excretion of 3-APB in adult non-pregnant sheep urine following a 100 mg labetalol administration (mean ± SEM; n=5). A: Cumulative excretion over 24 h; B: Excretion rate plot.  TABLE 7: URINARY EXCRETION OF 3-AMINO-1-PHENYL BUTANE LABETALOL 100 mg BOLUS: NON-PREGNANT SHEEP  1  Lin E/248.0. TIME (h) 0-0.5 0.5-1.0 1.0-1.5 1.5-2.0 2-3 3-4 4-5 5-8 6-8 8-10 10-12 12-24  CONC. VOLUME AMT EXC (ug) (mL) Ing/mL) 4.39 15 0.07 1138.49 16 18.22 1117.41 15 16.78 958.51 15 14.38 895.00 30 20.85 241.30 30 7.24 103.03 40 4.12 31.33 52 1.63 22.15 2.10 95 13.40 150 2.01 8.99 140 1.26 6.15 800 4.92  CUMULATIVE AMT. EXC. tug):  CONC. VOLUME AMT EXC (ug) (ml) (og/mL) ND 0 22 1.18 59.02 20 50.00 1.05 21 1.38 38.94 35 85 22.79 1.94 77 0.93 12.08 10.80 75 0.81 8.50 120 0.78 3.51 320 1.12 ND 330 0 ND 325 0 ND 225 0  ^  9.17  ND:^Not detected (< 1 ng/mL) ^ NS:^No Sample  CONC. VOLUME AMT EXC (ng/mL) (ml) (u9) 70.73 18 1.27 825.43 12.38 15 782.94 14 10.98 871.82 8.08 12 499.42 30 14.98 252.32 30 7.57 180.83 30 4.82 118.87 25 2.92 57.98 80 3.48 18.98 1.37 72 11.35 0.98 BB 8.01 480 2.78  CONC. (ng/mL) 24.05 68.79 284.45 273.31 272.77 250.85 167.80 85.18 32.04 12.58 4.53 4.46  VOLUME AMT EXC (ug) (ml) 12 0.29 3 0.20 18 4.78 8 2.19 4.91 18 15 3.78 15 2.52 14 1.19 48 1.54 70 0.88 88 0.39 380 1.69  ^  24.32  MEAN CUM. AMT. EXCRETED  DILEVALOL 25 mg BOLUS: NON-PREGNANT SHEEP E#105RR  E#248RR  ^  TIME (h) 0-0.5 0.5-1.0 1-2 2-3 3-4 4-5 5-6 8-8 8-10 10-12 12-24  CONC. VOLUME AMT EXC CONC. VOLUME AMT EXC (ng/mL) (ug) ImL) (ng/mL) (ml)^(ug) 1.11 20 0.02 4.57 58^0.27 564.83 19 10.73 14.48 84^1.22 208.75 28 5.79 2.40 110^0.28 91.70 27 2.48 2.19 100^0.22 45.36 1.50 1.93 33 110^0.21 35.56 0.71 2.51 20 112^0.28 23.64 37 0.87 1.83 125^0.23 14.75 90 1.33 ND 255^0.00 9.81 82 0.80 ND 285^0.00 8.35 107 0.68 ND 330^0.00 3.50 580 1.96 ND 2149^0.00  CUMULATIVE AMT. EXC. (ug): 1 .1.11687.1  E#316  E#617  E#543  ^  2.69  CONC. (ng/mL) NS NS NS 182.47 180.49 127.72 91.53 49.84 20.00 13.26 7.80 1.44  VOLUME AMT EXC tug) (mL) NS NS NS 20 3.85 3.81 20 3.32 28 30 2.75 40 1.99 84 1.68 105 1.39 115 0.90 825 0.90  92  excretion rate plot (0.123 ± 0.14 h I) was not significantly different from that of -  labetalol in plasma (0.134 ± 0.13 h I) (paired t-test), implying that the elimination -  rate constant of the metabolite (km) is much higher than that of the parent drug (K) (Gibaldi and Perrier, 1982). Application of the method of residuals provided an estimate of the apparent elimination half-life of the metabolite equal to 13.5 ± 3.8 min Evidence for glucuronidation and sulfation of 3-APB was also obtained (Fig 28). While only the glucuronide of the metabolite could be detected in urine samples, both glucuronide and sulfate were seen in the bile samples. Surprisingly, 3-APB was also detected in the free form in the bile samples. Urine and bile samples were further analyzed for the presence of the hydroxylated form of 3-APB and benzylacetone (Fig 29), which could result from oxidation of 3-APB, but neither of these compounds could be detected in any of the samples.  3.4.3. Hemodynamic Effects The observed changes in the hemodynamic parameters (heart rate, mean arterial pressure, femoral blood flow and vascular resistance) produced by labetalol administration are shown in Fig 30. A significant decrease in vascular resistance and increase in femoral blood flow were seen following the administration of labetalol. These vascular changes were associated with significant hypotension and the development of tachycardia. Maximum changes were seen between 1.5 - 2 h following drug administration. The mean arterial pressure remains decreased beyond 2 h, but the change was not statistically significant. 3.4.4. Metabolic Effects  93  ▪ =FREE & M TOTAL (free+glucuronide)  A  15012090  z 0  -  60 30  0  0  9  ^MI FREE, Ci FREE+GLUCURONIDE &^FREE+SULFATE  ir3 tbo  B 500 400 -  z  0 300  C-4 CD 0 CD  1111  100 -  2 0^MIN • 0.75^2.0^5.0^9.0  TIME (h)  FIG 28: Excretion of 3-APB conjugates following a 100 mg labetalol administration. A: Urine (amount) (E#105) and B: Bile (concentration) (E#248).  LABETALOL OH H2 N OC  HO  N-Dealkylation  3-AMINO-1-PHENYL BUTANE  CH 3  BENZYLACETONE  3-AMINO-1-(4-HYDROXYPHENYL)BUTANE  OH  CH  CH 3  FIG 29: Oxidative metabolism of labetalol. (Not exhaustive; dotted lines indicate potential pathways; structure of amphetamine is included for the sake of comparison; partly from Gal et al., 1988).  250  125  1-0, 1 IT^1, 0 1 1 TJ/1 ifl\c( ri I  :1.■^115  -  105 E—^95 (24  0  /0 1 1\ * \I *T * * ..--, ri 200^0 III 1`0-0. 0  .---...  .--^I I 1 I 150^  O  85 75  —1^2^5  ^  •  \T T  T T 0.0• 0- 0  I  1  I  100  50  11  '0 ' 0 '0-0  TIME (h)  —1  5  I  1 9.6.6 '0.()•0  o 1 1 "9^-O `o, -  ^  TIME (h)  11  110  Cm) U ,._,  tu) 100 X^T^ 0^  0  <•.4 . 0.75  E., E E^i^ ci) E 90^ v) w4 -^9^ 1^ 1 1 Ti?‘ (2 . 0,1 1,^ 10^C4 ,,,, 0.50 c4^ °I\T^po^ T ,6 9 \0I/I _6/1^ 80 Y, J).0/1\o' 1 i 1 T.-61\j) ^1 1 1^1^1^ ^,_C4 -cc 40 cn^ I I\ 0 9.•g I^1^ E i w*^ 0 - 0.25 I^ r24 70  l11  2.,^ 60  <C--->  —1^2^5  TIME (h)  11  FIG 30: Hemodynamic changes following labetalol bolus in adult nonpregnant sheep (mean ± SEM) (n=5). (Asterisks denote significant difference from control values).  11  96  Femoral arterial and venous blood gas changes induced by labetalol are shown in Fig 31. The marginal but significant decrease in pH and the more conspicuous decline in base excess suggest the development of metabolic acidosis over the first six hours following labetalol administration. The mean blood base excess value at 3 h, which represents the maximum change, shows a decrease from mean control value by 3.8 mEq/L in the arterial and by 4.7 mEq/L in the venous blood. Significant changes were also seen in arterial pCO 2 , which showed a maximum decrease of 13.9 ± 2.3% and venous p0 2 , which increased by 52.4 ± 6.8%. The calculated arterial and venous oxygen contents are shown in Fig 32. Both the arterial and venous oxygen contents show an increasing trend, but only the venous oxygen content changes were significant. Labetalol also causes significant hyperglycemia and lactic acidosis (Fig 33). The net uptake/output of labetalol, glucose and lactic acid by the hind limb was calculated from the respective arterio-venous concentrations and the femoral blood flow data using the Fick principle, i.e., [(CarteriarCvenous).* flow] and the results are shown in Figs 32 and 34. There was a marked and significant uptake of labetalol in the initial 15 min after drug injection, which however decreases rapidly and beyond 2 h, there was no significant uptake or release. There was also net uptake of glucose over the initial 4 h, after which there was a reversal of flux. In contrast, the venous lactic acid concentrations were consistently higher than or equal to that in the artery (Fig 33), so that there was sustained lactate release from the hind limb over 12 h (Fig 34). The total amount of lactate released was calculated as the AUC of the lactate output curve and averaged 6.25 ± 1.35 g (0.07 ± 0.015 M). The net uptake of oxygen before and after labetalol administration is shown in Fig 32. A significant increase in oxygen uptake across the hind limb at 15 min after the administration of labetalol was observed.  97  oARTERIAL  ^ •VENOUS  150 0^  r  0—o  9^1  0  o 9 01  e  Tr• •  ‘••••• •-•  • --•  30  \I  1\1 1\ te:t-i *** )  XI  8^11^24  TIME (h) FIG 31: Femoral arterial and venous blood gas changes following labetalol bolus in adult nonpregnant sheep (mean ± SEM) (n=5). (Asterisks denote significant difference from control values).  98  •—•  o-o ARTERIAL  VENOUS  20.0  41511Aei  0  •  a 15.0 m  i 6OTTT * ' 1 - 00,, T T^ ii c:6 0- t.^1 1 1 C)  (II i^Oi E--4 O • 0 ie•^ 11\^T. 0^T r • 0 0 C) -‹ I^'4? JA, T (Pi e/ 10.0 - I^.--•.1. ^ \r• ol-d .10 1. ^ I^•1 .I^  Cx1  ^1  .  O  .  ^  O  -  5.0  7^11^24  TIME (h)  B  1250  1000  750 11.  500  1\ ^r^T CL) ?‘ T/ C1)  C) >-+  250  0  T  ('?.  r T  .  0  0  I  0 7  11^24  TIME (h) FIG 32: Effect of labetalol on oxygen homeostasis in adult nonpregnant sheep (mean ± SEM) (n=5). (Asterisks denote significant difference from control values).  ^  99  0 ^ °ARTERIAL ^• ^ •VENOUS  •• N •t * \I .  p • , 'y** *gy0 lic\ i 4.0^I/0 Ttic^  *^0 .17 *\  ^2.0^  ''N.10  i 0  TIME (h) FIG 33: Labetalol bolus in adult nonpregnant sheep: Femoral arterial and venous blood glucose and lactate concentrations (mean ± SEM) (n=5). (Asterisks denote significant difference from control values).  100  TIME (h) 100  50  o^I o0  -50 Cr) 0 -100  -150 -1 0  8^11^24  TIME (h) 3.00  2.00  1.00 0  CT-1  0.00  Gn 2^3  4  TIME (h)  FIG 34: Changes in arterio-venous fluxes following labetalol administration in adult nonpregnant sheep (mean ± SEM) (n=5). Positive values indicate net uptake and negative values indicate net release from the hind limb. (Asterisks denote significant difference from control values).  101  3.5. Dilevalol Bolus Studies in Non-pregnant Sheep  Since these studies were conducted in only two animals, no statistical analysis was performed.  3.5.1. Pharmacokinetics  The disposition of dilevalol in arterial plasma (Fig 35) was best described by a triexponential equation. As a default, the concentrations were weighted by 1/C 2 . The estimates of the pharmacokinetic parameters for the two animals are listed in Table 8.  3.5.2. Metabolism  The cumulative urinary excretion of free dilevalol, glucuronide and sulphate conjugates are shown in Fig 36. The cumulative amounts of free drug, glucuronide and sulphate excreted over 24 h following drug administration in E# 105 were 0.47, 1.23 and 0.28 mg, respectively, representing 1.9, 4.92 and 1.12%, respectively. The corresponding values in E# 248 were 0.15, 1.09 and 0.20 mg, respectively, representing 0.58, 4.34 and 0.79%, respectively. The concentrations of free drug and conjugates in bile are shown in Fig 37. The plasma, urine and bile samples were analyzed for 3-APB, before and after incubation with P-glucuronidase and arylsulfatase preparations. The metabolite was detected only in the urine and bile samples. The cumulative amounts of 3-APB excreted in the urine over 24 h following dilevalol administration (E105RR and E248RR) are shown in Table 7. The cumulative amount recovered over 24 h in E#105 and E#248 were 26.87 and 2.69 pg,  0 ^ 0 E#105^• ^ • E#248  T I M 1-1 ( h) FIG 35: Disposition of dilevalol in adult nonpregnant sheep arterial plasma following a 25 mg i.v. bolus.  103  Table 8: Pharmacokinetics of Dilevalol (RR-isomer of Labetalol) in Adult Non-pregnant Sheep Following a 25 mg i.v. Bolus Administration.  PARAMETER  E# 105  E#248  Terminal elimination half-life (h)  5.87  3.70  AUC (mg*h/L)  0.162  0.141  AUMC (mg*h 2/L)  0.276  0.450  Total body clearance (mL/min/kg)  42.74  37.10  Mean residence time (h)  1.75  3.20  Vd ss (L/kg)  4.36  7.12  Vdarea (L/kg)  21.70  11.89  •  • FREE^0 ^ 0 SULPHATE^0 ^ A GLUCURONIDE  1250 O  0  O  1000  0 0  750  O  500  0  •  • ••  •^ •  A  250  6  ^ ^ 9 12  TIM ^ (h) FIG 36: Cumulative urinary excretion of dilevalol as unconjugated (free), glucuronide and sulphate in an adult nonpregnant sheep (E#105).  FREE  FREE+GLU^FREE+SUL  3.0  t=1° 2.0  a  7' 1.0  O U  0.0  2.5  ■ n^_, F6a^, _ ,.  4.0^6.0^10.0  TIMH (h) FIG 37: Concentrations of unconjugated (free) and conjugated dilevalol in bile of adult nonpregnant sheep following a 25 mg i.v. bolus (E#105).  106  respectively, representing 0.11 and 0.011%, respectively. The concentrations of free 3-APB and its conjugates in bile samples are shown in Fig 38.  3.5.3. Hemodynamic Effects  The changes in heart rate, mean arterial pressure, femoral vascular resistance and blood flow in E# 105 following a 25 mg dilevalol bolus are shown in Fig 39. The maximum changes seen include a 34.9% decrease in mean arterial pressure, 35.3% increase in heart rate, 66.7% decrease in femoral vascular resistance and a 150.6% increase in femoral blood flow.  3.5.4. Metabolic Effects  The changes in arterial and venous blood gas parameters (E#105) following dilevalol bolus are shown in Fig 40. The arterial pH and base excess values show an initial trend towards a decrease while no apparent changes were seen in arterial and venous p02 and pCO 2 . The arterial and venous blood glucose and lactate concentrations (E# 105) are shown in Fig 41. Dilevalol bolus appears to cause hyperglycemia and lactic acidosis. The area under the arterial lactate concentration curve is equal to 59.9 mMh. No marked changes were seen in the arterial and venous oxygen contents (Fig 42). The glucose, lactate and oxygen fluxes across the hind limb are shown in Figs 43 and 42. The glucose flux shows.a slight increase in hind limb uptake, which was maintained almost throughout the sampling period. A net release of lactate was seen in the control period. Following dilevalol administration, there was a reversal of flux, indicating net uptake, which continues for up to 150 min From 3 to 8 h, the net release of lactate from the hind limb was 2.13 g (23.67 mmol) (the area under the negative  A I^I FREE^PI; FREE + GLUCURONIDE  60.0 -  45.0  1'1  14  1,4 30.0 -) 0 ^15.0  0.0  B  P•4 P 4  ► •44 ► • • 14 • 14 • 4 ► • ►•► ••4 11 4 ► •4 14 • 14 • 11 •  1  P• 4  0  14 ► • •• • • ► • ► •44 ► 1•• 104 •  3  ► • • 14 •1 ► • 04 • ►•• •  ►  •  • • • •  14 ► •• • 14 • • 14 • ► 4 • •4 ► • • ► •4 14 ► •44 ► • • P4 • 14 •4 ► • ► • 11  • 14 •4 ► • 14 • 14 1. • 14 • • •4 ► •4 ► ►• • ► • • ► • ►• • • 14 • •  14 • 14 • 14 • • ► • ► • 04 • • 14 • ► • ► • ► • ► • ► • ► 4 4  11 ► • 14 ► •• 1•4  •  6  TIME. (h  ► • 4 4  P4  1"1 • 14 ►• •4 ► • ►• ► •4  ►4 •4 1• ► 4  ►• •  ►• •  •  ►  ► •4 •4 ► •  ►•  • 14 •• • 14 •4 ► • 14 • 14 • 14 4 ► 4 • • 14  ► • • ► •44 ► ► 4 ► 1 ► • • ► •  ► •  ► • •4 ► • • • • • ► • • • • 14 • 104 ►• • P4 • 14  '4•  4 14  • 14 •4 ► •4 ► • • 04 • 14  1^18  . FREE^I I FREE + GLU^CZJ FREE +SUL  400 -  300 za,0 200 z 0 100 -  0  I 2.0  4.0^6.0  171 10.0^24.0  TIME (h) FIG 38: Excretion of 3-APB and its conjugates following a 25 mg dilevalol administration in an adult nonpregnant sheep (E#105) A: Urine (amount) and B: Bile (concentration).  107  108  0-0 HEART RATE ^• — • ARTERIAL PRESSURE 125  125 to '  t=1  0.^/ 0 0.0.o. 0.0 \  100^0  100^r  0-0.0.0-0- 0  W^n  • • / •=4^ •  0•0-0 0 •-•-•\ igi, : ^cn cn \ /^‘0 •^p:1 • C.1 75^ -g---  • 1:^0-•-• •" 0  I •. W":,  Os^WO -  •  75 • \ / e,_,.... f24^ •^  50 -1  50  11  1500  /  •  • S.  •- • -•-•••  \ iv.  M " 10 0 -  o  '•-•- •, •  M 50 0  0  -1  2^5^8  11  2.00 C.) 1.50  124 tO  (/)  O  1.00 0  E 0.50  0.00  -1  .  .o^0 " , 0-0-0-0-0  2^5  TIME (h)  0 -0 °.°  -0. 0  /  11  FIG 39: Hemodynamic changes in an adult nonpregnant sheep (E#105) following a 25 mg dilevalol bolus.  0 — 0 ARTERIAL  • — • VENOUS 175  125  75  2  5^8 TIME (h)  5^8  ^ ^ 11  24  TIME (h)  FIG 40: Femoral arterial and venous blood gas changes in an adult nonpregnant sheep (E#105) following a 25 mg dilevalol bolus.  11  24  110  0 ^ 0 ARTERIAL  ^  • ^ • VENOUS  5.0  4.0  3.0  2.0  6.0  4.0  2.0  0.0  —1^2^5  11  24  TIME (h) FIG 41: Femoral arterial and venous blood glucose and lactate concentrations in an adult nonpregnant sheep (E#105) following a 25 mg dilevalol bolus.  