Application of Stable Isotope Labeled Diphenhydramine to Study the Pharmacokinetics and Metabolism of Diphenhydramine in Pregnant, Non- Pregnant, and Fetal Sheep. By George Roger Tonn B.Sc. (Pharm.), University of British Columbia, Vancouver, Canada, 1988. M.Sc. (Pharm.), University of British Columbia, Vancouver, Canada, 1990. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences) (Division of Biopharmaceutics and Pharmaceutics) We accept this thesis as conforming to the standard required THE UNIVERSITY OF BRITISH COLUMBIA February, 1995 © George Roger Tonn, 1995 advanced In presenting this thesis in partial fulfilment of the requirements for an Library shall make it degree at the University of British Columbia, I agree that the that permission for extensive freely available for reference and study. I further agree 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. Department of Pharmaceutical Sciences The University of British Columbia Vancouver, Canada 2 7—02-95 Date DE-6 (2188) 11 II. Abstract Diphenhydramine (DPHM), an antihistamine, has been used in pregnant women; however, information regarding its disposition in human pregnancy is lacking. Recently, detailed pharmacokinetic studies in pregnant sheep have demonstrated that DPHM readily crosses the ovine placenta, and is eliminated from the fetus by placental and non-placental pathways. The purpose of this study is to investigate the components of the fetal non-placental elimination (i.e., fetal renal and hepatic), and to compare these to the estimates obtained from adult sheep. Since stable isotope techniques were to be employed, synthesis of stable isotope labeled DPHM (i.e., {2H10]DPMA) was [2H10]DPHM) and its major metabolite diphenylmethoxyacetic acid (i.e., required. Next, gas chromatographic - mass spectrometric methods were developed to [2H10]DPHM, or DPMA and [2H10]DPMA. The simultaneously measure either DPHM and current study demonstrates that the measured fetal renal clearance of DPHM contributes only —2% to the observed fetal non-placental clearance. Overall, the total non-placental clearance of DPHM measured by direct methods (i.e., pulmonary [Yoo, 1989] and renal) can account for 10% of the non-placental clearance. Unlike adult sheep, where hepatic extraction of DPHM was —93%, no significant extraction of DPHM by the fetal liver following umbilical venous administration was observed. Therefore, fetal hepatic elimination is not likely to account for the remainder of the fetal non-placental clearance. However, fetal hepatic in vitro metabolism of DPHM (to form N-demethyl DPHM and DPMA) suggests that the fetal liver is capable of DPHM biotransformation. Thus, the liver and possibly other organs may contribute at least a portion of the fetal non-placental clearance via DPHM biotransformation. It appears that only a small fraction of the fetal non-placental clearance of DPHM can be accounted for by fetal renal 111 and pulmonary clearances. While the low renal clearance of intact DPHM is similar both in fetus and mother, large differences in the hepatic uptake andJor metabolism of DPHM were observed between mother and fetus. This suggests that the pathways for the non-placental elimination of DPHM differ in mother and fetus. Despite the advances made in this study, the components of the fetal non-placental clearance remains largely unknown, and requires further study. iv IV. Table of Contents II. Short Abstract jj ifi. Table of Contents iv IV. List of Tables ix V. List of Figures xii VI. List of Abbreviations xvi VII. Acknowledgments xxii 1. INTRODUCTION 1 1.1. General Introduction 1 1.2. Pharmacology of DPHM 2 1.3. Toxicology of DPHM 3 1.4. Clinical Applications of DPHM 4 1.5. Therapeutic Applications of Antihistamines In Pregnancy 4 1.6. Analysis Methods for DPHM 5 1.7. Pharmacokinetics of DPHM 6 1.7.1. Absorption of DPHM 6 1.7.2. Distribution of DPHM 7 1.7.3. Metabolism of DPHM 7 1.7.4. Excretion of DPHM and its Metabolites 9 1.8. DPHM Disposition in Pregnancy 10 1.8.1. Disposition and Fetal Effects of DPHM in Pregnant Sheep 11 1.9. Stable Isotopes 14 1.10. Rationale and Objectives 16 1.11. Hypothesis and Specific Aims 17 2. EXPERIMENTAL 20 2.1. Materials 20 2.1.1. Preparation of Stock Solutions and Buffers 22 2.2. Equipment and Instrumentation 24 2.2.1. Gas Chromatography with Nitrogen Phosphorus Specific Detection 24 2.2.2. Gas Chromatography with Mass