UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Differential inhibition of hepatic cytochromes P-450 by cimetidine in adult male rats Chang, Thomas Kwok Hung 1991

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1991_A1 C42_5.pdf [ 9.28MB ]
Metadata
JSON: 831-1.0100471.json
JSON-LD: 831-1.0100471-ld.json
RDF/XML (Pretty): 831-1.0100471-rdf.xml
RDF/JSON: 831-1.0100471-rdf.json
Turtle: 831-1.0100471-turtle.txt
N-Triples: 831-1.0100471-rdf-ntriples.txt
Original Record: 831-1.0100471-source.json
Full Text
831-1.0100471-fulltext.txt
Citation
831-1.0100471.ris

Full Text

DIFFERENTIAL INHIBITION OF HEPATIC CYTOCHROMES P-450 BY CIMETIDINE IN ADULT MALE RATS By THOMAS KWOK HUNG CHANG B . S c , The U n i v e r s i t y o f B r i t i s h Columbia, 1981 B.Sc. (Pharm.)/ The U n i v e r s i t y o f B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES ( F a c u l t y o f Pharmaceutical Sciences) ( D i v i s i o n o f C l i n i c a l Pharmacy) We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1991 © Thomas Kwok Hung Chang, 1991 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) Thomas K. H. Chang Department of Pharmaceutical Sciences The University of British Columbia Vancouver, Canada Date Ju ly 9, 1991 DE-6 (2/88) i i ABSTRACT The cytochrome P-450 enzymes are a family of hemoproteins that play an important role in drug metabolism in man and animals. Cimetidine is a histamine H2-receptor antagonist used in the treatment of peptic ulcers and other gastric acid-related disorders. It i s thought that this drug i s a general inhibitor of cytochrome P-450 enzymes. However, a detailed analysis of the literature indicates substantial, but indirect, evidence that certain cytochrome P-450 enzymes may not be inhibited by cimetidine. Also, i t is apparent that the observed inhibition of hepatic microsomal cytochrome P-450-mediated enzyme activities by in vitro cimetidine administration does not adequately explain the inhibition observed following the in vivo administration of the drug to intact animals or to humans. A major objective of the present study was to determine whether cimetidine, when administered in vivo, d i f f e r e n t i a l l y inhibits cytochrome P-450 enzymes in hepatic microsomes from adult male rats. Uninduced, phenobarbital-induced and dexamethasone-induced rats were sacrificed 90 min after a single intraperitoneal dose of cimetidine HCl (150 mg/kg) or saline. Based on the results from the in vivo cimetidine experiments using enzyme-specific substrates and immunoinhibition experiments with monospecific anti-cytochrome P450IIC11 antibody, i t was concluded that cimetidine administration to adult male rats inhibited i i i hepatic cytochrome P450IIC11. Indirect evidence also indicated that unidentified enzymes other than cytochrome P450IIC11 were inhibited by cimetidine in microsomes from uninduced adult male rats. However, the enzyme act i v i t i e s specific for cytochrome P450IIA1, cytochromes P450IIB1/2 and cytochromes P450IIIA1/2 were not affected by in vivo cimetidine. It i s possible that these enzymes are not inhibited by cimetidine or that the lack of effect i s related to the particular substrate used. In some cases, the extent of inhibition of enzyme ac t i v i t i e s by in vivo cimetidine administration depended on prior treatment with an inducer. This can be explained by the increasing contribution to such activities by inducible enzymes which were not subject to inhibition by cimetidine. Another objective was to determine whether the di f f e r e n t i a l inhibition of cytochrome P-450 by in vivo cimetidine i s observed when the drug i s administered in vitro. Cimetidine, at concentrations of up to 10 mM, did not affect the catalytic function of cytochrome P450IIA1. In contrast, i t did inhibit enzyme ac t i v i t i e s that were specific for cytochrome P450IIC11, cytochromes P450IIB1/2 and cytochromes P450IIIA1/2, with IC 5 0 values in the range of 1.0 - 7.4 mM. The discrepancy in the inhibition of cytochrome P-450 by in vivo and in vitro cimetidine administration was further characterized in enzyme kinetic experiments. Based on Lineweaver-Burk plots of the data, the cytochrome P450IIC11-mediated testosterone i v 2a-hydroxylase activity was inhibited non-competitively by in vivo cimetidine, but competitively by in vitro cimetidine. To further investigate the inhibition of cytochrome P-450 enzymes by in vivo and in vitro cimetidine, preincubation experiments were performed. Hepatic microsomes were preincubated with a low concentration (0.05 mM) of cimetidine and 1 mM NADPH for 15 min prior to the i n i t i a t i o n of substrate (testosterone) oxidation. Under these conditions, cimetidine resulted in the inhibition of the enzyme act i v i t i e s specific for cytochrome P450IIC11, but i t had no effect on those specific for cytochrome P450IIA1, cytochromes P450IIB1/2 and cytochromes P450IIIA1/2. This d i f f e r e n t i a l inhibition by in vitro cimetidine required the presence of NADPH in the preincubation medium, suggesting that a catalysis-dependent process i s involved. Thus, preincubation of hepatic microsomes with NADPH and a relatively low concentration (0.05 mM) of cimetidine in vitro results in a pattern of inhibition of cytochrome P-450 enzymes similar to that following the in vivo administration of cimetidine. V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES x i i LIST OF FIGURES xiv LIST OF ABBREVIATIONS x v i i i ACKNOWLEDGEMENTS XX 1. INTRODUCTION 1 1.1 CYTOCHROME P-450 1 1.1.1 Multiplicity 2 1.1.2 Nomenclature 3 1.1.3 Induction of Cytochrome P-450 5 1.1.4 Suppression of Cytochrome P-450 . . . . 8 1.1.5 Inhibition of Cytochrome P-450 10 1.1.5.1 Reversible Inhibition . . . . 13 1.1.5.2 Metabolite-Intermediate Complexation 14 1.1.5.3 Mechanism-Based Inactivation . 18 1.1.5.4 Single vs. Multiple Doses of an Inhibitor 20 1.1.5.5 Selective Inhibition of Cytochrome P-450 21 1.1.6 S p e c i f i c i t y of Microsomal Enzyme Activities 22 v i 1.2 CIMETIDINE 26 1.2.1 Chemical Structure 26 1.2.2 Pharmacology 26 1.2.3 Pharmacokinetics 28 1.2.4 Cimetidine Drug-Drug Interactions . . . 30 1.2.5 Differential Inhibition of Cytochrome P-450-Mediated Hepatic Drug Metabolism by Jn Vivo Cimetidine Treatment . . . . 32 1.2.5.1 C l i n i c a l Studies 32 1.2.5.2 Animal Studies 36 1.2.6 Inhibition of Cytochrome P-450 by In Vitro or Jn Vivo Cimetidine Treatment 39 1.3 OBJECTIVES 45 2. MATERIALS AND METHODS 46 2.1 CHEMICALS 46 2.2 ANIMALS 47 2.3 TREATMENT 47 2.3.1 Induction Protocol (Pretreatment) . . . 48 2.3.2 Inhibition Protocol (Treatment) . . . . 49 2.4 TIME OF SACRIFICE 50 2.5 PREPARATION OF HEPATIC MICROSOMES 50 2.6 DETERMINATION OF TOTAL CYTOCHROME P-450 CONTENT 51 2.7 MICROSOMAL PROTEIN ASSAY 52 2.8 ENZYME ASSAYS 52 2.8.1 Enzyme Assay Conditions . 52 2.8.2 Aminopyrine N-Demethylase Assay ...... 53 2.8.3 Pentoxyresorufin O-Dealkylase Assay . . 54 v i i 2.8.4 Erythromycin N-Demethylase Assay . . . . 56 2.8.5 E t h o x y r e s o r u f i n O-Deethylase Assay . . . 56 2.8.6 T e s t o s t e r o n e Oxidase Assay 57 2.9 IMMUNOINHIBITION STUDIES 60 2.10 STATISTICAL ANALYSES 61 3. RESULTS 62 3.1 STUDIES WITH IN VIVO CIMETIDINE 62 3.1.1 T o t a l Cytochrome P-450 Content 62 3.1.2 Aminopyrine N-Demethylase A c t i v i t y . . . 65 3.1.3 P e n t o x y r e s o r u f i n O-Dealkylase A c t i v i t y . 68 3.1.4 Erythromycin N-Demethylase A c t i v i t y . . 71 3.1.5 E t h o x y r e s o r u f i n O-Deethylase A c t i v i t y . 74 3.1.6 T e s t o s t e r o n e Oxidase A c t i v i t i e s . . . . 