111  0 ARTERIAL  0  15.0  • — • VENOUS  64%000 0 0 0 0 Z 0 Z 0 c:) 10.0 14, 0  ••ai `^\^Aft \ • •w• •  C.D > -1  o  '  er 5.0 -1  5^8  2  11  ^  24  TIME (h) 500 0 400 0 0  0  300  0 O  200 0  100  —1  ^ ^  2  5^8  ^  11  24  TIME (h)  FIG 42: Effect of dilevalol on oxygen homeostasis in an adult nonpregnant sheep (E# 105).  112  100  5 5  -  0^0 0  cbo  O  92D  U) 0 u -5 C  O -10 -1  ^// 5^8^11^24  2  10-  •  5 5  -  0  •  I•\ `• • .  tie^  E-1  -(C  u  -5 -  • 2  z ^ // 5^8^11^24  TIME (h) FIG 43: Changes in arterio-venous fluxes following dilevalol administration in an adult nonpregnant sheep (E#105). Positive values indicate net uptake and negative values indicate net release from the hind limb.  113  flux curve from 3 to 8 h). The net oxygen consumption by the hind limb, as shown by the oxygen flux, roughly doubles following dilevalol administration (Fig 41). 3.6. Labetalol Infusion in Non-pregnant Sheep  3.6.1. Disposition of Labetalol  The total amount of labetalol administered at the end of infusion in each of the five experiments was 280 mg. The arterial plasma labetalol concentrations during and after the infusion are shown in Fig 44. The slope of the straight line joining the concentrations from 90 min to 6 h (-0.016 ± 7.98E-3) was not significantly different from zero (one-sample, two tailed t-test; p<0.05), indicating that labetalol concentrations remained at steady-state over that period. The mean labetalol concentration during the steady-state ranged from 317.67 to 894.64 ng/mL (mean = 454.19 ± 111.05 ng/mL). The clearance of labetalol at steadystate, calculated as the ratio of infusion rate to steady-state plasma concentration, was 17.62 mL/minfkg. 3.6.2. Hemodynamic Effects  The changes in mean arterial pressure, heart rate, femoral vascular resistance and femoral blood flow before, during and after labetalol infusion are shown in Fig 45. There was prolonged hypotension associated with labetalol infusion and the change was significant from 4 h from the start of the infusion until the end of the experiment, at 10 h post-infusion. The hypotension was accompanied by transient, but significant, tachycardia, a significant decrease in  1000  o  qi) 6 69-ii I J. ----  .  -  n ... 0 -  0  \,  ctSi...-,.6 16-6  11^T -  0 I  1  0^3  6^9^12^24  TIME (h)  FIG 44: Arterial plasma labetalol concentrations following combined bolus (100 mg) and infusion (0.5 mg/min for 6 h) in adult nonpregnant sheep (mean ± SEM). (Solid line indicates infusion period).  TIME (h)  TIME (h) FIG 45: Hemodynamic changes during and after continuous infusion of labetalol in adult nonpregnant sheep (mean ± SEM) (n=5). Solid line denotes infusion period and asterisks denote significant difference from respective control values.  116  femoral vascular resistance and a significant and prolonged increase in femoral blood flow. 3.6.3. Metabolic Effects The arterial and venous blood gas changes are shown in Fig 46. The base excess and pCO2 showed significant decreases while the pH showed a decreasing trend. The arterial and venous oxygen contents are shown in Fig 47. While a trend towards an increase is seen in both the arterial and venous oxygen contents, only the latter was statistically significant. Significant hyperglycemia and lactic acidosis were also observed (Fig 48). The calculated arterio-venous fluxes of glucose and lactate across the hind limb are shown in Fig 49. A net uptake of glucose over the initial 2 h of the infusion is evident from the significant positive flux which, however, decreases quickly. The flux reversed before the end of infusion, signifying net release of glucose, and the negative flux was maintained for up to 18 h post-infusion. Similar changes were also seen in the case of lactate, which shows sustained net output from the hind limb beyond 2 h. The calculated area under the lactate flux curve, which corresponds to the net amount of lactate released from the hind limb is equal to 9.2 ± 3.12 g (0.102 ± 0.035 mol) of lactic acid. The mean oxygen consumption by the hind limb almost doubled immediately following labetalol administration, but the change was not significant (Fig 47). 3.6.4. Intra-arterial Norepinephrine Studies  Intra-arterial administration of NE caused decreases in femoral blood flow in a dose dependent manner (Fig 50), but the NE dose-response curve was not  ^  ▪^  0 ^ 7.500 -  °ARTERIAL  L I T _ z j .,..--VT^ Jeri)),^1 1 I f) 6 , 0 o____,__ 1, 1 0^ \ , •0, _,10,0^,•__,_ \o,•1 ,- I, T:1•, __• —T1 ill I ri 1 I i  if  0  QD e 7.450 -  Q-4 7.400 -  7.350  0  \I^ 0  •  •VENOUS  150.0  ,o  O t?D 120.0  /I^  r^T 0 \ (.4011'D 6—o„-,-o^T^o'—'0 0 \6,0  \  90.0  \ e  •  0.,^60.0  7.300 -  • —0^/I\ I — , • • -^ 1 1  •  30.0  7.0  45.0  Llo  cr^4.0  T,• 40.0  \Ill I^ i/ we • --  I  NT T^ 0^•  1.0  35.0  – 2.0  30.0  •^•  I 0-0 /\* , 0/1 I o _. 9^9 1 I ioi^N l^ 1 1^*  l  – 5.0^ 25.0 ^ –12^5^8^11^24^–1^2^5^8^11^24  TIME  (h)^  TIME (h)  FIG 46: Changes in femoral arterial and venous blood gas parameters during and after continuous infusion of labetalol in adult nonpregnant sheep (mean ± SEM). (Asterisks denote significant difference from control values; solid line indicates infusion period).  118  o — 0 ARTERIAL^• — • VENOUS  600  400  0^  -1  2  5^  6  TIME (h) FIG 47: Oxygen homeostasis in adult nonpregnant sheep before, during and after continuous infusion of labetalol (mean ± SEM). (Asterisks denote significant difference from control values; solid line indicates infusion period).  119 0 ^ 0 ARTERIAL^• -• VENOUS  9 00  III^I 1! ( .4•1 0 Or IP \ 1-141  6.00  T I\ • • N^•  3.00  0.00  0  GO  12.00  9.00  • Go  6.00  3.00 ^ —1^2^5^8^11^24  TIME (h) FIG 48: Effect of Labetalol on Arterial and Venous Blood Glucose and Lactate Concentrations (mean ± SEM). (solid line indicates infusion period; Arterial and venous blood lactate concentrations were significantly different from control between 30 min and 2 h post-infusion; arterial and venous glucose concentrations were significant between 3 h and 2 h post-infusion.).  120  10  , cp-0 „.o_.0 l  o --o oi  0  1 ' 1....'I  —10 ^  —1  2 .^I5^8^. 11 -  ^11 .  24  10-  1\ /?`(17- 1/^I1^I  ^0  ^0  0 ,^0  .^,• ^• . •^ # 2^5^ '^I ^6^. 11 24  TIME (h) FIG 49: Hind limb arterio-venous glucose and lactate flux before, during and after labetalol infusion in adult nonpregnant sheep (mean ± SEM). (Asterisks denote significant difference from control values; dashed line indicates infusion period).  0 ^ 0 CONTROL^• ^ • LABETALOL 60.0 -  40.0 -  20.0 -  0.0 ^ 0.01  0.10^  1.00  NOREPINEPHRINE DOSE (ug) FIG 50: Intra-arterial norepinephrine dose-response curve before and during labetalol infusion (mean ± SEM).  122  significantly shifted during labetalol steady-state. The calculated EC50 values for NE in the control period and during labetalol steady-state were 0.204 ± O. 041 and 0.172 ± 0.028 j.tg/kg, respectively, and were not statistically significant (paired ttest, p<0.05).  3.7. Intra-arterial Labetalol Studies  3.7.1. Studies in Adult Non-pregnant Sheep  Direct responses to labetalol were studied by injections of the drug via the femoral artery. Direct intra-arterial injection of labetalol caused dose-dependent vasodilation and no significant changes were seen in the response following phentolamine or propranolol infusion (Fig 51A and B). Also, the vasodilation caused by intra-arterial labetalol was virtually instantaneous, with the peak effect being achieved within 20 sec of injection (Fig 52). Injection of control saline had no effect on the flow.  3.7.2. Studies in Fetal Sheep  Direct responses to labetalol in the fetal lamb were studied by injections via the external pudendal epigastric artery (HLA) (Fig 2). No consistent changes were seen in the HLA blood flow and no meaningful dose-response relationship could be established (Fig 53).  3.8. Fetal Labetalol Bolus Studies  3.8.1. Experimental Details  123  A 80 -  • AFTER PHENTOLAMINE  E -  600  O  '4 rx4  z  400 0—'  20-  OH O 0 1E-4^1E-3^1E-2^1E-1 LABETALOL DOSE  1  (mg)  150- • AFTER PROPRANOLOL  0_  •  0  0  90-  30-  O 0 1E-4^1E-3^1E-2^1E 1^1 -  LABETALOL DOSE (mg)  FIG 51: A.: Intra-arterial labetalol dose-response relationship in adult nonpregnant sheep hind limb before and after phentolamine administration (E# 543). B.: Intra-arterial labetalol dose-response relationship in adult nonpregnant sheep hind limb before and after propranolol administration (E# 105).  I 24 1 min  TIME  ^  I  1 I  —400  A  l 1-0  1-240  i-200 11,1, 1 I I^1 1 1 1 1 .1 , 111.1 0  .1, ,1^.1.^111'41 , 1611 1 1 1 I I I 14 14. 11' 10,111111 I  r 400  3E3  Lo —240  2  As1,41,414,11411/2Qh:P-AirtidLIJJL,...Lbrit  , p,•1"-e'v  —60 200  lilt^I It^, , 0111 I1 1l I ,^I  „^, 1,1 NI l it^  , 0 1 1 , ,^..111,11,^11,11, „I I^Ltd IE1 d  —0  —400  •0 —200  01101101011,111011KINOSIMPOWINAtte  3 —0  r-240  2 -2  I 1^I  —60  FIG 52: Polygraph tracings showing the time course of hemodyamic changes corresponding to intra-arterial administration of A.: labetalol, B.: norepinephrine and C.: control saline. Vertical arrow on the time scale corresponds to the time of injection. [1: Femoral Blood Flow (mL/min), 2: Mean Arterial Pressure (mm Hg) and 3: Heart Rate (bpm)].  30 -  • • •  0 4-4 Q)  20  -  • •  10-  • 0 ^ lE 1E 2 —  • —  1^1^10^100^1 000  LABETALOL DOSE (ug) FIG 53: Intra-arterial (HLA) injection of labetalol in the fetal lamb (E#1154).  126  Table 9 lists the particulars about the animals used in the study. The gestational age of the ewes at the time of experiment in the labetalol group was 129.4 ± 2.2 days while in the control group it was 131.8 ± 1.5 days. The mean birth weight of the operated fetus was 3309 ± 296 g in the labetalol group and 3294 ± 364 g in the control group. The fetal and maternal cardiovascular and acid-base status were within the normal range at the beginning of each experiment. The mean pre-experiment values are listed in Table 3. 3.8.2. Pharmacokinetics  The disposition of labetalol in various sampling sites following a 4 mg fetal intravenous bolus administration is shown in Fig 54. The decline of labetalol concentration in fetal arterial plasma was best described by either a biexponential equation Ae - at + Be -ftt or a triexponential equation of the type Pert + Ae - at + Be -flt. The weighting factor chosen was 1/C 2 . The estimates of the fetal pharmacokinetic parameters obtained by nonparametric analysis of the arterial plasma concentration data are listed in Table 10. Labetalol appears in the tracheal fluid instantaneously and the concentrations are consistently higher than that in fetal plasma over the entire sampling period. In contrast, a gradual accumulation is seen in the amniotic fluid and beyond 3 h, the concentrations in the amniotic fluid were higher than that in fetal plasma. Labetalol was also detected in the maternal arterial plasma but at much lower concentrations. The peak maternal arterial plasma labetalol concentration (4.17 ± 0.18 ng/mL) was roughly 1/50th of that seen in fetal plasma (207.39 ± 27.66 ng/mL) and the maternal to fetal AUC ratio was 0.031 ± 0.002. The apparent terminal elimination half-life of labetalol in maternal plasma was 4.75 ± 0.15 h. The maternal and fetal  127  TABLE 9: FETAL BOLUS STUDIES: EXPERIMENTAL DETAILS  BODY EWE# WT. (kg)  GEST^# OF AGE^FETUS (days)  TERM (days)  BIRTH WT. OF FETUS (g)  LABETALOL EXPERIMENTS 116  80.0  130  2  139  3206*, 4162  489  88.9  125  2  138  3923*, 4235  540  75.1  136  2  142  3960*, 4494  608  89.3  124  2  132  2360*, 4957  722  71.1  132  2  139  3096*, 3330  CONTROL EXPERIMENTS 116  80.0  136  2  139  3206*, 4162  201  72.9  129  2  143  3910*, 4350  338  92.4  133  2  137  3500*, 3950  608  89.3  129  2  132  2360*, 4957  Weight of operated fetus.  0 ^ 0 FA  A  6'  AM  • ^ A TR,  ^  MA  -A  100  A A  '0 0  O H  0  0  7'4  O lE -  12^24  TIM FIG 54: Disposition of Labetalol in Pregnant Sheep following a 4 mg Fetal Intravenous Bolus Administration (concentrations are mean ± SEM). (FA: Fetal Arterial Plasma; AM: Amniotic Fluid; TR: Tracheal Fluid and MA: Maternal Arterial Plasma).  129 TABLE 10: Pharmacokinetics of labetalol in the fetal lamb and ewe following a 4 mg fetal i.v. bolus (mean ± SEM; n=5). ESTIMATE A. FETUS  PARAMETER CLf (mL/min/kg)a  50.45 ± 1.37 *  CL fp (mL/min/kg)a  7.27 ± 2.11 *  CL fa (mL/min/kg)a  43.18 ± 3.72 *  t to 0'0  4.35 ± 0.33 *  MRT (h)  4.72 ± 0.33 *  Vd„ (L/kg)a  14.28 ± 0.95 *  Vdarea (L/kg)a  19.01 ± 1.52*  [FAUC/MAUCIC  0.144 ± 0.015 B. EWE  Apparent t 1/2/3 (h)  4.75 ± 0.18  MAUC/FAUC  0.031 ± 0.002  CLmp (mL/min/kg)a  23.40 ± 8.99  CLmp (mL/min/kg)b  29.84 ± 17.3  CLm (mL/min/kg)b c  30.80 ± 3.83  b/w (h)c  2.79 ± 0.66  MRT (h)c  1.80 ± 0.25  ,  Vd„ (L/kg)b c  3.02 ± 0.18  ,  Vd area (L/kg)b c ,  6.49 ± 0.72  a^Normalised to an average fetal body weight of 3 kg. b^Normalised to the average maternal body weight of 73.8 kg. c^From maternal bolus administration studies. *^Significant difference from corresponding maternal values following maternal bolus administration.  130  transplacental and nonplacental clearances were calculated using data obtained from this study and the maternal bolus study and are included in Table 10. Estimates of fetal total body, placental and nonplacental clearances, elimination half-life and apparent volume of distribution were found to be significantly higher than the corresponding values for the ewe.  3.8.3. Metabolism  Conjugates of labetalol were analyzed by enzyme incubation of amniotic fluid samples. The results are shown in Fig 55. No sulfate conjugates could be detected, but a significant proportion of the dose was present as glucuronide conjugate. Fetal arterial plasma and amniotic fluid samples (up to 2 mL volume; obtained by pooling samples) were also analyzed for the presence of 3-APB (Fig 29), before and after incubation with glucuronidase and sulfatase preparations, but the metabolite could not be detected in any of the samples.  3.8.4. Hemodynamic Effects  The changes in fetal heart rate, mean arterial pressure, femoral blood flow and calculated hind limb vascular resistance ([mean arterial pressure-hind limb venous pressure] hind limb flow) following the administration of labetalol are shown in Figs 56 & 57. The heart rate tends to decrease over the first 2 h, followed by a gradual and significant increase. No significant changes were seen in arterial pressure, femoral blood flow and vascular resistance.  3.8.5. Metabolic Effects  FREE^L FREE+ GLUCURONIDE 1200  900 z  O  600 -  300 z C)^  U  0 0  3  a  9  12^24  TIME (h) FIG 55: Concentrations of labetalol and its glucuronide conjugate in the amniotic fluid following a 4 mg fetal intravenous bolus administration (mean ± SEM).^  W  132  180  160  °1 -1 0 I  T^0-° I T/1 I /1 1‘0^ 0^I \ I 0^I 16 -°- ? I 0.0/ 1  140  I 120  —1  i  2^5  8^11  60.0  T-,1I 1. C2? 0\Ij^ ,o j T o I I 0 1\0 _ 0I T^so, o il^T^0-6 1‘ o" 00  I 1^II  30.0 ^ —1  2  ^  ^T  1  0 ,T  ^  6/I 0  s  \p ^  5^8^11  TIME (h) FIG 56: Effect of a 4 mg fetal intravenous bolus on mean fetal heart rate and arterial pressure (mean ± SEM). Asterisk denotes significant difference.  