Spectrometry 25 2.2.3. Gas Chromatography/High Performance Liquid Chromatography - 25 Mass Spectrometry 2.2.4. Spectrophotometer 25 2.2.5. Physiological Monitoring 25 2.2.6. General Experimental Equipment 26 2.3. Chemical Synthesis of Standards and Metabolites of DPHM 27 [2Hiojbenzhydrol 27 2.3.1. Synthesis of [2H10]DPHM HC1 29 2.3.2. Synthesis of [2H10]DPHM 31 2.3.2.1. Characterization of V 2.3.3. Synthesis of N-demethyl DPHM HC1 and N,N-didemethyl DPHM HC1 31 2.3.3.1. Characterization of N-demethyl DPHM and N,N-didemethyl DPHM 31 {2H10]DPMA 2.3.4. Synthesis of DPMA and 32 [2H IO]DPMA 2.3.4.1. Characterization of DPMA and 33 2.3.5. Purity Assessment of Synthesized Standards. 35 2.4. Analytical Method Development 37 Development Analysis Method [2H10]DPHM 2.4.1. of an for DPHM and 37 2.4.1.1. Optimization of Mass Spectrometer Parameters 37 2.4.1.2. Optimization of Gas Chromatographic Parameters 37 2.4.1.3. Optimization of Extraction Procedure 38 2.4.1.4. Gas Chromatography - Mass Spectrometer Operating Conditions 38 [2H10JDPHM Extraction Procedure 2.4.1.5. DPHM and 39 2.4.1.6. Preparation of a Calibration Curve 40 Calculation of Extraction Recoveries [2H10]DPHM 41 2.4.1.7. of DPHM and 2.4.1.8. Sample Stability Assessment 41 2.4.1.9. Method Validation 42 Development of an [2H10]DPMA 43 2.4.2. Analysis Method for DPMA and 2.4.2.1. Optimization of Mass Spectrometer Conditions 43 2.4.2.2. Optimization of Gas Chromatograph Conditions 43 2.4.2.3. Optimization of Extraction 44 2.4.2.4. Optimization of Derivatization 44 2.4.2.5. Gas Chromatograph - Mass Spectrometer Operating Conditions 45 [2H10]DPMA Extraction Procedure 46 2.4.2.6. DPMA and 2.4.2.7. Preparation of a Calibration Curve 47 [2H10]DPMA 47 2.4.2.8. Calculation of Extraction Recovery of DPMA and 2.4.2.9. Sample Stability Assessment 47 2.4.2.10. Method Validation 48 2.5. Standard Procedures for Animal Experiments 48 2.5.1. Animal Handling 48 2.5.2. Surgical Preparation for Chronic Experimentation 49 2.5.2.1. Non-pregnant Sheep - Surgical Preparation 49 2.5.2.2. Pregnant Sheep - Surgical Preparation 51 2.5.3. Chronic Monitoring of Animals 55 2.5.3.1. Amniotic, Tracheal, and Vascular Pressures, Heart rate, Blood Flow, 55 ECoG and EOG. 2.5.3.2. Fetal Urine Measurements 55 2.5.3.3. Blood Gas Analysis 56 2.5.3.4. Glucose and Lactate measurements 56 2.5.4. Dosage Preparation 57 2.6. Experimental Protocols 57 2.6.1. Adult Non-Pregnant Studies 57 2.6.2. Adult Isotope Effect Studies 57 2.6.3. Fetal Isotope Effect Studies 58 2.6.3.1. FetalBolus 58 2.6.3.2. Fetallnfusion 58 vi 2.6.4. Adult First-Pass Metabolism 59 2.6.4.1. Adult First-Pass Metabolism During Normoxia 59 2.6.4.2. Adult First-Pass Metabolism During Mild Hypoxemia 60 2.6.5. Fetal Hepatic First-Pass Metabolism 60 2.6.5.1. Fetal Umbilical Venous Bolus 60 2.6.5.2. Fetal Umbilical Venous Infusion 61 2.6.6. Paired Fetal/Maternal Infusion 62 2.6.7. Sample Handling 63 2.7. In Vitro Experiments 63 2.7.1. DPHM Red Blood Cell Uptake 63 2.7.2. Adult and Fetal Hepatic Microsomal Metabolism Experiments 64 2.7.2.1. Preparation of Adult and Fetal Hepatic Microsomes 64 2.7.2.2. Protein Concentration and Cytochrome P450 Measurements 66 2.7.2.3. DPHM and N-demethyl DPHM Quantitation 66 2.7.2.4. Fetal and Adult Hepatic Microsomal Incubations 67 2.7.3. Plasma Protein Binding of DPMA 67 2.8. Data Analysis 69 2.8.1. Data Reduction 69 2.8.2. Calculation of In Utero Fetal Weight 69 2.8.3. Pharmacokinetic Data Analysis 70 2.8.4. Statistical Analysis 79 3. RESULTS 81 3.1. Development of Analytical Methodology 81 3.1.1. Capillary Gas Chromatographic-Mass Spectrometric Analysis of DPHM 81 [2H10]DPHM and 3.1.1.1. Optimization of Mass Spectrometer and Gas Chromatograph 81 Optimization of the Extraction Procedure for the DPHM [2H10IDPHM 85 3.1.1.2. and Analysis Method [2H10]DPHM Assay 3.1.1.3. Calibration Curve for the DPHM and 90 {2H10]DPHM 3.1.1.4. Extraction Recoveries of DPHM and 91 [2H10]DPHM 3.1.1.5. Sample Stability Assessment of DPHM 91 [2H10]DPHM Gas Chromatographic - 3.1.1.6. Validation of DPHM and Mass 93 Spectrometric Analysis Method Capillary Gas Chromatographic - Mass Spectrometric Analysis of DPMA 96 3.1.2. [2H10IDPMA and 3.1.2.1. Optimization of Mass Spectrometer and Gas Chromatography 96 3.1.2.2. Optimization of Extraction and Derivatization Procedures for DPMA 100 and [2H10JDPMA [2H10]DPMA 102 3.1.2.3. Calibration Curve for DPMA and {2H10]DPMA 3.1.2.4.