77 3.1.6.1 T e s t o s t e r o n e 2ct-Hydroxylase A c t i v i t y 80 3.1.6.2 T e s t o s t e r o n e 2fi- and 6{J-Hydroxylase A c t i v i t i e s . . . . 83 3.1.6.3 T e s t o s t e r o n e 7a-Hydroxylase A c t i v i t y 88 3.1.6.4 T e s t o s t e r o n e 16a-Hydroxylase A c t i v i t y 91 3.1.6.5 T e s t o s t e r o n e 16p"-Hydroxylase A c t i v i t y 94 3.1.6.6 Androstenedione Formation . . 97 3.1.6.7 Summary 101 v i i i 3.2 IMMUNOINHIBITION STUDIES WITH MONOSPECIFIC ANTI-CYTOCHROME P450IIC11 ANTIBODY 103 3.2.1 Aminopyrine N-Demethylase Activity . . . 103 3.2.2 Pentoxyresorufin O-Dealkylase Activity . 103 3.2.3 Erythromycin N-Demethylase Activity . . 106 3.2.4 Testosterone 2a-Hydroxylase Activity . 106 3.2.5 Testosterone 16a-Hydroxylase Activity . 109 3.2.6 Androstenedione Formation 109 3.2.7 Testosterone 2(3-, 60-, 7a- and 160-Hydroxylase Activities 112 3.3 STUDIES WITH IN VITRO CIMETIDINE 117 3.3.1 Pentoxyresorufin 0-Dealkylase Activity . 117 3.3.2 Erythromycin N-Demethylase Activity . . 117 3.3.3 Ethoxyresorufin 0-Deethylase Activity . 121 3.3.4 Testosterone 2a-Hydroxylase Activity . 121 3.3.5 Testosterone 20- and 60-Hydroxylase Activities . . 125 3.3.6 Testosterone 7a-Hydroxylase Activity . 128 3.3.7 Testosterone 16a-Hydroxylase Activity . 128 3.3.8 Testosterone 160-Hydroxylase Activity . 131 3.3.9 Androstenedione Formation 131 3.3.10 Enzyme Kinetics of the Inhibition of Testosterone 2cc-Hydroxylase Activity by Jn Vitro and Jn Vivo Cimetidine . . . . 134 ix 3.4 PREINCUBATION STUDIES WITH IN VITRO CIMETIDINE 137 3.4.1 Preliminary Experiments 137 3.4.1.1 Microsomes from Uninduced Rats 138 3.4.1.2 Microsomes from Phenobarbital-Induced Rats 141 3.4.2 Experiments With Individual Microsomal Samples 144 3.4.2.1 Testosterone 2cc-Hydroxylase Activity 144 3.4.2.2 Testosterone 2|3- and 6f5-Hydroxylase Activities . . 146 3.4.2.3 Testosterone 7a-Hydroxylase Activity 146 3.4.2.4 Testosterone 16a-Hydroxylase Activity 151 3.4.2.5 Testosterone 16p-Hydroxylase Activity 151 3.4.2.6 Androstenedione Formation . . 151 3.5 SUMMARY OF THE EFFECTS OF IN VIVO AND IN VITRO CIMETIDINE ON MICROSOMAL TESTOSTERONE OXIDATION 151 4. DISCUSSION . . . 158 4.1 DIFFERENTIAL INHIBITION OF CYTOCHROME P-450-MEDIATED ENZYME ACTIVITIES BY IN VIVO CIMETIDINE 158 4.1.1 Inhibition of Cytochrome P450IIC11 by In Vivo Cimetidine 158 X 4.1.2 Lack of Inhibition of Cytochromes P450IIB1/2, Cytochromes P450IIIA1/2 and Cytochrome P450IIA1 by Jn Vivo Cimetidine 161 4.1.3 Indirect Evidence for the Inhibition of Other Cytochrome P-450 Enzymes by Jn Vivo Cimetidine 164 4.1.4 Effect of Jn Vivo Cimetidine on Cytochrome P450IA1 and Cytochrome P450IA2 165 4.1.5 Effect of Pretreatment on Inhibition of Cytochrome P-450 by Cimetidine . . . 168 4.1.6 A Possible Effect of Substrate on the Inhibition of Cytochrome P-450-Mediated Enzyme Activities by Cimetidine . . . . 170 4.2 INHIBITION OF CYTOCHROME P-450-MEDIATED ENZYME ACTIVITIES BY IN VITRO CIMETIDINE 172 4.3 EFFECT OF PREINCUBATION ON THE INHIBITION OF CYTOCHROME P-450-MEDIATED ENZYME ACTIVITIES BY LOW CONCENTRATIONS OF CIMETIDINE 176 5. IMPLICATIONS 184 6. FUTURE STUDIES 186 6.1 STUDIES WITH RAT HEPATIC MICROSOMES 186 6.2 STUDIES WITH HUMAN HEPATIC MICROSOMES . . . . . 192 x i 7. SUMMARY AND CONCLUSIONS . . . . . 195 7.1 STUDIES WITH CIMETIDINE 195 7.2 STUDIES WITH MONOSPECIFIC ANTI-CYTOCHROME P450IIC11 ANTIBODY 198 8. REFERENCES 200 1 x i i L I S T OF TABLES Table Page 1 Nomenclature for hepatic cytochrome P-450 enzymes in rats 4 2 Major inducible hepatic cytochrome P-450 enzymes in rats 6 3 Contribution of rat hepatic cytochrome P-450 to microsomal enzyme activities 24 4 Inhibition of hepatic microsomal cytochrome P-450 enzyme act i v i t i e s by in vitro cimetidine in rats . 31 5 C l i n i c a l studies with cimetidine: lack of a drug-drug interaction 33 6 Effect of phenobarbital, dexamethasone and P-naphthoflavone on total microsomal cytochrome P-450 content . 63 7 Effect of phenobarbital, dexamethasone and (3-naphthof lavone on aminopyrine N-demethylase activ i t y 66 8 Effect of phenobarbital and dexamethasone on pentoxyresorufin O-dealkylase activity 70 9 Effect of phenobarbital and dexamethasone on erythromycin N-demethylase activity 73 10 Effect of |3-naphthof lavone on ethoxyresoruf in 0-deethylase activity . . . . . 76 11 Effect of phenobarbital and dexamethasone on testosterone 2cc-hydroxylase activity 82 12 Effect of phenobarbital and dexamethasone on testosterone 2|3-hydroxylase activity 84 13 Effect of phenobarbital and dexamethasone on testosterone 6(3-hydroxylase activity 85 14 Effect of phenobarbital and dexamethasone on testosterone 7a-hydroxylase activity 90 15 Effect of phenobarbital and dexamethasone on testosterone 16a-hydroxylase activity 93 x i i i 16 Effect of phenobarbital and dexamethasone on testosterone 16ft-hydroxylase activity 95 17 Effect of phenobarbital and dexamethasone on androstenedione formation 100 18 IC 5 0 values for the inhibition of pentoxyresorufin 0-dealkylase, erythromycin N-demethylase and ethoxyresorufin 0-deethylase act i v i t i e s by cimetidine in vitro 119 19 IC 5 0 values for the inhibition of testosterone oxidation by cimetidine in vitro 124 LIST OF FIGURES Figure Page 1 The c a t a l y t i c c y c l e of cytochrome P-4 50 11 2 The m e t a b o l i c pathways f o r c i m e t i d i n e i n r a t s . . 27 3 E f f e c t of in vivo c i m e t i d i n e on t o t a l microsomal cytochrome P-450 content 64 4 E f f e c t of in vivo c i m e t i d i n e on aminopyrine N-demethylase a c t i v i t y 67 5 E f f e c t of in vivo c i m e t i d i n e on p e n t o x y r e s o r u f i n O-dealkylase a c t i v i t y . . . 69 6 E f f e c t o f in vivo c i m e t i d i n e on erythromycin N-demethylase a c t i v i t y 72 7 E f f e c t of i n vivo c i m e t i d i n e on e t h o x y r e s o r u f i n O-deethylase a c t i v i t y 75 8 Summary of the e f f e c t s of in vivo c i m e t i d i n e on aminopyrine N-demethylase, p e n t o x y r e s o r u f i n O-dealkylase and erythromycin N-demethylase a c t i v i t i e s i n microsomes from uninduced, p h e n o b a r b i t a l - i n d u c e d and dexamethasone-induced r a t s 7 8 9 Summary o f the e f f e c t s o f in vivo c i m e t i d i n e on aminopyrine N-demethylase and e t h o x y r e s o r u f i n O-deethylase a c t i v i t i e s i n microsomes from uninduced and |3-naphthof lavone-induced r a t s . . . 79 10 E f f e c t o f in vivo c i m e t i d i n e on t e s t o s t e r o n e 2cc-hydroxylase a c t i v i t y 81 11 E f f e c t o f in vivo c i m e t i d i n e on t e s t o s t e r o n e 2p~hydroxylase a c t i v i t y 86 12 E f f e c t of in vivo c i m e t i d i n e on t e s t o s t e r o n e 6f3-hydroxylase a c t i v i t y 87 13 E f f e c t of in vivo c i m e t i d i n e on t e s t o s t e r o n e 7a-hydroxylase a c t i v i t y 89 14 E f f e c t o f in vivo c i m e t i d i n e on t e s t o s t e r o n e 16a-hydroxylase a c t i v i t y 92 15 E f f e c t o f i n vivo c i m e t i d i n e on t e s t o s t e r o n e 16(3-hydroxylase a c t i v i t y 96 X V 16 Lineweaver-Burk p l o t f o r the e f f e c t of in vivo c i m e t i d i n e on t e s t o s t e r o n e 16|3-hydroxylase a c t i v i t y 98 17 E f f e c t o f in vivo c i m e t i d i n e on androstenedione f o r m a t i o n 99 18 Summary o f the e f f e c t s o f in vivo c i m e t i d i n e on t e s t o s t e r o n e o x i d a t i o n 102 19 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 a n t i b o d y on aminopyrine N-demethylase a c t i v i t y . . 