133  60.0 -  T  T I O'l 0T 0-7^I 1^ 1 ^1T T^T -00, I 0'0 1" °1 I CI, 0. /1 0  45.0  0 -0  0 30.0 -  I  1  ^ I LI °I-°11 -44'1°-1 I I IT/1^ I I  2^5^8^11  I.  T. 0° J.  0  .0  T  1,0  T R \T T I ° \T T 0,T 0^ -0-0 / 01^,/, )-s- -0.1. T^P, T^ 1 i l^y-Y 1 ? - ? - 0-6'I ? - 6 ,a, ,Y I ? - 6 l'el3^yi c  -1^2  5^8^11  TIME (h) FIG 57: Effect of a 4 mg fetal intravenous bolus on mean fetal hind limb blood flow and vascular resistance (mean ± SEM).  134  Changes in the fetal arterial blood gas parameters following bolus administration of labetalol are shown in Fig 58. A gradual decrease in pH and base excess over the initial 4 h was seen, suggesting the development of metabolic acidosis. A maximum decrease of 0.096 ± 0.015 in pH and 5.7 ± 1.3 mEq/L in base excess were seen at 4 h after labetalol administration. No significant changes were seen in p0 2 and pCO 2 . Blood oxygen content showed an apparent (-50%) decrease, which, however, did not reach statistical significance. The maternal arterial blood gas parameters did not show any significant change. The effects of labetalol on glucose and lactate concentrations in HLA, HLV and amniotic fluid are shown in Fig 59 & 60. The mean glucose concentrations before the administration of labetalol in HLA, HLV and amniotic fluid were 0.87 ± 0.11, 0.65 ± 0.05 and 0.13 ± 0.03 mM, respectively, while the mean lactate concentrations were 1.58 ± 0.14, 1.62 ± 0.16 and 1.71 ± 0.08 mM, respectively. Marginal increases in the glucose concentration lasting up to 5 h were observed in all three sampling sites. The changes in lactate concentrations, however, were more prominent, with peak concentrations in HLA and HLV representing roughly a four-fold elevation over control values. Significant accumulation of lactic acid was seen in the amniotic fluid. Lactate concentrations returned to pre-treatment (control) values in HLA and HLV by 24 h, while it remained significantly increased in the amniotic fluid. A transient and marginal increase in maternal arterial lactate concentration was also observed, while there were no significant changes in the maternal glucose concentrations (Fig 61). No significant changes in glucose and lactate concentrations were seen in the control experiments (Fig 62). The changes in the net arterio-venous flux of labetalol, glucose and lactate across the fetal hind limb were calculated by the application of the Fick principle [(HLA concentration - HLV concentration) * flow] (Fig 63). Net release of  ^  •  •LABETALOL  0—oCONTROL 30.0  7.375  Q1.1 7.325  T r)  24.0  0  •  - -  27.0  E 21.0  ON  7.275  TI \ . /I^I  Q. 18.0  II  1  °  ?  re\e^•>‹4; 1401 "'?' 10 :NI  •  15.0  7.225  12.0  ^, ,_.  C 0.0  0 cr)  Cuii;\ Cu -2.0 U^  I 7,  .  .,-.*--1 I  —6.0^*^I  9.0  55.0  0.0 r  -  50.0  12.0  I  11,  ^4:3,AI^ 41,14110,^0,0_..0 I^1 • /?\ /I^I  CCV 45.0 •  40 0  N _ ,_ ^•  Tr!^/ I 7 1  .1\T.N...,,I---  cil w -4.0^ cn^1111 a 0:1  0 1^II^I^11 • •0 100 I^I^I^I  0^0^.  II9  •- •^  1 •  T 2  6. . •  ,  .---T  ^  8^II^24  TIME (h)  3.0  I I  2  11^24  TIME (h)  FIG 58: Effect of labetalol and control (saline) fetal bolus administration on fetal arterial blood gas parameters (mean ± SEM). Asterisks denote significant difference.  0 ^ 0 FA^• —• HV^z—z\ AM  1.0 -  0.5 H  0  —1^2^5^8^11^24  TIME (h) FIG 59: Effect of a 4 mg fetal intravenous labetalol bolus on glucose concentrations (mean ± SEM). (FA: Fetal arterial blood; HV: Hind limb venous blood and AM: Amniotic fluid).  0 ^ oFA^• ^ •HV^L ^ LAM 8  6  4  U  -  DL  2  —1^2^5^8^11^24  TIME (h  )  FIG 60: Effect of a 4 mg fetal intravenous labetalol bolus on lactic acid concentrations (mean ± SEM). (FA: Fetal arterial blood; HV: Hind limb venous blood and AM: Amniotic fluid). FA and HV concentrations were statistically significant between 90 min and 12 h and AMN concentrations between 2-24 h.  0 ^ 0 GLUCOSE^• ^ • LACTATE  I  3 0 .  -3.0 T  O 0 U Cf) O U  0  O  .  •I 144.11 4411  I  1 0 .  . —1  0 0  H -2.0  2 0  T  • T  T  2^5^8^11  TIM H ice  o  O z  .  -^  c)  •  o  -1.0  0.0  )  FIG 61: Glucose and lactate concentrations in maternal arterial blood following a 4 mg fetal intravenous bolus of labetalol (mean ± SEM).  139  A-A  • ^ • FA  2.0  4,A 1  HV 0-0 MA  A ^ A AMN  ill^I • -0  0  1 --  z  • A4iAA -A -A -A^A ^ A ^ A A L^^  0  z  z 0  1.0  06 0^--- 0 ^ 0 ^ 0  1.4 <4  0  <4 0.0  3  .0  1 1, i  1  0 000-  0 124 2.0  E-■  111  I  111  I  I  0 C.11 C/) 0  1.0  0  ^A A A A 00A ^ -  TYTT  1  T^ I A ! INAT I • T^ T  8^11  ^  A  24  TIME (h ) FIG 62: Changes in glucose and lactate concentrations following control (saline) fetal intravenous bolus administration (mean ± SEM). (FA: Fetal arterial blood; HV: Hind limb venous blood; MA: Maternal arterial blood and AM: Amniotic fluid).  140  TIME (h)  TIME (h) FIG 63: Effect of a 4 mg fetal intravenous bolus of labetalol on hind limb arterio-venous labetalol, glucose and lactate fluxes (mean ± SEM). Asterisks denote significant difference from control values.  141  labetalol from the hind limb in the first 2 h, with a momentary net uptake at 15 min was observed. But beyond 2 h, there was no appreciable net uptake or release of labetalol. The mean control values for hind limb glucose and lactate uptakes were 0.570 ± 0.224 mmol/h and 0.085 ± 0.227 mmol/h. The net uptake of glucose by the hind limb was unchanged following the administration of labetalol. While there was also net uptake of lactate by the hind limb prior to and for the first 4 h post-dose, a significant change resulting in net release was observed thereafter. The total lactate release from the hind limb, calculated as the AUC of the release curve from 4 to 24 h, averaged 3.85 ± 2.05 g (0.043 ± 0.023 M). There were no changes in hind limb glucose and lactate fluxes in the control experiments.  142  4. DISCUSSION 4.1. Development of a Microbore HPLC Assay with Fluorescence Detection for the Quantitation of Labetalol in Biological Fluids.  Several reverse phase HPLC assay procedures using ultraviolet (Dusci and Hackett, 1979; Woodman and Johnson, 1981; Hidalgo and Muir, 1984), fluorescence (Meredith et al., 1981; Oosterhuis et al., 1981; Alton et al., 1984; Luke et al., 1987; Bates et al., 1987; Ostrovska et al., 1988), electrochemical (Wang et al., 1985; Abernethy et al., 1986) and thermospray mass-spectrometric (Lant et al., 1987) detection systems have been described for the determination of labetalol in human plasma. To date, no microbore HPLC assay has been reported for the trace level quantitation of labetalol. Also, there has been no report on the quantitation of labetalol in species other than humans. Successful analysis of drugs and metabolites in the various biological fluids obtained from pregnant sheep (maternal and fetal plasma, amniotic and fetal tracheal fluid) following a single dose drug administration pose a few challenges: (a) The various biological fluids differ considerably in their composition (concentration of proteins, phospholipids, electrolytes etc.) and matrix consistency (e.g., fetal tracheal fluid is much more viscous than plasma) so that their extraction  behaviour and chromatography could be different and hence have to be treated individually. (b) Due to repetitive sampling from multiple sites over a short period of time (see sections 2.7.1. and 2.7.5.), the sample volume obtainable at each point is generally low (e.g. ---250^for fetal plasma). Hence, it is essential to have an assay that could separate the analyte of interest from the endogenous substances and at the same time provide sufficient sensitivity and precision in  143  quantitation. The method described here has been developed to fulfill these objectives. The octadecylsilane (C 18 ) stationary phase and phosphate buffer mobile phase were chosen to begin with because most of the previously published assays have employed them. The molarity and pH of the phosphate buffer were optimized to provide satisfactory peak shape and retention time. The upper end of the molarity range studied was set at 20 mM based on the HPLC manufacturer's recommendations for a microbore system. The higher and lower limits of pH (8.0 and 2.5 respectively) used were in accordance with the range suggested by the column manufacturer. The occurrence of broad peaks (peak width > 2 min) at pH values of the phosphate buffer greater than 4.0 is probably due to labetalol existing in both ionized and unionized form (pKa of labetalol = 7.4). Decreasing the pH of the buffer from 7.0 to 2.5 probably causes progressively increasing ionization of labetalol, resulting in the elution of a mostly single moiety (the ionized form) and hence the improvement in peak shape and symmetry. Whereas both ether and ethyl acetate provided comparable extraction recoveries (Fig 4), the latter was chosen in preference to the former based on considerations of safety and ease of handling. The two-step extraction (extraction with ethyl acetate followed by re-extraction of the organic layer with dilute phosphoric acid) was found necessary to overcome interference from endogenous components (Fig 5). This improved selectivity was accompanied by a slight loss of recovery, which in absolute terms, decreased from about 81% to about 75%. However, this procedure eliminates the need for drying the samples and hence shortens the time for sample preparation. The ratio of the volume injected (60 !IL) to the volume of the aqueous phase at the end of the second step of extraction (600 1.11,) was 0.1, which enables multiple injections to be made from the same sample.  144  The primary objective of this assay is to provide adequate mass sensitivity, that would enable quantitation of a few nanograms of labetalol present in — 200300iLtL. It has been shown that the use of a microbore column could significantly enhance mass sensitivity (Wong, 1989) and minimum detection limit (Simpson and Brown, 1987) and thus is ideal for applications involving restricted samplevolume. Hence, in this assay, a microbore column (2.1 mm i.d.) and connections (0.12 mm i.d.) (between injector, column and detector) were employed. The length of the tubing that connects the column outlet to the detector was restricted to 100 mm, to minimize post-column diffusion. Also, the potential peak diffusion that could occur at the detector was overcome with the use of low volume flow cell (5 fit capacity). The use of an emission cut-off (370 nm) filter resulted in significant reduction in baseline noise at emission wavelength equal to 412 nm. Further optimisation of the signal/noise ratio was achieved with the use of a 2 mm wide excitation slit and 4 mm wide emission slits instead of the standard 1 mm and 2 mm wide slits, respectively. Even though the wider slits increase the bandwidth of excitation and emission wavelengths and thus compromise selectivity, no interfering peaks were found under the conditions employed.  4.2. Development of a GC-MSD Assay for the Identification and Quantitation of 3-APB, an Oxidative Metabolite of Labetalol in the Biological Fluids of Sheep  The possibility that labetalol undergoes oxidative biotransformation was first suggested by Gal et al., 1988. They identified 3-APB and its p-hydroxy derivative in urine sample from a patient on labetalol therapy using GC-MS with negative chemical ionization after pentafluoro-propionyl derivatization. Subsequently, the same group of authors reported a quantitative stereoselective  145  assay for 3-APB, which involves negative ion CI of the (S)-a-methoxy-atrifluoromethylphenylacetic acid derivatives (Changchit et al., 1991). The assay for 3-APB presented here has been developed with an objective to identify and quantitate this compound in biological fluids using a bench-top Gas Chromatograph with Mass Selective Detector (GC-MSD) using electron impact ionization. Satisfactory chromatography in terms of peak shape and resolution between the analyte (3-APB) and the internal standard (1-methyl-2-phenoxyethylamine) derivatives has been obtained by "cold trapping" of the sample, produced by a 100 °C gradient between the injection temperature and initial column temperature. While underivatized 3-APB shows good chromatography, derivatization is essential to overcome interference from the parent compound (labetalol), which undergoes decomposition to yield trace levels of 3-APB under the GC conditions employed. No such interference could be seen following HFBA derivatization possibly because labetalol is not readily derivatized. The EI mass spectra of the HFBA derivatives of 3-APB and the internal standard (Fig 13B & 13C, respectively) show that the m/z 254 fragment is much more intense in the mass spectrum of the internal standard, possibly due to the presence of an ether linkage in 1-methyl-2-phenoxyethylamine (structure shown in Fig 13A). Despite extensive fragmentation of the 3-APB derivative (Fig 13B), the molecular ion (m/z 345) could still be detected in the total ion chromatogram, thus enhancing the diagnostic utility of the assay. Two ions for each of the two compounds (m/z 345 and 132 for 3-APB and m/z 347 and 134 for internal standard) were selected for the quantitative SIM mode. The molecular ions were included in preference to the most intense ions (m/z 117 in the case of 3-APB and 254 with the internal standard) to enhance the selectivity of the method. While the abundance of m/z 117 in 3-APB mass spectrum was roughly 20% higher than that of m/z 132, the  146  latter provided a better signal-to-noise ratio in the SIM mode and hence was chosen as the second ion. The dwell time chosen for each group of ions was 200 msec so that each peak (width = 6 sec) is sampled about 12 times. The sensitivity of quantitation was appreciably improved by tuning with target ions of mass 69, 218.95 and 264 of PFTBA and also by selecting the optimal fractional mass of the ion, correct to a decimal place. It should be noted that the MSD used in this method is not truly a high resolution mass spectrometer and that specifying a fractional mass simply helps to narrow the scan window, which in some cases can lead to an appreciable increase in sensitivity. Thus m/z 345.4 provided the best signal/noise ratio for 3-APB derivative. Hexane was chosen as the extraction solvent over ether, primarily because of safety considerations while sample pH adjustment with 5N sodium hydroxide provides sufficient alkalinization of biological fluid sample (1 mL) with 200-300pL volume, thus providing near optimal aqueous/organic phase ratio (roughly 1:4). Triethylamine was added to the reconstitution solvent (toluene) to act as sequestering agent and minimize adhesion of 3-APB to glass surfaces. The absolute recovery of 3-APB was consistently high (range: 90-107%), over the concentration range studied. 4.3. Maternal Bolus Studies  Labetalol is used in the treatment of various hypertensive disorders in pregnancy (Ashe et al., 1987; Goa et al., 1989), but there is little information in the literature regarding the in utero fetal exposure and fetal effects of labetalol. The maternal bolus studies were conducted to obtain this vital information in the pregnant sheep model. 