104 20 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 a n t i b o d y on p e n t o x y r e s o r u f i n 0 - d e a l k y l a s e a c t i v i t y 105 21 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 a n t i b o d y on erythromycin N-demethylase a c t i v i t y . 107 22 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 a n t i b o d y on t e s t o s t e r o n e 2ct-hydroxylase a c t i v i t y 108 23 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 a n t i b o d y on t e s t o s t e r o n e 16a-hydroxylase a c t i v i t y 110 24 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 a n t i b o d y on androstenedione f o r m a t i o n I l l 25 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 a n t i b o d y on t e s t o s t e r o n e 2|3-hydroxylase a c t i v i t y . 113 26 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 a n t i b o d y on t e s t o s t e r o n e 6f5-hydroxylase a c t i v i t y . 114 27 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 ant i b o d y on t e s t o s t e r o n e 7a-hydroxylase a c t i v i t y 115 28 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 a n t i b o d y on t e s t o s t e r o n e 16(3-hydroxylase a c t i v i t y 116 2 9 E f f e c t o f in vitro c i m e t i d i n e on p e n t o x y r e s o r u f i n 0 - d e a l k y l a s e a c t i v i t y 118 30 E f f e c t o f in vitro c i m e t i d i n e on er y t h r o m y c i n N-demethylase a c t i v i t y 120 31 E f f e c t o f in vitro c i m e t i d i n e on e t h o x y r e s o r u f i n 0-deethylase a c t i v i t y 122 32 E f f e c t o f in vitro c i m e t i d i n e on t e s t o s t e r o n e 2a-hydroxylase a c t i v i t y 123 x v i 33 E f f e c t of in vitro c i m e t i d i n e on t e s t o s t e r o n e 2 0-hydroxylase a c t i v i t y 126 34 E f f e c t o f in vitro c i m e t i d i n e on t e s t o s t e r o n e 6 0-hydroxylase a c t i v i t y 127 3 5 E f f e c t o f in vitro c i m e t i d i n e on t e s t o s t e r o n e 7a-hydroxylase a c t i v i t y 129 36 E f f e c t o f in vitro c i m e t i d i n e on t e s t o s t e r o n e 16a-hydroxylase a c t i v i t y 130 37 E f f e c t o f in vitro c i m e t i d i n e on t e s t o s t e r o n e 160-hydroxylase a c t i v i t y 132 3 8 E f f e c t o f in vitro c i m e t i d i n e on androstenedione f o r m a t i o n , 133 39 Lineweaver-Burk p l o t f o r the i n h i b i t i o n o f t e s t o s t e r o n e 2a-hydroxylase a c t i v i t y by in vitro c i m e t i d i n e 135 40 Lineweaver-Burk p l o t f o r the i n h i b i t i o n o f t e s t o s t e r o n e 2ct-hydroxylase a c t i v i t y by in vivo c i m e t i d i n e 136 41 T e s t o s t e r o n e 2a- and 6 0-hydroxylase a c t i v i t i e s i n microsomes p r e i n c u b a t e d w i t h NADPH and c i m e t i d i n e 139 42 T e s t o s t e r o n e o x i d a t i o n i n microsomes p r e i n c u b a t e d f o r v a r i o u s times w i t h NADPH i n the absence o f c i m e t i d i n e 140 43 T e s t o s t e r o n e 2a-hydroxylase a c t i v i t y i n microsomes p r e i n c u b a t e d w i t h c i m e t i d i n e i n the presence and absence o f NADPH 142 44 T e s t o s t e r o n e 2a- and 6 0-hydroxylase a c t i v i t i e s i n microsomes p r e i n c u b a t e d f o r v a r i o u s times w i t h 0.05 mM c i m e t i d i n e and NADPH 143 45 T e s t o s t e r o n e 2a-, 60- and 160-hydroxylase a c t i v i t i e s i n microsomes p r e i n c u b a t e d f o r 15 min w i t h c i m e t i d i n e and NADPH . 145 4 6 E f f e c t o f p r e i n c u b a t i o n o f microsomes w i t h c i m e t i d i n e and NADPH on t e s t o s t e r o n e 2a-hydroxylase a c t i v i t y 147 47 E f f e c t o f p r e i n c u b a t i o n o f microsomes wi t h c i m e t i d i n e and NADPH on t e s t o s t e r o n e 2 0-hydroxylase a c t i v i t y 148 x v i i 4 8 Effect of preincubation of microsomes with cimetidine and NADPH on testosterone 6P-hydroxylase activity . . . . 149 4 9 Effect of preincubation of microsomes with cimetidine and NADPH on testosterone 7a-hydroxylase activity 150 50 Effect of preincubation of microsomes with cimetidine and NADPH on testosterone 16a-hydroxylase activity 152 51 Effect of preincubation of microsomes with cimetidine and NADPH on testosterone 16f3-hydroxylase activity 153 52 Effect of preincubation of microsomes with cimetidine and NADPH on androstenedione formation 154 53 Summary of the effects of in vivo and in vitro cimetidine on testosterone oxidation in microsomes from uninduced rats 156 54 Summary of the effects of in vivo and in vitro cimetidine on testosterone oxidation in microsomes from phenobarbital-induced rats 157 LIST OF ABBREVIATIONS A androstenedione APND arainopyrine N-demethylase BNF R-naphthoflavone cm c e n t i m e t e r c y c l i c AMP c y c l i c adenosine 35'-monophosphate DEX dexamethasone EDTA e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d EMND erythromycin N-demethylase EROD e t h o x y r e s o r u f i n O-deethylase g gram h hour(s) HEPES (N-[2-hydroxyethy1]piperaz ine-N'-[2-e t h a n e s u l f o n i c a c i d ] ) HPLC hig h performance l i q u i d chromatography kg k i l o g r a m M molar mg m i l l i g r a m min minute mL m i l l i l i t e r s ) mm m i l l i m e t e r mM m i l l i m o l a r NADPH ^ - n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate nm nanometers PB p h e n o b a r b i t a l PROD p e n t o x y r e s o r u f i n 0 - d e a l k y l a s e TRIS (Tris[hydroxymethyl]amino-methane) ixg microgram u.L microliter urn. micrometer uM micromolar v/v volume per unit volume w/v weight per unit volume X X ACKNOWLEDGEMENTS I wish to thank my mentors, Dr. Marc Levine and Dr. Gail Bellward. Their wisdom and foresight have been a guiding light, while their patience and encouragement have helped me overcome many hurdles. Special thanks to Dr. Helen Burt, Chairperson of my Supervisory Committee, for her leadership qualities. I also wish to thank the other commmitte members, Dr. Peter Jewesson, Dr. James Orr and Dr. David Seccombe, for their interest and constructive criticism. Great appreciations to Dr. Stelvio Bandiera for generously providing the antibodies and for contributing to many thoughtful discussions, Edith Lemieux for her excellent technical assistance and constant encouragement, and Mike Lane for his s k i l l f u l care of the animals. 1 INTRODUCTION 1.1 CYTOCHROME P-450 Many drugs are e l i m i n a t e d by metabolism i n the l i v e r . Among the h e p a t i c m e t a b o l i c enzymes are the cytochrome P-450 monooxygenases, a f a m i l y o f c l o s e l y - r e l a t e d hemoproteins. The p r o s t h e t i c group i n each of these hemoproteins i s i r o n p r o t o p o r p h y r i n IX. The term "cytochrome P-450" was c o i n e d by Omura and Sato (1964). Carbon monoxide binds t o the f e r r o u s form of the hemoprotein and y i e l d s a s p e c t r a l peak at a p p r o x i m a t e l y 450 nm. The components o f the h e p a t i c cytochrome P-450 enzyme system a r e : cytochrome P-450, the membrane-bound t e r m i n a l oxidase which c a t a l y z e s s u b s t r a t e o x i d a t i o n by the a d d i t i o n o f an oxygen atom from m o l e c u l a r oxygen; and NADPH-cytochrome P-450 r e d u c t a s e , which reduces cytochrome P-450 by the t r a n s f e r of two e l e c t r o n s ( O r t i z de M o n t e l l a n o , 1986). Cytochrome P-450 enzymes are found mainly i n the l i v e r , but a l s o e x i s t i n e x t r a h e p a t i c t i s s u e s , i n c l u d i n g the kidney, lung, b r a i n and i n t e s t i n e s (Adesnik and A t c h i s o n , 1986). They are most abundant i n the endoplasmic r e t i c u l u m o f the c e l l , which i s i s o l a t e d i n the microsomal f r a c t i o n by d i f f e r e n t i a l u l t r a c e n t r i f u g a t i o n . In a d d i t i o n , these enzymes are a l s o found i n the n u c l e a r membrane and m i t o c h o n d r i a (Astrom and D e P i e r r e , 1986). 2 Cytochrome P-450 enzymes play a c r i t i c a l r o l e i n the oxidative metabolism of exogenous compounds such as drugs and environmental pollutants (Conney, 1982). They also p a r t i c i p a t e i n the b i o a c t i v a t i o n of prodrugs to t h e i r pharmacologically active forms (LeBlanc and Waxman, 1989) and i n the formation of reactive intermediates that r e s u l t i n t o x i c , mutagenic and carcinogenic products (Conney, 1982). Cytochrome P-450 enzymes are involved i n the biosynthesis and metabolism of endogenous compounds such as s t e r o i d s , f a t t y acids and prostaglandins (Kupfer, 1980; Waterman et a l . , 1986). 1.1.1 Mu l t i p l i c i t y The existence of multiple cytochrome P-450 enzymes had been hypothesized since the i n i t i a l studies on microsomal drug metabolism. Conney et a l . , (1959) reported that the administration of benzo[a]pyrene to rats e i t h e r increased, decreased or had no e f f e c t on . the hepatic microsomal metabolism of several drugs. Subsequent evidence of broad and overlapping substrate s p e c i f i c i t y and r e s u l t s from s p e c t r a l studies led to the conclusion that there were at l e a s t two enzymes of cytochrome P^ -450 i n rat l i v e r s . The enzyme inducible by phenobarbital and phenobarbital-like inducers was c a l l e d cytochrome P-450, and the enzyme inducible by 3-methylcholanthrene and p o l y c y c l i c aromatic hydrocarbon-like inducers was c a l l e d cytochrome P-448 3 (Alvares et al., 1967). Data from kinetic studies also provided evidence for the existence of more than one enzyme of cytochrome P-450. Examples include the biphasic decay of radiolabelled cytochrome P-450 heme (Levin et al., 1975) and biphasic Lineweaver-Burk plots of kinetic data from studies of the metabolism of xenobiotics by hepatic microsomes (e.g. Pederson and Aust, 1970). Indeed, since the f i r s t report of the pa r t i a l purification of "cytochrome P-450" and "cytochrome P-448" from rat hepatic microsomes by Lu and Levin (1972), many cytochrome P-450 enzymes have been isolated and purified to apparent homogeneity from several species, including rats, rabbits and man (e.g. Waxman, 1986; Guengerich, 1987, 1989; Ryan and Levin, 1990). It is not known exactly how many cytochrome P-450 enzymes exist, but there may be as many as 200 of them (Renton, 1986). 1.1.2 Nomenclature The purification of the different cytochrome P-450 enzymes by different investigators has resulted in the development of different nomenclatures. The nomenclatures used by the four major research groups involved in rat hepatic cytochrome P-450 purification are presented in Table 1. The recently recommended gene designation for cytochrome P-450 w i l l be used in this dissertation (Nebert et a l . , 1989). Each cytochrome P-450 is encoded by a different gene. In this classification system, genes which 4 TABLE 1 NOMENCLATURE FOR HEPATIC CYTOCHROME P-450 ENZYMES IN RATS G e n e 1 G u e n g e r i c h 2 L e v i n 3 Schenkman* Waxman 5 Designation P450IA1 BNF-B c - BNF-B P450IA2 ISF-G d - ISF-G P450IIA1 UT-F a RLM2b 3 P450IIA2 - - RLM2 -P450IIB1 PB-B b PBRLM5 PB-4 P450IIB2 PB-D e PBRLM6 PB-5 P450IIC6 PB-C k RLM5a PB-1 P450IIC7 - f RLM5b -P450IIC11 UT-A h RLM5 2c P450IIC12 UT-I i f RLM4 2d P450IIC13 - g RLM3 -P450IIE1 - j RLM6 -P450IIIA1 PCN-E p - PB-2a P450IIIA2 _ — 2a 6 Due t o space r e s t r i c t i o n , the nomenclature from o n l y the f o u r major groups i n v o l v e d i n r a t h e p a t i c cytochrome P-450 p u r i f i c a t i o n i s shown. The recommended gene d e s i g n a t i o n w i l l be used i n the t h e s i s . ^rom: Nebert e t al., 1989. 2From: Guengerich e t al., 1982a; L a r r e y e t al., 1984. 3From: Ryan e t al., 1979, 1980, 1982a, 1984, 1985; Ban d i e r a e t al., 1986; Wrighton e t al., 1985a. 4From: Cheng and Schenkman, 1982, Backes et a l . , 1985; Jansson e t al., 1985; Favreau et al., 1987. 5From: Waxman and Walsh, 1982, 1983; Waxman e t al., 1983; Waxman, 1984; Waxman e t al., 1988b. 5 encode proteins that have at least 3 6% similarity in their amino acid sequences are in the same family. If the similarity i s at least 70%, then they are in the same subfamily. In each designation, the Roman numeral indicates the family, the capital letter indicates the subfamily, and the Arabic numeral indicates the gene (see Table 1 for examples). It has been recommended that this system be used when referring to a particular cytochrome P-450 (gene product) (Nebert et a l . , 1989). 1.1.3 Induction of Cytochrome P-450 Many of the cytochrome P-450 enzymes are subject to induction as a consequence of exposure to xenobiotics or altered physiologic states. The term induction refers to increased de novo protein synthesis (Tukey and Johnson, 1990). In some instances, the term i s used by investigators to mean increased de novo protein synthesis and/or stabilization of existing protein. The major inducible hepatic cytochrome P-450 enzymes in the rat are cytochromes P450IA1, P450IA2, P450IIB1, P450IIB2, P450IIE1, P450IIIA1 and P450IVA1. The preferential inducing agents for these enzymes are shown in Table 2. The level of cytochrome P450IIE1 is also increased in altered physiological states such as diabetes (Bellward et a l . , 1988) and fasting (Johansson et a l . , 1988; Ma et a l . , 1989). TABLE 2 MAJOR INDUCIBLE HEPATIC CYTOCHROME P-450 ENZYMES IN RATS Enzyme P r e f e r e n t i a l Inducer References P450IA1 3-methylcholanthrene Dannan et al., Thomas et al., 1983 1983 P-naphthoflavone Guengerich et Thomas et al., Waxman et al., al., 1982a 1983 1985 P450IA2 Iso s a f r o l e Guengerich et Thomas et al., Waxman et al., a l . , 1982a 1983 1985 P450IIB1 Phenobarbital Guengerich et Thomas et al., Waxman et al., al., 1982a 1983 1985 P450IIB2 Phenobarbital Guengerich et Thomas et al., Waxman et al., al., 1982a 1983 1985 P450IIE1 Iso n i a z i d Thomas et al., 1987 Ethanol Thomas et al., 1987 P450IIIA1 Dexamethasone Heuman et al., 1982 PCN Guengerich et Waxman et al., al., 1982a 1985 TAO Wrighton et al ., 1985a P450IVA1 C l o f i b r a t e Gibson et al., 1982 A b b r e v i a t i o n s : PCN, pregnenolone 1 6 a - c a r b o n i t r i l e ; TAO, t r i a c e t y l o l e a n d o m y c i n 7 A c h a r a c t e r i s t i c o f the i n d u c i b l e cytochrome P-450 enzyme i s t h a t one i n d u c e r can induce more than one enzyme, and a s i n g l e enzyme can be induced by many compounds. For example, although p h e n o b a r b i t a l p r e f e r e n t i a l l y induces cytochromes P450IIB1 and P450IIB2 i n r a t s , i t a l s o can induce cytochromes P450IIA1, P450IIC6 and P450IIIA1, alth o u g h t o d i f f e r e n t e x t e n t s (Guengerich e t al., 1982a; Heuman e t al., 1982; Thomas et al., 1983; Waxman et al., 1985). On the o t h e r hand, cytochrome P450IIIA1 i s i n d u c i b l e i n r a t s by a v a r i e t y o f s t r u c t u r a l l y d i v e r s e c h e m i c a l s , i n c l u d i n g dexamethasone, pregnenolone 1 6 c t - c a r b o n i t r i l e , t r i a c e t y l o l e a n d o m y c i n , c l o t r i m a z o l e , k e t o c o n a z o l e , and p h e n o b a r b i t a l (Guengerich et a l . , 1982a; Heuman et