4.3.1. Pharmacokinetics  147  There are no reports in the literature of any detailed pharmacokineticpharmacodynamic investigations of labetalol in pregnant animals. The chronically instrumented pregnant sheep model offers several advantages including repeated sampling from the mother, fetus and other fluid compartments in utero as well as continuous physiological and biochemical monitoring. Labetalol shows a triexponential or biexponential decline in the maternal plasma with a high total body clearance of 135.86 ± 16.93 L/h (30.8 ± 3.83 mIlmin/kg), a value that approximates the hepatic blood flow, which in pregnant sheep is about 150-300 L/h (Katz and Bergman, 1969). This value of clearance also represents about 27% of the normal cardiac output in near-term ewes (Rosenfield, 1977). The rapid distribution seen in the first 15 min could be explained by the high apparent volume of distribution (VD area = 477.5 ± 53.0 L or 6.48 ± 0.72 L/kg and VD ss = 222 ± 12.98 L or 3.02 ± 0.18 L/kg). Table 5 compares the pharmacokinetics of labetalol in pregnant sheep with the reported values (Rubin et al., 1983; Michael, 1979) for pregnant women. With the exception of fetal drug exposure, where the indices (AUC vs concentration ratio at the time of delivery) are different, the pharmacokinetic parameters show good agreement, which suggests that the pregnant sheep is a suitable model, at least for the maternal pharmacokinetics of labetalol. However, it should be noted that the studies by Rubin et al., 1983 and Michael, 1979 were conducted in pre-eclamptic women while normal, healthy sheep were used in this study. The fetal/maternal plasma labetalol AUC ratio in the pregnant sheep was much lower than the cord blood/maternal venous plasma concentration ratio reported in humans (0.144 vs 0.5). This could be due to one or more of the following reasons. Firstly, the epitheliochorial sheep placenta differs from the hemochorial human placenta in having a lower permeability to hydrophilic polar substances (Faber and  148  Thornberg, 1983). Secondly, the human data are based on a single point determination of fetal-to-maternal drug concentration ratio, which changes constantly with respect to the time of dosing in non steady-state conditions (Anderson et al., 1980b). Thirdly, the extensive maternal and fetal hemodynamic changes that occur during and immediately after delivery may affect the disposition of the drug in the mother and/or the fetus (Hamshaw-Thomas et al., 1984) and hence extrapolation of the concentration ratio at delivery to the antepartum intrauterine conditions may be erroneous. Finally, the pathological changes in the placental structure and function that are known to occur in preeclampsia (Dadak et al., 1984) may affect placental transfer processes. Transfer of labetalol across the sheep placenta was rapid as evidenced by the peak concentration in the fetal plasma at 3 min (Fig 16). The significantly longer apparent elimination half-life of labetalol in the fetus when compared to the ewe could be due to extensive binding of labetalol to and consequent slow release from proteins and/or tissues in the fetus or recirculation of labetalol from the fetal tracheal, amniotic and allantoic fluid compartments back to the fetus. In a clinical case study, Haraldsson and Geven, 1989, have reported the apparent half-life of labetalol in a premature infant, whose mother had received oral labetalol for ten weeks prior to delivery, to be approximately 24 h. From this study, it is clear that labetalol accumulates in the amniotic and tracheal fluids and persists much longer in these sites (24-48 h) than it does in the maternal or fetal plasma. Similar observations have been made with other drugs in the pregnant sheep following maternal administration (Rurak et al., 1991), but in each case with different fetalto-maternal half-life relationships. The disposition half-life in the fetus was significantly higher than that in the ewe for metoclopramide, the same with  149  diphenhydramine and significantly lower in the case of ritodrine. Thus, other factors like the kinetics of protein and tissue binding may be involved. 4.3.2. Hemodynamic Effects  The hypotensive action of intravenous labetalol is generally believed to be mediated primarily by peripheral alpha s -blockade (van Zwieten, 1990), but the hemodynamic response to labetalol and in particular the preponderance of either alpha or beta-antagonism can vary considerably in different experimental situations depending on the balance of autonomic influences (Brittain and Levy, 1976). In our study, no consistent maternal cardiovascular changes were observed. The two animals that showed a trend towards hypotension and simultaneous tachycardia were ENTG and E 105. Since ENTG had a low body weight (Table 2) and hence received a higher labetalol dose/kg (2.47 mg/kg while the mean value is 1.42 ± 0.13 mg/kg), it is possible that the maternal cardiovascular changes are dose dependent. In the study of Eisenach et aL, 1991 in pregnant sheep, a statistically significant change in maternal mean arterial pressure was seen with a dose of 3 mg/kg of labetalol but not with the lower doses of 0.5 and 1.0 mg/kg, implying that a minimum dose is required to elicit any hemodynamic response. However, the AUC and other pharmacokinetic data in our study (Table 4) suggest the lack of any labetalol concentration related phenomenon. In contrast to our observations and that of Eisenach et al., 1991, a significant fall in maternal mean arterial pressure in normotensive pregnant sheep was reported by Mohan et al., 1990, following a 100 mg infusion of labetalol over 5 min. It could be that this difference is due to the mode of administration - slow infusion as opposed to an instant bolus. The lack of any apparent change in the fetal hemodynamics in this study is similar to that reported by Eisenach et al. (at a  150  dose of 1 mg/kg) and Mohan et a/.,1990. The lack of any cardiovascular response in the fetal lamb may be at least in part due to the low fetal exposure to maternal labetalol. 4.3.3. Metabolic Effects  The metabolic effects of labetalol, unlike its hemodynamics, have not received much attention. In particular, the effect of labetalol on carbohydrate and lipid metabolism in pregnancy has not been studied in detail. Our results suggest that labetalol exerts very significant metabolic effects in the pregnant sheep. In both the mother and fetus, the decrease in arterial blood pH is accompanied by a concomitant change in base excess, implying that the acidosis is of metabolic origin. Maternal pH and base excess show only marginal changes, with the values quickly returning to control state. This is probably a result of respiratory compensation in the form of hyperventilation, which in the adult, is a fairly robust compensatory mechanism. The initial fall in maternal pCO2 and the subsequent upward trend in p02 (the latter not statistically significant) is consistent with hyperventilation. The fetus, in the absence of any respiratory compensatory mechanisms, shows sustained deterioration in pH and base excess. The sharp fall in fetal oxygen saturation and content is most likely due to the Bohr effect, i.e., a shift in the oxyhemoglobin dissociation curve to the right, caused by the drop in pH.  Several clinical studies of labetalol have reported variable changes in blood glucose levels. Andersson et al., 1976, observed a significant increase in fasting blood sugar without any change in insulin levels or glucose tolerance in hypertensive men following labetalol administration, but the authors did not provide any explanation for these observations. But, Barbieri et al., 1981,  151  observed only slight elevations in blood glucose level in their study and attributed the effect to the a l -blocking action of labetalol since phentolamine, a pure aadrenergic blocker has been shown to augment the increase in glucose during exercise (Galbo et al., 1977) and prazosin, a selective post-synaptic a r adrenergic blocker, causes an increase in glucose in hypertensive men (Barbieri et al., 1980). While the extent of a and r3-blockade caused by labetalol in this study was not investigated, studies in adult nonpregnant sheep (see section 4.6.4.) show that labetalol does not cause any significant a-blockade. On the other hand, there is substantial evidence to show that labetalol has significant beta 2 -agonist activity (Riley, 1981; Baum and Sybertz, 1983). Increases in c-AMP levels caused by labetalol has also been reported in an in vitro study using isolated pregnant rat uterus (Chimura, 1985). Beta 2 -agonists are known to cause elevated maternal glucose levels in humans (Schreyer et al., 1980) and sheep (Bassett et al., 1985; van der Weyde et al., 1992). Bassett et al., 1985, observed that maternal infusion of ritodrine, a beta2 -agonist, causes significant (2-fold) increase in the maternal glucose concentrations, while van der Weyde et al., 1992, found a similar effect on fetal glucose levels with fetal infusion of ritodrine. Hence, it appears that 13 2 agonism is responsible for the maternal hyperglycemia observed in this study, although contribution from oc i -blockade cannot be completely ruled out. The fetal hyperglycemia (Fig 20) appears to be a direct result of the maternal hyperglycemia since the changes in the fetal glucose concentrations paralleled those in the mother. In the fetal lamb, under normoxic conditions, almost the entire supply of glucose is obtained from the maternal circulation and there is very little glucogenesis (Hay et al., 1981). About 60% of fetal glucose is oxidized, accounting for about 30% of the net fetal oxygen uptake (Hay et al., 1983). Glucose in the fetus is also converted into lactate and fructose as well as transferred back into the maternal circulation (Prior, 1980). The transfer of  152  glucose across the placenta is by passive diffusion along the concentration gradient and Hay and Meznarich, 1989, have shown that in sheep, the fetal glucose concentration shows a statistically significant linear relationship to maternal glucose concentrations over a wide range (1-9 mM or about 20-160 mg/100 mL). The observation of significant fetal acidemia, as indicated by the blood pH and base excess in our initial experiments, prompted us to monitor the lactic acid changes in the latter experiments. The results (Fig 20) show that the fetal acidemia is largely, if not completely, due to the lactic acidosis. In the fetus, the peak lactate concentration, the total area under the lactate concentration curve (AUC) and the incremental increase in the lactate AUC following labetalol administration (calculated as ([Lc](ty[Lc](0)) dt) were consistent in all the four animals in which lactate concentrations were measured, with mean values of 5.9 ± 0.26 mM, 110.4 ± 6.18 mMh and 67.8 ± 7.26 mMh, respectively. The corresponding values for the maternal lactate concentrations were 3.5 ± 0.77 mM, 53.6 ± 1.38 mMh and 34.4 ± 2.62 mMh, respectively. Lactate in the amniotic fluid was possibly derived from fetal urine since the permeability of the chorioamnion to lactic acid is low (Britton et al., 1967). This would also mean that similar high concentrations of lactate should be present in the allantoic fluid (not sampled in this study) as well, since the fetal urine is almost equally distributed between the amniotic and allantoic fluid cavities (Wlodek et aL, 1988). To the best of our knowledge, there has been no report in the literature on the effect of labetalol on lactate homeostasis in any species - pregnant or nonpregnant. There does not appear to be any simple explanation for the acute lactic acidosis that we have observed, especially in the fetus, since the fetal exposure to maternal labetalol is only about 14%. Since the sheep placenta has a very low permeability to lactate (Britton et al., 1967; Sparks et al., 1982; Kitts and  153  Krishnamurti, 1982), the fetal lactic acidosis induced by labetalol is unlikely to have been due to the maternal lactic acidosis. This view is supported by the fetal labetalol bolus studies (see section 4.8.4.), where there was a rise in fetal lactate concentrations with minimal changes in the mother. The low placental lactate permeability could also explain the persistence of this substrate in the fetus, since transplacental clearance is a major component of fetal elimination for a number of compounds (Rurak et al., 1991). The persistence of lactate in the fetal lamb for several hours following an initial rapid increase as observed in this study has been reported in other studies involving physiological (hypoxia; Britton et al., 1967) and pharmacological (ritodrine administration; Bassett et al., 1985 and van der Weyde et al., 1992) perturbations. Thus, the elevation in fetal lactate concentration following maternal labetalol administration likely resulted from increased production or decreased elimination of lactate or a combination of both. Studies in normoxic fetal lambs have demonstrated that circulating lactate originates from production by fetal tissues and by the placenta, with the latter organ accounting for about 35% of total lactate production (Sparks et al., 1982). Fetal lactate is produced from both glucose and non-glucose (e.g. alanine) sources (Kitts and Krishnamurti, 1982). Approximately, 75% of the lactate is oxidized (Hay et al., 1983) and this accounts for about 25% of fetal oxygen consumption (Burd et al., 1975). The rate of lactate oxidation has been found to be concentration dependent with increased oxidation occurring at higher concentrations (Hay et al., 1983). Although hyperglycemia can lead to increased blood lactate concentrations, it is unlikely that the fetal lactic acidosis is consequent to maternal or fetal hyperglycemia since Hay and Meznarich, 1989, have found that following a fourfold increase in maternal glucose concentration caused by the glucose-clamp technique, the fetal lactate increased only marginally (from 1.57 mM to 2.07 mM)  154  even though uteroplacental glucose uptake showed a three-fold increase. Sustained mild hypoxemia (fall in p0 2 by 5 mm Hg) in the fetus can result in elevated lactic acid levels (Towell et al., 1987). However, the modest and transient changes in fetal p0 2 observed in this study do not suggest a similar mechanism. The role of adrenergic blockade in the metabolic status of fetal lamb has been investigated by Jones and Ritchie, 1978. They found that infusion of phentolamine or propranolol alone does not cause any significant change in any of the fetal metabolic indices including lactic acid, which rules out the possibility of fetal adrenergic blockade being the primary mechanism behind the lactic acidosis in this study. Labetalol has also been shown to cause significant elevations in plasma norepinephrine concentrations