al., 1982; Waxman e t al., 1985; Wrighton et al., 1985a, 1985b; H o s t e t l e r et al., 1989). The mechanism o f i n d u c t i o n of the d i f f e r e n t cytochrome P-450 enzymes i s s t i l l not w e l l understood. However, i n c r e a s e d t r a n s c r i p t i o n has been shown t o oc c u r (Okey, 1990). The i n d u c t i o n o f cytochromes P450IA1 and P450IA2 by 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n and r e l a t e d halogenated aromatic hydrocarbons r e q u i r e s the b i n d i n g o f the i n d u c e r t o an endogenous c y t o s o l i c r e c e p t o r (Poland e t al., 1976), named the Ah r e c e p t o r (Okey e t a l . , 1979). The i n d u c e r -r e c e p t o r complex e n t e r s the nucleus, and the subsequent i n t e r a c t i o n between the complex and the n u c l e a r DNA s t i m u l a t e s t r a n s c r i p t i o n . R e c e n t l y , Poland's group p u r i f i e d 8 the Ah receptor to apparent homogeneity and determined the N-terminal amino acid sequence (Bradfield et a l . , 1991). It remains to be determined whether receptors are involved in the induction of the other cytochrome P-450 enzymes. It has been shown that transcriptional activation does not occur in the "induction" of cytochrome P450IIE1 in adult rats (Koop and Tierney, 1990). The observed increase i n the level of cytochrome P450IIE1 appears to be due to increased stabilization of mRNA, increased translation of synthesized mRNA and/or decreased protein degradation (Koop and Tierney, 1990). 1.1.4 Suppression of Cytochrome P-450 Several of the major cytochrome P-450 enzymes in the uninduced rat have been shown to be resistant to enzyme induction following exposure to known inducing agents. These include cytochrome P450IIA2 (Waxman et al., 1988b), cytochrome P450IIC11 (Guengerich et a l . , 1982a; Dannan et al., 1983; Waxman, 1984; Waxman et al., 1985; Yeowell et al., 1987, 1989; Emi and Omura, 1988; Shimada et a l . , 1989), cytochrome P450IIC12 (Waxman et al., 1985) and cytochrome P450IIC13 (Bandiera et al., 1986). The amount (per mg of microsomal protein, per g of li v e r or percent of total cytochrome P-450) of these enzymes i s decreased following the administration of known inducing agents to rats and this effect i s called suppression (Guengerich, 1987). 9 The level of the cytochrome P450IIC11 enzyme in the adult male rat i s suppressed following the chronic administration of known inducing agents such as phenobarbital, 3-methylcholanthrene, R-naphthoflavone, dexamethasone and triacetyloleandomycin (Guengerich et al., 1982a, Dannan et al., 1983; Waxman, 1984; Waxman et al., 1985; Yeowell et al., 1987; Miura et al., 1989; Shimada et al., 1989). The level of cytochrome P450IIC11 is also suppressed by the chronic administration of cisplatin (LeBlanc and Waxman, 1988) and cyclophosphamide (LeBlanc and Waxman, 1990). The decline in the level of cytochrome P450IIC11 is accompanied by a decrease in i t s catalysis of microsomal testosterone 2a-hydroxylation (Waxman, 1984; Yeowell et al., 1987; LeBlanc and Waxman, 1988, 1990; Shimada et al., 1989). It has been well-established that cytochrome P-450-mediated hepatic drug metabolism is impaired following the administration of interferon and interferon inducers to experimental animals (Renton, 1986). Recently, Craig et al. (1990) reported that cytochrome P450IIIA2, which is a male-specific cytochrome P-450, i s suppressed following the administration of a recombinant interferon or a naturally-derived interferon. Morgan and Norman (1990) reported that the interferon-inducing agents, polyriboinosinic acid, polyribocytidylic acid and R11-877DA, a tilorone analog, suppressed the content of hepatic cytochrome P450IIC11 in adult male rats. 10 The mechanism of the suppression of cytochrome P-450 enzymes i s not known. The decrease in the level of the protein is accompanied by a decrease in the level of the corresponding mRNA. For example, the decline in the content of the cytochrome P450IIC11 protein after the administration of 3-methylcholanthrene, 3,4,5,3',4',5'-hexachlorobiphenyl or cyclophosphamide to adult male rats i s accompanied by a decrease in the level of cytochrome P450IIC11 mRNA (Yeowell et a l . , 1987, 1989; LeBlanc and Waxman, 1990). In some instances, multiple mechanisms may be involved. With interferon-inducing agents, both pre- and post-translational mechanisms have been suggested (El Azhary et a l . , 1980; Morgan and Norman, 1990; Renton et al., 1991). 1.1.5 Inhibition of Cytochrome P-450 The cytochrome P-450 enzymes are subject to inhibition, which i s defined in this dissertation as a transient or permanent impairment of the catalytic function of the hemoproteins. This i s different from suppression, which is a decrease in the quantity of the hemoprotein. As a result of inhibition, the observed enzyme activity i s decreased. The cytochrome P-450 catalytic cycle is shown in Figure 1. The steps in the cycle are: 1) binding of the substrate to the fer r i c form of the hemoprotein; 2) electron transfer from NADPH-cytochrome P-450 reductase to produce the ferrous hemoprotein-substrate complex; 3) binding of 11 F i g u r e 1 The c a t a l y t i c c y c l e o f cytochrome P-450. (From Murray and Reidy, 1990). R O H F<i I O 3 + H 2 0 2 H + F « 3 * - R H R H [ F O 3 - . R H ] NAOPH/P4SO REDUCTASE / [ F . ^ . R H ] \ F « 3 + - R H I NAOPH / P4SO REDUCTASE . Of NAOH / b 5 REOUCTASE /'b 5 12 molecular oxygen to the ferrous hemoprotein-substrate complex; 4) reduction of the ferrous hemoprotein-dioxygen complex by the addition of a second electron from NADPH-cytochrome P-450 reductase; 5) drug oxidation by the addition of an oxygen atom from molecular oxygen and reduction of the other oxygen atom to water; and 6) release of the product and recovery of the f e r r i c hemoprotein (Ortiz de Montellano and Reich, 1986). Each step in the catalytic cycle i s potentially susceptible to interference. One approach in classifying the inhibitors of cytochrome P-450 is based on the type of inhibition as determined by enzyme kinetic analysis; for example, competitive, non-competitive or mixed (competitive and non-competitive) inhibition. However, this c l a s s i f i c a t i o n does not identify the mechanism involved (Testa and Jenner, 1981). Non-competitive inhibition can occur as a result of reversible ligand binding by a parent compound (Lesca et al., 1979) or irreversible ligand binding by a metabolite to form a metabolite-intermediate complex (Franklin, 1977). A cla s s i f i c a t i o n scheme that reflects the mechanism of inhibition i s more commonly used. The major mechanisms of inhibition of cytochrome P-450 are reversible inhibition, metabolite-intermediate complexation and mechanism-based inactivation (Testa and Jenner, 1981; Ortiz de Montellano and Reich, 1986; Murray and Reidy, 1990). 13 1.1.5.1 R e v e r s i b l e I n h i b i t i o n Reversible inhibition may occur by substrate (hydrophobic) binding and/or ligand binding. In substrate binding, two compounds compete for the same binding sites on the hemoprotein, resulting in alternate substrate inhibition (Testa and Jenner, 1981). The observed inhibition is competitive and is a function of the relative concentrations of the competing substrates, the l i p o p h i l i c i t y of the inhibitors, and the relative a f f i n i t y of the two compounds for the binding s i t e . In ligand binding, the interaction is between an electron-donating group of a ligand and the fe r r i c or ferrous iron of the hemoprotein (Testa and Jenner, 1981). Oxygen can no longer bind to the heme iron and drug oxidation is impaired. This type of binding depends on the relative a f f i n i t y of the two competing ligands. Binding occurs with a ligand with a higher a f f i n i t y for the heme iron. Steric factors are also important in ligand binding. Carbon monoxide is a ligand that can bind to the ferrous hemoprotein and this forms the basis for the standard spectral determination of the total microsomal cytochrome P-450 content (Omura and Sato, 1964). More commonly, a reversible inhibitor acts by both substrate binding and ligand binding. 