in hypertensive men (Lin et al., 1983; Christensen et al., 1978), which has been attributed to the intrinsic sympathomimetic activity of the compound. Exogenous norepinephrine has been known to cause increases in plasma glucose and lactate in adults (Himms-Hagen, 1967) and in fetal sheep (Jones and Ritchie, 1978). Hence, there exists a possibility that the fetal and maternal metabolic changes are at least in part, secondary to the rise in norepinephrine concentrations, which were not measured in this study. It is well recognized that P 2 -agonists like ritodrine, which are used in the management of premature labour, cause significant changes in the carbohydrate and lipid metabolism including elevations in glucose and lactic acid concentrations in humans (Lenz et al., 1979) and maternal and fetal sheep (Bassett et al., 1985; van der Weyde et al., 1992). Hence it is possible that P r agonism is involved in the pharmacodynamics of labetalol. Whether the sympathomimetic effect is derived from the intrinsic activity of labetalol (Riley, 1981) or actually mediated through an active metabolite cannot be ascertained from the data obtained in this study. Hence, the oxidative metabolism of labetalol was investigated in  155  subsequent studies involving labetalol administration in adult nonpregnant ewes (see section 4.4.) and in fetal lambs (see section 4.8.). It is possible that the release of lactate occurred from the fetal and maternal  tissue and/or from the placenta since the sheep placenta is known to possess beta2 receptors (Padbury et al., 1981). The rise in lactate concentrations resulting from the administration of ritodrine, a 13 2 -agonist, appears at least in part due to increased placental production (van der Weyde et al., 1992; Wright, 1992). However, studies conducted in adult nonpregnant sheep and fetal lambs (see sections 4.4. and 4.8., respectively) to assess the extent of release of lactate from the carcass in general, and the hind limb in particular, in response to labetalol administration, suggest that these tissues could also be involved in the increased lactate production. A simple concentration-effect relationship does not exist between the maternal/ fetal labetalol concentrations and any of the observed effects. This is understandable since most of the effects (e.g. acidemia, glucose, lactate concentration changes) presumably involve a number of events in sequence that precede the observed "effect". We did not attempt any pharmacokineticpharmacodynamic modelling in terms of an effect compartment (Holford and Sheiner, 1981) or a biophase (Veng-Pedersen and Gillespie, 1988), concepts that are applied in the minimization of the hysteresis loop, simply because the mechanism(s)/events involved in the observed effects are not understood and a model, under such circumstances, would be of dubious value. The pharmacokinetic and pharmacodynamic data from this study underline the limitations of using a strictly concentration-based fetal safety index (maternal/fetal concentration or AUC ratio) since a low index of drug exposure (AUCfetal/AUCmaternal 14%) was associated with marked fetal effects (e.g. prolonged metabolic acidemia). Thus, for a given concentration-based fetal drug  156  exposure index, it is possible to obtain a much different fetal/maternal effect ratio and this ratio in turn could be different for different pharmacodynamic endpoints. Hence, it may be necessary to assign one or more "pharmacodynamic indices", which when applied with the AUC ratio will result in a more meaningful measure of fetal safety. The relevance of the findings in this study to clinical obstetric practice remains to be established. Much of the data on the fetal effects of labetalol in humans relates to uteroplacental hemodynamics (Nylund et al., 1984; Joupilla et al., 1986; Harper and Murnaghan, 1991; Pirnohen et al., 1991) and/or neonatal  follow-up (MacPherson et al., 1986; Sibai et al., 1987; Pickles et al., 1989). There are limited data on blood gas and fetal metabolic effects of labetalol in the literature. The dose of labetalol used in the clinical management of pregnancyinduced hypertension depends on the severity of the condition, but it also varies considerably due to the large inter-individual variability in the hypotensive response to the drug. Doses of up to 200 mg as intravenous infusion (Michael, 1986; Ashe et al., 1987) and 1200 mg p.o. (Michael, 1982) have been used to control hypertensive emergencies in pregnancy. Thus the labetalol dose used in this study (100 mg or 1.4 mg/kg) is within the clinically relevant dosing range. In terns of fetal exposure to labetalol in humans, the cord blood labetalol concentration at the time of delivery ranges from about 10-260 ng/mL (Rogers et al., 1990; Michael, 1979). In this study, following maternal administration of  labetalol, the peak labetalol concentration in ovine fetal plasma ranged from 23-66 ng/mL, which suggests that the human fetus is exposed to relatively higher concentrations of labetalol than the fetal lambs in this study. However, no significant deterioration of fetal acid-base or oxygenation status due to labetalol therapy has been reported in any of the clinical studies. Pirnohen et al., 1991, observed that following a 0.8 mg/kg intravenous bolus to normotensive pregnant  157  women at 38 weeks gestation, the cord blood gas parameters were within the normal range and that in all the cases, the umbilical artery pH was above 7.15 and the umbilical venous pH was above 7.25. But, assessment of fetal blood gas status in clinical studies is normally based on the cord blood gas parameters obtained immediately after delivery and cord clamping. In a recent study, Khoury et al., 1991, compared the blood gas values obtained in the intact fetal circulation just before elective cesarean section with those obtained immediately after delivery and found that pH, p02 and base excess were significantly decreased while pCO2 was significantly increased after delivery. This indicates that the blood gas values obtained at delivery do not reflect the prenatal situation. Further, if the maximum changes in the blood gas parameters seen in the fetal lambs in this study were to be observed in the human fetus at delivery, they would still be considered to be in the normal range (Rurak et aL, 1987). Moreover, in the presence of preeclampsia, any labetalol induced fetal blood gas changes would likely be ascribed to the underlying clinical problem rather than to an effect of the drug. Further investigation to assess the metabolic consequences of maternal labetalol in the human fetus is thus necessary.  4.4. Labetalol Bolus Studies in Nonpregnant Sheep  The primary objectives of this study were to assess the contribution of skeletal muscle and other carcass components to the lactic acidosis observed following labetalol administration (section 3.4.3.) and to explore the possibility of involvement of active metabolite(s) in the mediation of labetalol induced metabolic effects. The hind limb was chosen as a representative tissue of the carcass due to the ease of arterio-venous catheterization and blood flow measurement in that region.  158  4.4.1. Pharmacokinetics  The total body clearance, apparent terminal elimination half-life and the volume of distribution of intravenous labetalol in adult nonpregnant sheep were not significantly different from the estimates obtained in pregnant sheep (Table 6) suggesting that pregnancy does not alter the pharmacokinetics of labetalol in sheep. Similar conclusions were reached in human studies as well (Rubin et aL, 1983). Also, the estimates of pharmacokinetic parameters obtained in this study (mean total body clearance equal to 29.00 ± 2.67 mL/min/kg and terminal elimination half-life equal to 2.41 ± 0.30 h) are similar to the reported values in normotensive nonpregnant women (mean total body clearance in that group was 33.8 mL/min/kg and terminal elimination half-life was 2.1 h) (Rubin et al., 1983). The percentage of the dose excreted in the urine as unchanged drug was less than 2%, which is similar to that reported in humans (0-5%) (Martin et al., 1976). Significant biliary excretion of the unchanged drug was also seen (Fig 25) and the biliary concentrations were consistently higher than the corresponding concentrations in plasma, suggesting the involvement of an active transport mechanism. 4.4.2. Metabolism  Glucuronidation of labetalol at both the phenolic and secondary hydroxy groups has been reported (Martin et al., 1976; Niemeijer et al., 1991). The results from enzyme hydrolysis of urine and bile samples in this study show that labetalol undergoes significant glucuronidation in the sheep (11.5%) and the cumulative amount excreted in the urine as glucuronides was roughly ten-fold larger than the  159  amount excreted as unchanged drug. In sheep, it appears that labetalol also undergoes sulfation, in contrast to the observations by Martin et al., 1976, in rats, rabbits, dogs and humans. These apparent differences may be species related and also dependent upon the enzyme preparations used for conjugate hydrolysis. In contrast to the extent of glucuronidation, sulfate conjugation was quantitatively less significant (1.5%) and in two of the animals, the sulfate conjugates could not be detected in the urine. Both the glucuronide and sulfate conjugates were excreted in the bile and the glucuronide/sulfate concentration ratio in the bile was between 0.5-2.0 while it was around 8-10 in urine, suggesting preferential biliary excretion of sulphate conjugate. Quantitative assessment of biliary excretion was not made since continuous collection of bile was not feasible. The oxidative metabolism of labetalol has not received much attention in the past. Recently, Gal et al., 1988, suggested that labetalol undergoes Ndealkylation (Fig 29) to yield 3-APB and its hydroxylated derivative, which they identified in urine samples obtained from patients on labetalol therapy. However, the quantitative relevance of N-dealkylation or the pharmacodynamic contribution of the metabolites formed was not clarified. We developed a sensitive and selective EI GC-MSD assay for the identification and quantitation of 3-APB in biological fluids (section 4.2.). Following positive identification of the compound in sheep urine and bile samples, a quantitative assessment of the excretion of 3APB in urine was made (27A & B). The rate of excretion of 3-APB in the urine suggests rapid formation of the metabolite in the first 60 min following drug administration. The short apparent elimination half-life of 3-APB (13.5 ± 3.8 min) prompted us to explore whether this metabolite might undergo further conjugative/oxidative biotransformation. Both glucuronide and sulfate conjugates of 3-APB were found in the bile, while only the glucuronide could be detected in the urine samples, which suggests preferential biliary excretion of sulfate  160  conjugate in the bile, as was observed with labetalol. It seemed surprising that free (unconjugated) 3-APB (MW=149) was present in the bile in relatively high concentrations 200 ng/mL) while it remained undetectable in plasma, particularly since low molecular weight organic cations (92-236 daltons) have been shown to undergo insignificant biliary excretion in several species including sheep (Abou-El-Makarem et aL, 1967). However, hydrolysis of the conjugates in bile in vivo or under the assay conditions, could not be ruled out. No evidence for oxidative metabolism of 3-APB, including p-hydroxylation (Fig 29), was found.  4.4.3. Hemodynamic Effects The hemodynamic response obtained in nonpregnant sheep were more consistent than that observed in pregnant sheep study (see section 4.3.2.) and significant changes were seen in the mean heart rate and arterial pressure values after labetalol administration. The significant increase in femoral blood flow along with the hypotension and delayed onset of tachycardia suggest peripheral vasodilation and the development of reflex tachycardia. The tachycardia may further suggest that labetalol does not cause significant f3 1 -antagonism in the sheep. Indeed, the adrenoreceptor blocking actions of labetalol have been shown to vary, depending upon the experimental situation and the balance of autonomic influences involved. Hypotension and reflex tachycardia with no significant change in stroke volume were observed in conscious, chronically instrumented dogs whereas decreases in myocardial contractility and heart rate were seen in anesthetized dogs following intravenous labetalol (Brittain and Levy, 1976). The exact mechanism involved in peripheral vasodilation, i.e., a-receptor blockade, 13agonism or direct vasodilation, was subsequently investigated (see sections 4.6.4.  161  and 4.7.1.). However, active metabolite contribution to the hemodyamic effects of labetalol remains to be studied.  