9-Hydroxyellipticine inhibits rat hepatic microsomal cytochrome P-450-mediated enzyme act i v i t i e s competitively and non-competitively (Lesca et a l . , 1979). The competitive inhibition is attributed to 14 substrate binding, and the non-competitive inhibition to ligand binding (Testa and Jenner, 1981). Other compounds which are thought to inhibit by both substrate binding and ligand binding include metyrapone (Jonen et a l . , 1974) and the 1-substituted imidazole derivatives such as clotrimazole and ketoconazole (Rodrigues et al., 1987). The inhibitory potency of these inhibitors is determined by the l i p o p h i l i c i t y of the inhibitor, a f f i n i t y of the ligand for the heme iron, and steric hindrance by substituents on the inhibitor (Murray and Ryan, 1983). 1.1.5.2 Metabolite-Intermediate Complexation A compound can inhibit cytochrome P-450 indirectly by the formation of a metabolite-intermediate complex with the enzyme. In this case, the parent compound may have l i t t l e or no inhibitory effect. However, once i t i s oxidized by cytochrome P-450, a metabolite forms a complex with the ferrous and/or f e r r i c hemoprotein by a ligand interaction. As a result, oxygen can not bind to the hemoprotein, rendering the enzyme functionally inactive. The time-dependent formation of a metabolite-intermediate complex occurs both in vivo and in vitro and proceeds under the same conditions as those required for catalysis by cytochrome P-450. In order for the complex to be generated in vitro, the microsomes must be preincubated aerobically with the parent compound and NADPH for a f i n i t e period of time. 15 Several classes of compounds are known to form metabolite-intermediate complexes. These include: 1) the methylenedioxybenzenes such as isosafrole; 2) dioxolanes such as 4-n-butyldioxolane; 3) nitrogenous compounds such as amphetamine, SKF 525-A (diethylaminoethyl-2,2-diphenyl-valerate), propoxyphene, orphenadrine, erythromycin, triacetyloleandomycin and amiodarone; and 4) hydrazines such as N-aminopiperidine (Pershing and Franklin, 1982; Larrey et a l . , 1986; Reidy et al., 1989). The inhibition of cytochrome P-450 by metabolite-intermediate complexation has been described as reversible by Testa and Jenner (1981) and irreversible by Ortiz de Montellano and Reich (1986). The complex formed by dioxolane breaks down relatively quickly after formation (Dahl and Hodgson, 1979). In this case, the inhibition is reversible. However, according to Ortiz de Montellano and Reich (1986), with many of the compounds in this class, once the complex is formed in vivo or in vitro, i t is stable and the inhibition that results is irreversible. The degree of s t a b i l i t y i s demonstrated by the fact that a metabolite-intermediate complex formed in vivo remains intact after microsomal preparation. However, the complex formed in vivo can be displaced or dissociated in vitro. In the ferric state, the metabolite-intermediate-cytochrome P-450 complex formed with a methylendioxybenzene derivative such as isosafrole i s unstable (Franklin, 1977). It can be 16 displaced by li p o p h i l i c agents such as cyclohexane (Thomas et al., 1983) or substrates such as 7-ethoxycoumarin (Ryan et al., 1980) and androstenedione (Murray et al., 1986). A nitrogenous compound such as SKF 525-A forms a stable metabolite-intermediate complex with the ferrous iron of the hemoprotein (Buening and Franklin, 1976). This type of complex can be dissociated by potassium ferricyanide, which is an oxidant. Once the complexed cytochrome P-450 has been displaced or dissociated, i t i s catalytically active. Thus, displacement and dissociation i l l u s t r a t e the quasi-irreversible nature df the binding of the metabolite-intermediate to the iron atom of the hemoprotein (Ortiz de Montellano and Reich, 1986). The mechanism by which a metabolite-intermediate complex is broken down in vivo i s not known. A characteristic of the in vivo inhibition of cytochrome P-450 by metabolite-intermediate complex formation is that the inhibition lasts much longer than can be explained by the elimination h a l f - l i f e of the parent compound in the body. The observed in vitro inhibition of a cytochrome P-450-mediated enzyme activity by a compound that forms a metabolite-intermediate complex can be competitive, non-competitive or mixed (competitive and non-competitive), depending on the experimental conditions. Without the aerobic preincubation of microsomes with the parent compound and NADPH, the inhibition of the enzyme activity can be 17 competitive. This is attributed to alternate substrate inhibition by the parent compound (Franklin, 1977; Testa and Jenner, 1981). However, with preincubation, either non-competitive or mixed (competitive and non-competitive) inhibition may be observed, depending on the concentrations of the parent compound used in a given experiment (Franklin, 1977). At low concentrations, non-competitive inhibition is observed and this is due to the ligand binding of a metabolite to the heme iron (Franklin, 1977; Testa and Jenner, 1981). At high concentrations, both competitive and non-competitive inhibition occurs and this i s due to both ligand binding by a metabolite and alternate substrate inhibition by the parent compound (Franklin, 1977; Testa and Jenner, 1981). The characteristics of a metabolite-intermediate complex are as follows. F i r s t , complex formation i s a time-dependent process. Second, the complex shows a spectral peak at 448-456 nm, depending on the particular compound. This allows for the formation of the complex to be observed in vitro. Third, the complexed cytochrome P-450 is ca t a l y t i c a l l y inactive, whereas the uncomplexed form is cat a l y t i c a l l y active. Fourth, the complexed cytochrome P-450 can not be measured spectrally by the standard dithionite-reduced carbon monoxide-binding method of Omura and Sato (1964). Therefore, in order to measure the total (complexed and uncomplexed) cytochrome P-450 content, the 18 metabolite-intermediate complex must f i r s t be dissociated or displaced. 1.1.5.3 Mechanism-Based Inactivation Mechanism-based inactivation i s an enzyme-mediated and irreversible process (Rando, 1984). The f i r s t step in mechanism-based inactivation is the formation of a reactive intermediate from the parent compound by a cytochrome P-450 enzyme. The second step involves the inactivation of that enzyme by the reactive intermediate (Ortiz de Montellano, 1988). Therefore, the enzyme initiates i t s own destruction. An agent which inhibits a cytochrome P-450 in this manner is called a mechanism-based inactivator (Rando, 1984) or a suicide substrate (Walsh, 1982). Mechanism-based inactivation occurs as a result of covalent binding of a reactive intermediate to the prosthetic heme group or the apoprotein of a cytochrome P-450 enzyme (Ortiz de Montellano and Reich, 1986). Agents that form reactive intermediates which bind covalently to the prosthetic heme group of cytochrome P-450 include: terminal olefins such as allylisopropylacetamide and secobarbital; terminal acetylenes such as ethinyl estradiol and danazol; and heterocyclic compounds such as 1-aminobenzotriazole and 3,5-diethoxycarbonyl-l,4-dihydro-2,4,6-trimethylpyridine (Ortiz de Montellano and Correia, 1983)'. In these cases, enzyme inactivation occurs as a 19 result of the N-alkylation of the prosthetic heme group by a reactive intermediate. The same molar amount of heme and apoprotein is lost and the alkylated heme moiety can be isolated. Agents that form reactive intermediates which bind covalently to the apoprotein of cytochrome P-450 include: halogenated compounds such as chloramphenicol; and sulfur-containing compounds such as parathion and carbon disulfide (Ortiz de Montellano and Reich, 1986). The detailed mechanism of enzyme inactivation by many of these compounds is not well known. However, in the case of chloramphenicol, the oxamyl intermediate of chloramphenicol formed by cytochrome P-450 acylates a lysine residue in the active center of the protein (Halpert, 1981). This modification impairs the transfer of electrons from NADPH-cytochrome P-450 reductase to cytochrome P-450 (Halpert et al., 1985b) and substrate (drug) oxidation i s impaired. The observed in vitro inhibitory effect of a mechanism-based inactivator on cytochrome P-450-mediated enzyme act i v i t i e s depends on the experimental conditions. Both reversible and irreversible inhibition have been observed with chloramphenicol when i t is added in vitro to microsomes. Reversible inhibition i s observed when chloramphenicol is added immediately prior to the i n i t i a t i o n of the enzymatic reaction and this is due to alternate substrate inhibition by the parent compound (Grogan et al., 1972; Reilly and Ivey, 1979). Irreversible inhibition is 20 observed when chloramphenicol i s p r e i n c u b a t e d w i t h the microsomes p r i o r t o the i n i t i a t i o n o f s u b s t r a t e o x i d a t i o n ( H a l p e r t et al., 1983). The p r e i n c u b a t i o n a l l o w s f o r the fo r m a t i o n o f the r e a c t i v e m e t a b o l i t e t h a t binds c o v a l e n t l y t o the a p o p r o t e i n o f cytochrome P-450 ( H a l p e r t , 1981). I r r e v e r s i b l e i n h i b i t i o n o f cytochrome P-450-mediated enzyme a c t i v i t i e s i s a l s o observed when ch l o r a m p h e n i c o l i s a d m i n i s t e r e d in vivo t o r a t s ( H a l p e r t e t al., 1983, 1985a). 1.1.5.4 S i n g l e v s . M u l t i p l e Doses o f an I n h i b i t o r A compound may a c t as an i n h i b i t o r and an i n d u c e r o f cytochrome P-450, depending on the d u r a t i o n o f treatment. SKF 525-A and t r i a c e t y l o l e a n d o m y c i n i n h i b i t cytochrome P-450-mediated enzyme a c t i v i t i e s f o l l o w i n g a s i n g l e i n j e c t i o n t o r a t s , w i t h the major mechanism being m e t a b o l i t e - i n t e r m e d i a t e complexation (Buening and F r a n k l i n , 1976; Pessayre e t al., 1981). However, a f t e r m u l t i p l e i n j e c t i o n s o f these agents over s e v e r a l days, the l e v e l o f cytochrome P-450 enzymes and t h e i r c a t a l y t i c f u n c t i o n are i n c r e a s e d (e.g. Schenkma'n e t al., 1972; Buening and F r a n k l i n , 1976; Wrighton et al., 1985a; Murray, 1988). The f u l l e x t e n t o f these i n c r e a s e s i s observed f o l l o w i n g d i s s o c i a t i o n o r displacement of the m e t a b o l i t e - i n t e r m e d i a t e complex in vitro. The observed e f f e c t w i t h m u l t i p l e d o s i n g i s due t o both i n c r e a s e d de novo p r o t e i n s y n t h e s i s and decreased d e g r a d a t i o n of e x i s t i n g p r o t e i n s (Watkins e t al., 21 1986). Therefore, in experiments to study the in vivo inhibitory action, the compound should be administered as a single dose to avoid this complication, unless the intent is to increase the amount of the enzyme present as an enzyme-metabolite-intermediate complex. 1.1.5.5 Selective Inhibition of Cytochrome P-450 Cytochrome P-450 enzyme activities appear to be subject to d i f f e r e n t i a l inhibition. For example, 9-hydroxy-e l l i p t i c i n e inhibits ethoxyresorufin O-deethylase activity in microsomes from rats pretreated with 3-methylcholan-threne, but not ethylmorphine N-demethylase activity in microsomes from rats pretreated with phenobarbital (Phillipson et al., 1985). Metyrapone i s more potent in inhibiting p-nitroanisole O-demethylase activity than aniline hydroxylase activity in microsomes from rats pretreated with phenobarbital (Jonen et a i . , 1974). An aspect of research in the f i e l d of cytochrome P-450 has been the design and synthesis of selective inhibitors. Selective cytochrome P-450 enzyme inhibition has been shown with several compounds, including certain derivatives of 1-aminobenzotriazole (Mathews and Bend, 1986), chloramphenicol (Stevens and Halpert, 1988; Halpert et al., 1990), progesterone (Halpert et al., 1989a), pregnenolone (Halpert et al., 1989b) and 3,5-diethoxycarbonyl-l,4-dihydro-2,4,6-trimethylpyridine (Riddick et al., 1990). The 22 identification of relatively selective inhibitors has been fa c i l i t a t e d by the use of enzyme-specific substrates. The most powerful approach involves the use of one of the following substrates: testosterone, androstenedione, progesterone or warfarin (Kaminsky et a l . , 197 9; Waxman, 1988). These compounds are hydroxylated in a regioselective and stereoselective manner. An advantage of using one of these compounds as a substrate is that, in some instances, a particular cytochrome P-450 is the major or sole catalyst in the formation of a metabolite. Another advantage i s that the multiple metabolites formed by the same microsomal preparation can be analyzed simultaneously. To date, not one inhibitor has been shown to be enzyme-specific; that i s , one which inhibits only a single enzyme. An enzyme-specific inhibitor used in conjunction with an enzyme-specific substrate would be very useful in studying the function of a particular cytochrome P-450. 1.1.6 Specificity of Microsomal Enzyme Activities The role of a cytochrome P-450 enzyme in the metabolism of a substrate can be determined in microsomes by immunoinhibition experiments. At saturating concentrations of a monospecific antibody preparation, the contribution of a given enzyme to a substrate reaction can be estimated by determining the percent inhibition of the enzyme activity by the antibody. The purification of cytochrome P-450 enzymes 23 and the preparation of monospecific antibodies have fa c i l i t a t e d the identification of enzyme act i v i t i e s that are specific for particular cytochrome P-450 enzymes. To date, several specific microsomal enzyme act i v i t i e s have been identified using this approach. Some of these are shown in Table 3. The specificity of an enzyme activity for a cytochrome P-450 enzyme depends on factors such as prior drug treatment and the type of tissue under examination. Cytochromes P450IIB1/2 account for more than 90% of the pentoxyresorufin O-dealkylase activity in hepatic microsomes from phenobarbital-treated rats (Lubet et al., 1985; Waxman et a l . , 1987; Dutton and Parkinson, 1989), but do not contribute to this activity i n microsomes from uninduced rats (Waxman et al. , 1987). Testosterone 16ct-hydroxylase activity