4.4.4. Metabolic Effects  The significant metabolic effects observed following labetalol administration in adult nonpregnant ewes include hyperglycemia, lactic acidosis, a fall in arterial pCO 2 , rise in femoral vein p0 2 and oxygen content and an increase in oxygen consumption across the hind limb. Similar metabolic effects were observed in pregnant sheep (see section 3.4.3.) suggesting that pregnancy does not significantly alter the metabolic consequences of labetalol administration. The decrease in arterial pCO 2 is probably due to acidemia induced hyperventilation while the increases in venous oxygen tension and content are likely a sequel to a decrease in percent oxygen extraction across the hind limb, in turn caused by significant increase in femoral blood flow. The increase in hind limb oxygen uptake suggests a labetalol induced rise in metabolic rate by carcass components. The initial hind limb uptake of labetalol (Fig 34) is probably a distribution phenomenon. Initial net uptake of glucose may suggest increased consumption of glucose by the hind limb carcass while the reversal of flux may be due to glycogenolysis and sparing of glucose. The consistent and substantial net output of lactate underlines the significant role played by the hind limb in the lactic acidosis induced by labetalol. The hind limb lactate output data can be extrapolated to the entire carcass based on carcass weight and the fraction of the carcass weight represented by the hind limb. In sheep, each hind leg constitutes about 15% of the total carcass weight, which in turn is roughly 50% of the whole body weight (Gerrard, 1977). Assuming uniform distribution of cardiac output within the carcass, the net lactate output from the carcass is approximately 41 g  162  (0.46 mol) or 0.11 g/100 g of carcass. The amount of lactate produced by the total body can be calculated as the product of arterial lactate AUC and the clearance value for lactate in sheep, based on principles of elimination kinetics (Rowland and Tozer, 1989). Lactate kinetics in nonpregnant sheep has been studied by Reilly and Chandrasena, 1978, following infusion of 14 C-lactate to steady-state. Using the lactate clearance estimate from that study in conjunction with the arterial blood lactate AUC from the current investigations, a total lactate production of about 22 g (0.24 mol) can be calculated. This estimate assumes that the metabolic clearance of lactate is independent of the concentration of lactate (i.e., linearity in lactate kinetics). While both the estimates have several  assumptions and extrapolations involved in their calculations, it would nonetheless appear that the lactate output from the carcass fully accounts for the elevation in lactate concentration seen following labetalol administration. It is possible that the glucose and lactate homeostasis across the hindlimb are interrelated, but studies with radio-labelled tracer substrates would be required to assess this. The exact mechanism involved in the metabolic effects induced by labetalol are not clear. But there are reasons to suspect the involvement of active metabolite(s) of labetalol. Firstly, following maternal labetalol administration, the lactic acidosis in the fetal lamb was much more pronounced than that in the mother, despite marginal fetal exposure to maternal labetalol (see section 4.3.3.). Secondly, the metabolic effects in both pregnant and nonpregnant sheep suggest potent 13-agonist activity. The identification of 3-APB in the urine and bile samples is particularly significant because of the close structural similarity of 3APB to d-amphetamine (Fig 29), a potent central stimulant. Not much is known about the pharmacological effects of 3-APB, but it appears that in terms of sympathomimetic activity, this compound is at least as potent as d-amphetamine (Larsen, 1938). The pharmacology and toxicology of d-amphetamine are well  163  documented and in particular, its metabolic effects in both animals and humans following lethal doses have been reported (Zalis and Parmley jr., 1963; Zalis et al., 1967). The toxicity of d-amphetamine had been attributed to the development  of a generalized hypermetabolic state, which includes hyperventilation, transient hyperglycemia, lactic acidosis, increased skeletal muscle blood flow and increased oxygen consumption, changes which were observed in this study following labetalol administration. Further, coadministration of propranolol offered protection against acute d-amphetamine intoxication (Davis et al., 1974), which suggests the involvement of 13-agonism in the amphetamine-induced metabolic effects. The systemic concentrations of d-amphetamine that were associated with these effects are not clear, but the peak plasma concentrations observed following 15-25 mg of d-amphetamine in humans (normal therapeutic dose is 10. mg; Imes and Nickerson, 1965) are reported to be in the range of 1-50 ng/mL (Beckett et al., 1969; Vree and Henderson, 1980). This along with an equal or higher potency of 3-APB (Larsen, 1938) may suggest that 3-APB could be pharmacologically active at doses that do not result in detectable plasma concentrations. Further experiments involving direct administration of 3-APB will be necessary to clearly elucidate the contribution of this metabolite towards the pharmacodynamics of labetalol. 4.5. Dilevalol Bolus Studies in Nonpregnant Sheep  Of the four isomers of labetalol, dilevalol, the R, R-isomer, has been the most extensively studied (Chrisp and Goa, 1990) and is also the only isomer which we were able to obtain in sufficient quantities to perform animal studies. However, little is known about the metabolic effects of dilevalol, which contributes to most of the 13 -adrenergic activities of labetalol (Gold et al., 1982).  164  This preliminary study described here, was undertaken to compare the hemodynamic and metabolic effects of dilevalol with that of labetalol and also to investigate its conjugative and oxidative metabolism in adult nonpregnant sheep. Extensive analysis of the pharmacokinetic and pharmacodynamic observations was not possible since the data was obtained from only two animals. 4.5.1. Pharmacokinetics  The terminal elimination half-life and total body clearance of dilevalol in the two animals (Table 8) were within the range observed with labetalol in pregnant and nonpregnant sheep (Tables 4 and 6, respectively). But the estimates of volume of distribution (Vd ss and Vd area) of dilevalol appear to be higher than that of labetalol. Clinical pharmacokinetic studies with dilevalol (Kramer et al., 1988; Tenero et al., 1989) have also shown the estimate of volume of distribution of dilevalol (16.6 - 24.6 L/kg) to be higher than the range reported for labetalol (2.5 - 15.7 L/kg) (Goa et al., 1989). The differences in the volume of distribution may be related, at least in part, to the protein binding differences. In humans, about 75% of dilevalol is bound to plasma proteins while the corresponding figure for labetalol is only 50% (Donnelly and Macphee, 1991). The fraction of dilevalol dose excreted unchanged in the urine (1.9 and 0.58%) was similar to that of labetalol (1.61 ± 0.38%). 4.5.2. Metabolism  Not much is known about the metabolism of dilevalol. The  p-  glucuronidase and arylsulphatase enzyme incubation studies with the urine and bile samples obtained following dilevalol administration show that the isomer  165  undergoes both glucuronidation and sulphation (Figs 36 and 37) like labetalol. However, the extent of glucuronidation of dilevalol (4.92 and 4.34%) appears to be roughly half of that observed in the case of labetalol (11.46 ± 2.82%). Since labetalol is glucuronidated at two sites - the phenolic and secondary hydroxy groups (Martin et al., 1976; Niemeijer et al., 1991), it could be that dilevalol is glucuronidated at only one of the sites, i.e., stereoselectivity in the glucuronidation of labetalol isomers. However, such a trend is not apparent in sulphation in that both dilevalol and labetalol seem to undergo sulphation to similar extents (about 1%)  .  The pattern of biliary excretion of the conjugates of dilevalol appears to be somewhat different from that of labetalol in that the ratio of glucuronide to sulphate conjugate concentration was about 10 in the case of dilevalol while it was between 0.6 - 3.0 with labetalol. This observation coupled with the urinary excretion profiles of the glucuronides of dilevalol and labetalol may suggest that the glucuronide conjugate of dilevalol is excreted preferentially in the bile as compared to the glucuronide conjugate of labetalol. There has been no report to date in the literature concerning the oxidative metabolism of dilevalol. Results from this study show that 3-APB was formed in adult sheep following dilevalol administration. It is interesting to note that the ratios of the 24 h cumulative urinary excretion of 3-APB in E# 105 and E#248 following dilevalol (26.87 and 2.69 mg, respectively) and following labetalol administration (93.55 and 9.17 pg) (Table 7) were roughly equal to about 25%, i.e., the ratio of the doses administered, suggesting the lack of significant stereoselectivity. However, data from at least two other isomers are required to verify this.  4.5.3. Hemodynamic Effects  166  Dilevalol causes hypotension, transient tachycardia and increase in femoral blood flow (Fig 39) in adult nonpregnant sheep similar to that observed with labetalol. It is interesting to note that the magnitude of these hemodynamic changes caused by 25 mg dilevalol bolus was similar to that caused by 100 mg labetalol bolus in adult nonpregnant sheep (Fig 30). Thus, it appears that the hemodynamic changes observed following labetalol administration are almost completely attributable to one isomer, i.e., dilevalol. But since dilevalol is devoid of any a-adrenergic activity (Gold et al., 1982), it would follow that the appreciable increase in femoral blood flow induced by dilevalol is caused either by its partial 13-agonism or by a direct mechanism. Further, while dilevalol is considered to be four times more potent in its 13-blocking activity than labetalol (Sybertz et al., 1981), the appreciable tachycardia observed in this study suggests that dilevalol does not exert significant p i -blockade in adult nonpregnant sheep. The progressive increase in heart rate, resulting in a distinct lag time in the maximum effect observed, may indicate that the tachycardia is mediated by a reflex or other indirect mechanism and not due to the partial sympathomimetic activity of dilevalol. 4.5.4. Metabolic Effects  Notable hyperglycemia and lactic acidosis were observed following dilevalol administration (Fig 41). The magnitude of lactic acidosis seen after dilevalol (59.9 mMh) was similar to that observed with a 100 mg labetalol bolus in pregnant (53.6 ± 1.38 mMh) and nonpregnant sheep (42.7 ± 4.9 mMh) implying that the observed metabolic effects of labetalol could be totally attributed to the effects of dilevalol. However, in contrast to the magnitude of arterial lactate  167  elevation, the hind limb lactate flux changes after dilevalol appear to be different from that observed after labetalol. Firstly, the lactate flux in the initial 3 h following dilevalol administration shows net uptake by the hind limb (cf. changes with labetalol, section 4.4.4.). Secondly, the net release of lactate from the hind limb (2.13 g) was roughly one-third of that observed after labetalol administration despite similar arterial lactate AUCs. These differences might indicate that a considerable amount of lactate was released in response to dilevalol administration from a site other than hind limb. However, a larger sample size will be required to confirm these differences. 4.6. Labetalol Infusion in Nonpregnant Sheep  Labetalol is used in both short term and long term clinical management of hypertension of various etiologies including pre-eclampsia (Goa et al., 1989), but the metabolic consequences of continuous administration of labetalol have not been studied. This is especially important in the light of observations in sheep following a single intravenous bolus administration, which suggest profound metabolic disturbances. The two main objectives of this study were (a) to assess the hemodynamic and metabolic consesquences of continuous infusion of labetalol in adult nonpregnant sheep and (b) to determine the contribution of oc-blockade to peripheral vasodilation, which appears to be largely responsible for the hypotensive action of labetalol in adult nonpregnant sheep. 4.6.1. Disposition  The priming bolus dose and the infusion rate of labetalol used in this study were estimated from pharmacokinetic parameters obtained in the pregnant sheep  168  (see section 3.3.2.) to obtain average steady-state concentrations reported in clinical situations (about 300 ng/mL) following mutiple oral administrations of labetalol (Sanders et al., 1980; McNeil et al., 1982; Chung et al., 1986). The infusion was continued for 6 h to accommodate NE dose ranging experiments in duplicate. The mean steady-state concentration achieved (454.19 ± 111.05 ng/mL) appears to be higher than the target concentration of 300 ng/mL, although the difference was not significant (two-tailed, one-sample t-test). The apparent high steady-state concentration could be due to altered pharmacolcinetics during infusion. The clearance of labetalol following infusion (17.62 ± 3.02 mL/min/kg) was significantly lower than that obtained following a single bolus administration (30.80 ± 3.83 mL/min/kg) (paired t-test). Similar observations have been made in humans (McNeil et al., 1982; Chauvin et al., 1987). The decreased clearance at steady-state could be due to saturable first-pass hepatic extraction, a phenomenon noted with other 13-blockers like propranolol (Evans and Shand, 1973). 4.6.2. Hemodynamic Effects  The hemodynamic effects of continuous infusion of labetalol to steady-state were qualitatively similar to that observed following a single bolus (see section 3.4.4.) and include hypotension, tachycardia and increase in femoral blood flow.  However, the hypotension was sustained for up to 10 h post-infusion, with the arterial pressure remaining significantly decreased even after the femoral blood flow had returned to control values, suggesting the possibility of a different mechanism in the maintenance of hypotension as opposed to initiation, through peripheral vasodilation. It is also interesting to note that the tachycardia was transient and the heart rate gradually returns to baseline values by 4 h into the infusion despite continued vasodilation and hypotension. It could be that the  169  reflex tachycardia is strictly an acute response to peripheral vasodilation and hence it could be inferred that long teim/continuous administration of labetalol in sheep will produce sustained hypotension without causing tachycardia. 4.6.3. Metabolic Effects  The metabolic effects in this study (increase in glucose and lactate concentrations, increase in venous oxygen content and a trend towards increased oxygen consumption in the hind limb), which are qualitatively similar to that observed following bolus administration, underline the potential of labetalol to cause significant metabolic effects including lactic acidosis at therapeutic concentrations. The net amount of lactate released from the hind limb (9.2 ± 3.12 g) in response to labetalol infusion is roughly 50% higher than that observed following a 100 mg i.v. bolus (see section 4.4.4.). The glucose and lactate concentrations reached a peak during the infusion and started to decline well before the end of infusion (Fig 48). This may be due to desensitization of the Preceptors (Hausdorf et al., 1990) following continuous administration, a phenomenon observed with continuous infusion of ritodrine, a f3 agonist, in sheep -  (Bassett et al., 1985).  The implications of these results in clinical practice are not known at this stage, but investigation of the metabolic effects of acute and chronic administration of labetalol in humans is warranted. 4.6.4. Intra-arterial Norepinephrine Studies  The cornerstone of the observed hemodynamic effects of labetalol in sheep has been the significant increase in femoral blood flow. The peripheral  170  vasodilatory effect of labetalol has been previously demonstrated in dogs (Baum et al., 1981; Dage and Hsieh, 1980), rats and isolated rabbit portal vein strips  (Johnson et al., 1977), but reports about the mechanism involved are conflicting. Suggested mechanisms include 132-agonism (Baum et al., 1981), a-blockade (Sweet et al., 1979) and direct vasodilation (Dage and Hsieh, 1980; Johnson et al., 1977). These differences could be at least in part due to the differences in the experimental situations involved in the above studies (species employed and differences in experimental protocol). The contribution of peripheral a-blockade was assessed in this study with intra-arterial NE, at doses that do not cause any detectable systemic effects (an example shown in Fig 52). The transient vasoconstriction caused by NE was dose dependent as well as readily reproducible (Fig 50). However, steady-state concentrations of labetalol did not truncate or shift the dose-response relationship of NE, which implies that labetalol does not produce any significant a-blockade in sheep. Similar conclusions were reached from studies conducted in dogs by Sybertz et al., 1981. 4.7. Intra-arterial Labetalol Studies  The intra-arterial NE studies clearly show that a-blockade is not involved in the observed femoral vasodilation, at least in adult sheep. Studies with intraarterial administration of labetalol were conducted primarily to determine whether labetalol has a direct vascular effect. 4.7.1. Studies in Adult Nonpregnant Sheep  The immediate (Fig 52) and dose dependent vasodilation (Fig 51) caused by intra-arterial injections of labetalol suggest that labetalol has a direct  171  vasodilatory effect in the adult sheep hind limb. That a-blockade is not the primary mechanism of vasodilation is also seen in the intra-arterial injections following phentolamine infusion (Fig 51 A), which is in agreement with the results obtained from intra-arterial NE studies. Propranolol failed to abolish the vasodilatory response (Fig 51 B) suggesting that [3-agonism is not the principal mechansim, at least in sheep. This is in contrast to the observations by Baum et al., 1981, who reported that propranolol attenuated the vasodilatory effect of intra-  arterial labetalol in dogs. Intravenous administration of ritodrine, a f3 2 -agonist, does not cause any significant change in femoral blood flow in sheep (Rurak, D.W., Kwan, E., Hall, C., Wright, M.R., Szeitz, A., and Axelson, J.E., unpublished observations). Thus it appears that the vasodilation of labetalol in sheep is unrelated to its adrenergic activities. Contribution in part, from other indirect mechanisms, such as mediation of this effect through an active metabolite (Gal et al., 1988) could not be ruled out and remains to be studied. 4.7.2. Studies in Fetal Sheep  In striking contrast to the observations in adult sheep, in which labetalol causes dose-dependent femoral arterial vasodilation, direct administration of labetalol via the external pudendal epigastric artery in the fetal lamb (0.1 to 500 Ix g) failed to produce any consistent vasodilatory effect. However, these results are in agreement with the lack of significant changes in hind limb blood flow following direct fetal intravenous bolus administration (see section 3.8.4.). The disparity in the hind limb flow responses in adult and fetal sheep to intra-arterial labetalol are probably indicative of some fundamental differences in the fetal vasculature as compared to the adult. It is unlikely that these differences are related to receptor differentiation in the fetus, especially [3-receptors, since Van  172  Petten and Wiles, 1970 have demonstrated that the response to isoproterenol and propranolol in the ovine fetus in late gestation is similar to that of the ewe in terms of changes in arterial pressure, heart rate and total peripheral resistance. A better understanding of the exact mechanism involved in the labetalol induced vasodilation in the adult sheep is necessary to identify the differentiating feature in the fetus. 4.8. Fetal Labetalol Bolus Studies  The dose of labetalol used in the clinical management of pre-eclampsia and subsequent fetal exposure to this drug varies considerably due to the large interindividual variability in the hypotensive response to labetalol. Doses of up to 200 mg in the form of intravenous infusion (Michael 1986; Ashe et al., 1987) and 1200 mg p.o. (Michael, 1982) have been used and the reported cord blood concentration of labetalol at the time of delivery ranges from 10-260 ng/mL (Michael, 1979; Rogers et al., 1990). As previously mentioned, the single point cord blood concentrations do not accurately reflect the in utero fetal exposure to the drug (Anderson et al., 1980b) and hence it is likely that the human fetus is exposed to concentrations higher than that suggested by the cord blood concentrations. The present study was undertaken primarily to assess the effects of labetalol in the fetus following exposure to clinically relevant concentrations and to investigate the ability of the fetus to metabolise and eliminate labetalol. Direct fetal administration of labetalol was used because of the restricted placental transfer of labetalol following maternal administration (see section 3.3.2.). The chronically instrumented fetal hind limb was used as a representative of the fetal carcass to assess whether those tissues, which receive about 40% of fetal  173  combined ventricular output (Rudolph and Heymann, 1970), could be involved in any labetalol induced fetal lactic acidosis. 4.8.1. Pharmacokinetics  The disposition of labetalol in the fetal lamb was characterized by extensive uptake in the tracheal fluid (Fig 54), a phenomenon observed with other amino compounds like metoclopramide and diphenhydramine (Riggs et al., 1987) and ritodrine (Wright et al., 1991b). Fetal tracheal fluid is formed at a rate of 4 mL/h in the late gestation ovine fetus primarily by the active transport of chloride ion across the pulmonary epithelium (Harding et al., 1984). While the Na+ and K+ concentrations of tracheal fluid are similar to that of plasma, the chloride concentration is much higher and the protein concentration much lower than that of plasma (Olver and Strang, 1974). In this study, the C TR/C FA ratio for labetalol was 3.84 ± 0.21 and is similar to that of ritodrine and diphenhydramine (ratio 4), but lower than that of metoclopramide (ratio of about 15). It appears that in the adult, amphiphilic weakly basic amine drugs accumulate and persist in lung tissue to a greater extent than neutral or basic compounds (Philpot et al., 1977; Benford and Bridges, 1986; Okumura et al., 1978). While the exact mechanism involved in the uptake of amino drugs in the adult lung is not clear, both passive diffusion and carrier-mediated active transport processes across the pulmonary endothelial cells have been suggested (Philpot et al., 1977; Bend et al., 1985). The accumulation of drug in fetal tracheal fluid may  result, at least in part, from drug uptake by the fetal lung and subsequent release into the tracheal fluid (Rurak et al., 1991). In addition, compounds with 13 2agonist activity cause significant decrease in the production of fetal tracheal fluid and the resorption of this fluid across the fetal lung (Warburton et al., 1987 a and  174  b), which in turn may increase the apparent concentration of the drug in tracheal fluid (Wright, 1992). Such a mechanism is possible in the case of labetalol since following maternal or fetal administration in pregnant sheep as well as in nonpregnant sheep, labetalol appears to exert potent I3 2 -agonism (see sections 4.3.3., 4.4.4. and 4.8.4.). Administration of amphiphilic amine drugs, which accumulate and persist in lung tissue, to animals at high doses or over prolonged periods of time results in pulmonary phospholiposis. But the exact developmental significance of accumulation of labetalol in fetal tracheal fluid and the consequent exposure of fetal lung to high concentrations of labetalol remains to be explored. The terminal elimination half-life, the body weight-normalised total body clearance and the volume of distribution of labetalol in the fetus were significantly higher than that observed in the pregnant ewe following maternal administration (see section 3.3.2.), suggesting distinct differences between the fetus and adult in the distribution and elimination of labetalol. The longer apparent elimination halflife in the fetus (following maternal or fetal administration) could be due to accumulation of the drug in amniotic and tracheal fluids and possible recirculation from these sites, although preferential binding to fetal tissues and proteins cannot be ruled out. Placental transfer of labetalol from the fetus to the mother was rapid since peak maternal concentrations were reached almost instantaneously (Fig 54). Although the maternal to fetal plasma AUC concentration was just 3.01 ± 0.2%, the calculated amount of labetalol transferred across the placenta (calculated as the product of mean maternal total body clearance obtained from maternal bolus studies and the total maternal AUC observed in this study) is equal to 1.88 ± 0.7 mg, i.e., 47.01 ± 17.5% of the fetal dose. The apparent maternal half-life of labetalol observed in this study (4.75 ± 0.15 h) was not significantly different from the terminal elimination half-life in the fetus (4.35 ± 0.33 h), but significantly higher than that observed in the ewe following maternal administration (2.79 ±  175  0.66 h). This suggests that the apparent maternal elimination half-life of labetalol following a fetal bolus is determined by the continuous input of labetalol from the fetal circulation at a rate equal to the rate of elimination in the fetus. From the transplacental and nonplacental clearance estimates, it appears that the higher fetal total body clearance (CL f = 50.45 ± 1.37 mL/min/kg and CL m from maternal bolus study = 30.8 ± 3.83 mL/min/kg) is due to its nonplacental contribution (CL f„), which is significantly higher than that of the mother (CL„ m) (Table 10). However, unlike the case with most other drugs for which data are available (Rurak et al., 1991), the calculated CL fr, of labetalol (7.27 ± 2.11 mL/min/kg) is much lower than CL mp (23.4 ± 8.9 mL/min/kg), the reasons for which are not clear but may involve the fact that some of the data used to calculate the fetal and maternal clearances (i.e., CLm, FAUC/MAUC) were obtained from a previous study using a different group of sheep. Labetalol induced changes in uteroplacental/fetoplacental hemodynamics or differences between maternal and fetal protein binding of labetalol could also be involved. In common with the other drugs that have been studied in pregnant sheep (Rurak et al., 1991), the percentage of total labetalol clearance due to transplacental clearance is higher in the fetus (14.4 ± 1.54 %) than in the ewe (3.1 ± 0.2 %). The ovine fetal transplacental clearance of labetalol (7.27 ± 2.11 mL/min/kg) is less than that reported for morphine (19 ± 2 mL/min/kg), methadone (168 ± 29 mL/min/kg), metoclopramide (103 ± 13 mL/min/kg), diphenhydramine (124 ± 22 mL/min/kg) and acetaminophen (31 ± 2 mL/min/kg), but higher than that for ritodrine (5 ± 2 mL/min/kg) (Rural( et al., 1991). The factors which influence the transplacental drug transfer, in general, include the physico-chemical properties of the drug (e.g., molecular weight, pKa, partition coefficient), maternal and fetal protein binding, maternal and fetal placental blood flows and the extent of placental drug metabolism (Levy and Hayton, 1973; Reynolds and Knott, 1989; Rurak et al.,  176  1991). However, protein binding is not likely to be the major factor since the plasma protein binding of labetalol (about 50% in humans; Martin et al., 1976) is similar to that reported for metoclopramide (49.5% in pregnant ewe; Riggs et al., 1990) and ritodrine (38% in humans; Kuhnert et al., 1986). Among the physicochemical properties, the molecular weights of labetalol (MW = 329), metoclopramide (MW = 300) and ritodrine (MW = 287) are quite similar, suggesting that molecular size per se is not a critical determinant of the extent of placental transfer for these drugs. However, the partition coefficient of these compounds do differ appreciably with labetalol having a value of 1.2 (Riley, 1981), which is intermediate between that of metoclopramide (12.0; Okumura et al., 1978) and ritodrine (<0.01; Nandakumaran et al., 1982). Thus, the extent of placental transfer and transplacental clearance of drugs appear to depend markedly on lipid solubility of the compounds (Rurak et al., 1991).  4.8.2. Metabolism  The results from the enzyme incubation of amniotic fluid samples (Fig 55) shows that labetalol undergoes extensive glucuronidation in the fetal lamb, with a mean glucuronide/free drug concentration ratio (over 24 h) of 11.44 ± 2.82. The conjugates are likely of fetal origin since the ovine placenta has insignificant permeability to glucuronides (Wang et al., 1985). Although labetalol also undergoes sulfate conjugation in adult sheep (see section 3.4.3.1.), no evidence for sulfation was found in the present study and this is similar to results recently reported for ritodrine in fetal lamb (Wright et al., 1991b). The amniotic fluid concentration of glucuronide at 12 h (820.5 ± 77.1 ng/mL) represents roughly 20% of the administered dose (assuming a normal amniotic fluid volume of 1 L) and since fetal urine is equally divided between amniotic and allantoic fluid cavities  177  (Wlodek et al., 1988), it is likely that similar concentrations were present in the allantoic fluid as well. Thus, about 40% of the fetal labetalol dose appears to be glucuronidated, which underlines the significance of conjugation in the fetal nonplacental clearance of labetalol. The oxidative metabolite of labetalol, 3-APB could not be detected in the plasma or amniotic fluid samples. The extremely short apparent elimination half-life of the metabolite (estimated to be about 13.5 minutes in adult sheep), the low fractional turnover (about 0.05% of labetalol dose in adult sheep) and the possible increase in amniotic fluid volume due to fetal lactic acidosis (Powell and Brace, 1991) may explain the absence of 3-APB in amniotic fluid. However, quantitative or even qualitative differences in the metabolism of labetalol between the fetus and adult cannot be ruled out.  4.8.3. Hemodynamic Effects The observed hemodynamic changes suggest that labetalol does not cause any significant cardiovascular effects in the fetal lamb unlike that in the adult sheep. The significant changes in arterial pressure and heart rate induced by labetalol in the adult sheep appear to be secondary to the significant peripheral vasodilation (see section 4.4.3.). Thus, the lack of any significant hypotension or tachycardia in the fetal lamb after labetalol administration is likely consequent to the absence of direct vasodilatory effect of labetalol in the fetus, the reasons for which are not clear. The marginal bradycardia in the initial 2 h followed by a progressive tachycardia could be due to the lactic acidosis since exogenous lactic acid administration has been shown to cause similar changes in ovine fetal heart rate (Bocking et al., 1991). 4.8.4. Metabolic Effects  178  The changes in the blood pH, base excess and lactic acid concentrations indicate the development of fetal metabolic acidosis, similar to that observed following maternal labetalol administration (see section 3.4.5.). And there was also a similar rise in fetal glucose concentration. However, in this case, there were no significant changes in maternal glucose concentrations, indicating that the fetal hyperglycemia resulted from increased fetal glucose supply/production and/or decreased utilization. As was discussed previously (p. 151), i3 2 -adrenergic agonists like ritodrine, induce hyperglycemia in the ewe and fetus, and in the latter, at least, this appears to be due to increased glycogenolysis (Warburton et al., 1988). Hence, the labetalol induced fetal hyperglycemia may be via the same  mechanism. Concentrations of labetalol, glucose and lactate in HLA and HLV were monitored to understand the role of skeletal muscles and other carcass constituents in the perturbation of carbohydrate metabolism induced by labetalol. The net uptake of labetalol seen in the initial 60 min is probably due to rapid tissue distribution of the drug. While there are no changes in glucose uptake following labetalol administration, the lactate flux shows a delayed net release from the hind limb. The net lactate output from the hind limb (3.85 ± 2.05 g) can be extrapolated to the entire carcass based on fetal carcass and hind limb weights. The ovine fetal hind limb (weight —300 g) represents roughly 10% of total fetal body weight while the entire carcass constitutes about 70% of body weight (Wilkening et al., 1988). Assuming, for simplicity, that lactate production and utilization is the same throughout the carcass, the net output of lactate from the entire carcass following labetalol administration will be about 27.0 g (0.30 moles). The total lactate produced in the body can also be calculated from the metabolic clearance value of lactate in the fetal lamb and the incremental increase in arterial  179  lactate AUC following labetalol administration based on basic principles of elimination kinetics (Rowland and Tozer, 1989) as applied in adult nonpregnant sheep studies (see section 4.4.4.). An estimate of lactate clearance in the fetal lamb can be calculated from the data provided by Sparks et al., 1982, and is equal to 35 mL/min/kg. The total amount of lactate produced is calculated as the product of the incremental increase in arterial lactate AUC following labetalol administration (61.1 mMh) and the mean lactate clearance and it amounts to 34.4 g (0.38 moles). This calculation assumes that lactate kinetics is linear over the concentrations involved. From these calculations, it would appear that carcass plays a significant role in the development of lactic acidosis in response to fetal labetalol administration. The reason(s) for the distinct lag time (4-5 h) seen in lactate release from the hind limb (Fig 63) is not clear but may suggest an indirect or mediated effect. The observed delay in hind limb flux may also suggest that lactate release over the initial 4 h occurs from a site other than the hind limb since arterial lactate concentrations do not exhibit a similar lag time (Fig 60). As discussed earlier (p. 155), one potential site is the placenta. The marginal increase seen in maternal arterial blood lactate concentration (net increase in AUC equal to 5.31 ± 5.28 mMh) is likely due to labetalol induced release of lactate in the mother and/or increased placental lactate production since the ovine placenta is nearly impermeable to lactic acid (Sparks et al., 1982). Fetal exposure to labetalol in this study, determined by plasma AUC, is roughly four times higher than that observed following maternal administration of labetalol (441.76 ± 11.52 vs 111. 86 ± 11.14 p.g*h/L) and the peak plasma concentration was about six times higher (207.39 ± 27.66 vs 33.7 ± 5.8 ng/mL). But, the magnitude of changes in the various pharmacodynamic parameters (decrease in fetal arterial blood pH by 0.096 ± 0.015, base excess by 5.7 ± 1.3 mEq/L and net increase in lactate AUC by 61.11 ± 15.59 mMh) were not  180  significantly different from that observed following maternal administration of labetalol in pregnant sheep (0.100 ± 0.011, 7.0 ± 1.3 mEq/L and 67.8 ± 7.26 mMh respectively) (two-sample t-test). This pharmacokinetic-phannacodynamic dissociation may suggest that the metabolic effects are actually mediated by active metabolite(s) and that the governing factor is fetal exposure to the metabolite(s). It could be hypothesized that the fetal exposure to the active metabolite(s) following maternal administration of labetalol is higher than that resulting from direct fetal administration. It could be speculated at this stage that 3-APB contributes to the pharmacological/toxicological effects of labetalol in the fetal lamb, since this metabolite is present in adult sheep urine and bile, but further experiments involving direct administration of 3-APB to the ewe and fetal lamb are required to clearly elucidate the role of this metabolite in the observed metabolic effects.  181  5. SUMMARY AND CONCLUSIONS  5.1. Quantitation of Labetalol in Biological Fluids  A sensitive assay using microbore high-performance liquid chromatography and low-dispersion fluorescence detection, has been developed for the quantitation of labetalol in various biological fluids of the pregnant sheep (Yeleswaram  et al.,  1991). The method represents significant improvement over previously published assays in terms of volume of sample required, precision of quantitation and minimum quantitation limit The sensitivity of the method has been optimized by i)  the use of microbore column and connections to minimize the dead volume in the system,  ii)  reducing the baseline noise with an appropriate cut-off filter,  iii)  enhancing the signal intensity by using wider excitation and emission slits and  iv)  by using a low-dispersion fluorescence cell.  The intra-day coefficients of variation were less than 10% in all cases, the mean extraction recovery of labetalol from various biological fluids was between 70 to 76% and the minimum quantitation limit of the assay was 30 pg injected. 5.2. Analysis of 3-amino-l-phenylbutane in Biological Fluids  A sensitive and specific gas chromatographic assay with electron-impact ionization and mass selective detection has been developed for the identification and quantitation of 3-amino-l-phenylbutane, an oxidative metabolite of labetalol (Yeleswaram et al., 1992b). This method represents the first assay for this metabolite involving mass spectrometry with electron-impact ionization. The  182  samples were extracted with n-hexane, derivatized with heptafluorobutyric anhydride, chromatographed on a cross-linked 5% phenylmethylsilicone stationary phase and subjected to electron-impact ionization. Identification of the metabolite was accomplished with an authentic standard. Quantitation was performed by selectively monitoring two ions of the derivative (m/z=345 and 132), including the molecular ion (m/z=345). The coefficients of intra-sample variation were less than 12% in all cases, the extraction recovery was between 98 to 103% and the minimum limit of quantitation of the assay was 2 pg (amount injected). 5.3. Maternal Bolus Administration of Labetalol  In this study, labetalol was administered as a 100 mg i.v. bolus to the ewe and serial samples were obtained from maternal and fetal artery as well as amniotic and tracheal fluid to characterize the disposition of labetalol. Maternal and fetal cardiovascular and blood gas parameters were monitored before and for up to 24 h following labetalol administration. Fetal exposure to labetalol, determined as fetal/maternal arterial plasma AUC ratio, was found to be 14.4 ± 1.5% (n=8). Labetalol persisted in the amniotic and tracheal fluid compartments for up to 24 h. The apparent elimination half-life of the drug in the fetus (3.7 0.5 h) was significantly higher than that in the mother (2.8 0.7 h). There were no significant changes in maternal or fetal cardiovascular parameters (heart rate and mean arterial pressure), but marked changes were seen in the fetal blood gas parameters. The fetal blood pH, base excess and oxygen content decreased significantly. Only minimal changes were observed in the maternal blood pH and base excess. The significant blood gas changes in the initial experiments led to the monitoring of maternal and fetal glucose and lactic acid concentrations in subsequent experiments, as markers of carbohydrate metabolism. The results  183  indicate significant, albeit reversible, hyperglycemia and lactic acidosis in both the fetus and ewe. The metabolic effects produced by labetalol resembled that of a potent P 2 -agonist. Also, the elevation in lactic acid concentration was more pronounced as well as lasted longer in the fetus than in the mother despite marginal placental transfer of labetalol, suggesting an indirect or mediated effect.  5.4. Labetalol Bolus in Adult Nonpregnant Sheep  This study was conducted to assess the contribution of carcass in general and in particular the hind limb, to the development of lactic acidosis in response to labetalol administration and to investigate the metabolism of labetalol. Following a 100 mg intravenous bolus, labetalol, glucose and lactate concentrations in femoral artery and vein, along with femoral blood flow, heart rate and arterial pressure were monitored. Urine and bile samples were obtained for the study of conjugative and oxidative metabolism. A significant increase in femoral blood flow, hypotension, reflex tachycardia, hyperglycemia, lactic acidosis with a substantial output of lactate (6.25 ± 1.35 g) from the hind limb were observed. Glucuronide and sulfate conjugates of labetalol were present in the urine and bile samples. The cumulative urinary excretion of labetalol as unchanged drug, glucuronide and sulfate were found to be 1.61 ± 0.38, 11.46 ± 2.83 and 1.47 ± 0.74% of the dose respectively. The oxidative metabolite, 3-amino-lphenylbutane, was identified in urine and bile samples, with a cumulative urinary excretion equivalent to 0.044 ± 0.016% of labetalol dose. Glucuronide conjugates of the metabolite in urine samples and glucuronide and sulfate conjugates of the metabolite in bile samples were also present. The structural similarity of 3-amino1-phenylbutane and amphetamine on the one hand, and the close similarity between the reported metabolic effects of amphetamine and that observed in sheep  184  following labetalol administration on the other hand, may suggest a possible involvement of the metabolite in the mediation of labetalol pharmacodynamics. However, experiments involving direct administration of the metabolite are necessary to establish the cause-effect relationship unequivocally. 5.5. Dilevalol Bolus in Adult Nonpregnant Sheep  The pharmacokinetics and pharmacodynamics of dilevalol, the RR-isomer of labetalol, were studied in two animals. With the exception of the apparent volume of distribution, which was higher, the estimates of pharmacokinetic parameters of dilevalol were within the respective ranges observed with labetalol. The cardiovascular and metabolic effects of dilevalol in adult non-pregnant sheep were also similar to those of labetalol, suggesting that the observed pharmacological effects of labetalol in sheep were actually elicited by one of the four isomers, i.e., dilevalol. But, further experiments are required to confirm this. 5.6. Labetalol Infusion to Steady-state in Adult Nonpregnant Sheep  The hypothesis that the mechanism of vasodilation caused by labetalol is ablockade was examined with intra-arterial injections of norepinephrine, before and during continuous infusion of labetalol to steady-state. No significant shift in the intra-arterial norepinephrine dose-response relationship was caused by steady-state concentrations of labetalol, thus implying that a-blockade is not the primary mechanism involved in the vasodilation caused by labetalol. 5.7. Intra-arterial Administration of Labetalol  185  In the adult sheep hind limb, intra-arterial injections of labetalol ranging from 0.1 pg to 1.0 mg, resulted in spontaneous and dose-dependent vasodilation. Further, administration of phentolamine, an a-adrenoceptor blocker, or propranolol, a 13-adrenoceptor blocker, did not cause any significant change in the vasodilatory response to intra-arterial administration of labetalol. These results suggest that labetalol has direct vasodilatory effect and that a-blockade or  p-  agonism are not involved in the vasodilation. In the fetal lamb hind limb, infraarterial administration of labetalol failed to produce any consistent, dosedependent change in hind limb blood flow. This observation may suggest that the fetal vasculature is different from that of the adult.  5.8. Labetalol Bolus Administration in the Fetal Lamb The disposition, metabolism and pharmacodynamics of labetalol in the fetal lamb were studied following exposure to clinically relevant labetalol concentrations, achieved with a 4 mg direct intravenous fetal bolus administration. The total body clearance (50.45 ± 1.37 mL/min/kg), elimination half-life (4.35 ± 0.33 h) and apparent volume of distribution at steady-state (14.28 ± 0.95 L/kg) of labetalol in the fetal lamb were significantly higher than the corresponding values in the ewe following a 100 mg bolus. The ratio of maternal to fetal plasma AUC was equal to 0.031 ± 0.002. The calculated fetal transplacental clearance was 7.27 ± 2.11 mL/min/kg while the fetal nonplacental clearance was 43.18 ± 3.72 mL/min/kg. Glucuronide conjugates were present in the amniotic fluid samples and the amount of the conjugates at 12 h represents about 20% of the administered dose. The oxidative metabolite, 3-amino-l-phenylbutane, could not be identified in fetal plasma or amniotic fluid samples. No significant changes in fetal heart rate, arterial pressure and hind limb blood flow, but significant increases in arterial  186  and hind limb venous blood glucose and lactate concentrations were observed. The net output of lactate from the fetal hind limb was equal to 3.85 ± 2.05 g. The magnitude of fetal lactic acidosis (incremental increase in the area under the arterial lactate concentration-time curve) (61.11 ± 15.59 mMh) was not significantly different from that observed following maternal administration (67.8 ± 7.26 mMh), despite a roughly four-fold higher fetal exposure to labetalol (as determined from area under the plasma labetalol concentration-time curve values). The following conclusions could be reached from these studies: (1) Labetalol undergoes rapid but limited placental transfer in the pregnant sheep. (2) Following maternal or fetal administration, labetalol causes significant metabolic effects in the fetus, including lactic acidosis. But the magnitude of the metabolic effects were not proportional to the extent of fetal exposure to labetalol. (3) The hind limb in particular and the carcass in general, appear to release lactic acid in response to labetalol administration in both the adult and fetal sheep. (4) The hypotensive effect of labetalol in adult nonpregnant sheep appears to be caused primarily by a significant peripheral vasodilation. The vasodilation seems to be through a direct effect of labetalol and it does not involve a-blockade or P-agonism. In contrast, labetalol does not cause any significant vasodilation or hypotension in the fetal lamb following similar doses. 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