is specific for cytochrome P450IIC11 in hepatic microsomes from uninduced rats (Waxman, 1984; Waxman, 1987), but not in microsomes from phenobarbital-treated rats (Thomas et al., 1981; Reik et al., 1985; Waxman et al., 1987). Recently, Sesardic et al. (1990a) demonstrated that the high-affinity phenacetin O-deethylase activity was specific for cytochrome P450IA2 in microsomes prepared from the l i v e r , kidney and gut, but not those from the lung. These examples i l l u s t r a t e the importance of and need for verifying the specificity of a given cytochrome P-450-24 TABLE 3 CONTRIBUTION OF RAT HEPATIC CYTOCHROME P-450 TO MICROSOMAL ENZYME ACTIVITIES Enzyme Activi t y Inducer Treatment % Enzyme Reference Erythromycin N-Demethylase DEX 55-60 P450IIIA1/2 Wrighton et a l . , 1985a Ethoxyresorufin O-Deethylase 3MC 3MC 82 > 90 P450IA1 P450IA1 Kelley et a l . , 1987 Dutton and Parkinson, 1989 None 6 P450IA1 Kelley et a l . , 1987 3MC 27 P450IA2 Kelley et a l . , 1987 None 78 P450IA2 Kelley et a l . , 1987 Pentoxyresorufin O-Dealkylase PB PB > 90 > 90 P450IIB1/2 P450IIB1/2 Lubet et al., 1985 Waxman et a l . , 1987 PB > 90 P450IIB1/2 Dutton and Parkinson, 1989 None 0 P450IIB1/2 Waxman et a l . , 1987 Testosterone Hydroxylase 2a None > 85 P450IIC11 Waxman, 1984 Waxman et al., 1987 2p, 60 None PB DEX > 85 > 85 > 85 P450IIIA1/2 P450IIIA1/2 P450IIIA1/2 Halvorson et a l . , 1990 Halvorson et al., 1990 Halvorson et a l . , 1990 7a None PB > 97 80 > 98 P450IIA1 P450IIA1 P450IIA1 Levin et a l . , 1987 Waxman et a l . , 1988b Arlotto and Parkinson, 1989 DEX > 96 > 98 > 96 P450IIA1 P450IIA1 P450IIA1 Arlotto and Parkinson, 1989 Levin et al., 1987 Arlotto and Parkinson, 1989 16a None None > 85 14 0 0 P450IIC11 P450IIB1/2 P450IIB1/2 P450I1B1/2 Waxman, 1984 Waxman et al., 1987 Thomas et a l . , 1981 Reik et a l . , 1985 Waxman et a l . , 1987 PB 66 77 60-70 P450IIB1/2 P450IIB1/2 P450IIB1/2 Thomas et a l . , 1981 Reik et a l . , 1985 Waxman et a l . , 1987 PB 30 P450IIC11 Waxman, 1984 Waxman et a l . , 1987 up PB PB 89 > 90 P450IIB1/2 P450IIB1/2 Reik et a l . , 1985 Waxman et a l . , 1987 Abbreviations: PB, phenobarbital; DEX, dexamethasone; 3MC, 3-methylcholanthrene mediated microsomal enzyme activity under the experimental conditions. 26 1.2 CIMETIDINE 1.2.1 Chemical S t r u c t u r e C i m e t i d i n e i s a 4 , 5 - s u b s t i t u t e d i m i d a z o l e d e r i v a t i v e w i t h a cyano group a t t a c h e d t o i t s s i d e c h a i n . I t s chemical s t r u c t u r e i s shown i n F i g u r e 2. 1.2.2 Pharmacology The b a s o l a t e r a l membrane o f the g a s t r i c mucosal and p a r i e t a l c e l l s c o n t a i n r e c e p t o r s f o r his t a m i n e , g a s t r i n s and a c e t y l c h o l i n e . These p a r t i c u l a r r e c e p t o r s f o r histamine are c a l l e d h i s tamine H 2-receptors (Black e t al., 1972). S t i m u l a t i o n o f histamine H 2 - r e c e p t o r s a c t i v a t e s adenylate c y c l a s e , which i n t u r n i n c r e a s e s the i n t r a c e l l u l a r c o n c e n t r a t i o n o f c y c l i c AMP ( H i l l , 1990). As a r e s u l t , s p e c i f i c c y c l i c AMP-dependent p r o t e i n k i n a s e s are a c t i v a t e d , the p r o t o n pump, a H +,K +-ATPase, i s s t i m u l a t e d , and the s e c r e t i o n o f hydrogen i o n s i n t o the stomach i s i n c r e a s e d (Wolfe and S o i l , 1988). I t has been shown t h a t s t i m u l a t i o n o f t h e r e c e p t o r s f o r g a s t r i n s and a c e t y l c h o l i n e a l s o r e s u l t s i n t he s e c r e t i o n o f hydrogen i o n s ( H i l l , 1990). C i m e t i d i n e i s a c o m p e t i t i v e histamine H 2-receptor a n t a g o n i s t (Brimblecombe e t al., 1975). As r e s u l t o f h i s t a m i n e H 2 - r e c e p t o r blockade by c i m e t i d i n e , both the b a s a l Figure 2 The metabolic pathways for cimetidine i n r a t s . (From: Taylor et a l . , 1978; Zbaida et a l . , 1984)-H 3 C C H 2 S C H 2 C H 2 N H C N H C H 3 X J-CN H N i i N N C N cimetidine ^ o t H . C C H j S C H j C H j N H C N H C H , HN^ N N C N cimetidine tulphoaide H O C K , C H 2 S C H , C H 2 N H C N H C H , H N , . N N C N C.H,0« I H,C -CHJSCHJCHJNCNHCH, HN M NCN NCN .NH-C-NH, H 3 C t 1 H N ^ N Aw'-dcimclhylcimctidin« metabolite H , C C H , S C H 2 C H , N H C N H C H , >=< » H N .N N C N H j cimetidine quanylurea H 1 C S . C H , S C H , C H , N H C N H C H j H N ^ N N H c t n i e l t d t n e - N * - q l u c u r o n i d e c t f l i e t i d i n e q u a n i d m e 28 and stimulated secretion of gastric acid are reduced (Pounder, 1984). In 1977, cimetidine was approved for c l i n i c a l use in Canada. It is used therapeutically in the treatment of peptic ulcers and other gastric acid-related disorders (Feldman and Burton, 1990b). 1.2.3 Pharmacokinetics Cimetidine i s rapidly absorbed after oral administration in man. Following the ingestion of a 4 00 mg tablet of cimetidine, the peak serum concentration i s approximately 10 uM and occurs at 90 min after dosing (Griffiths et al., 1977). The absolute bioavailability after an oral dose ranges from 58-89% (Lin, 1991). In rats, more than 90% of an oral dose is absorbed (Taylor et al., 1978). In man, cimetidine is distributed to the kidney, gallbladder, stomach, l i v e r and skeletal muscle (Schentag et al., 1981). In addition, cimetidine penetrates the blood-brain barrier and distributes into the cerebrospinal f l u i d (Jonsson et al., 1982). The distribution of cimetidine in rats i s similar to that in man except that there i s a lack of penetration into the central nervous system (Cross, 1977). Cimetidine in plasma is only 13-25% bound to plasma proteins in man (Taylor et al., 1978; Somogyi et al., 1980) 29 and 10-23% i n r a t s ( T a y l o r et al. , 1978; Adedoyin e t al., 1987a) . The e l i m i n a t i o n h a l f - l i f e o f c i m e t i d i n e i s a pproximately 2 h i n h e a l t h y human v o l u n t e e r s and u l c e r p a t i e n t s (Somogyi and Gugler, 1983) and 30-45 min i n r a t s (Weiner and Roth, 1981; Adedoyin e t al., 1987a). C i m e t i d i n e i s e l i m i n a t e d mainly by r e n a l t u b u l a r s e c r e t i o n and glomerular f i l t r a t i o n (Somogyi e t al., 1980; Weiner and Roth, 1981). In a 24 h p e r i o d f o l l o w i n g the i n g e s t i o n of a s i n g l e o r a l dose of c i m e t i d i n e i n man, 70-80% o f t h e dose i s recovered i n u r i n e (Burlahd e t al., 1975; T a y l o r e t al., 1978; M i t c h e l l et al., 1982). In r a t s , the r e c o v e r y i s 60-70% i n the same p e r i o d ( T a y l o r e t al., 1978). In both c a s e s , 50-75% of the t o t a l r e c o v e r y i s the parent compound ( T a y l o r et al., 1978; M i t c h e l l et al., 1982; Dixon et al., 1985; Adedoyin e t al., 1987a). The metabolism of c i m e t i d i n e i n man and r a t s i n v o l v e s g l u c u r o n i d a t i o n , o x i d a t i o n and h y d r o l y s i s ( F i g u r e 2 ) . The most abundant m e t a b o l i t e of c i m e t i d i n e i n man i s c i m e t i d i n e -N ' - g l u c u r o n i d e , which accounts f o r 24% of the t o t a l u r i n a r y e x c r e t i o n i n the f i r s t 24 h a f t e r d o s i n g ( M i t c h e l l et al., 1982). The o t h e r m e t a b o l i t e s of c i m e t i d i n e are c i m e t i d i n e s u l f o x i d e (7-19%), 5-hydroxymethylcimetidine (4-5%), c i m e t i d i n e guanylurea ( 2 % ) , and c i m e t i d i n e g u a n i d i n e (<0.1%) (B u r l a n d e t al., 1975; G r i f f i t h s e t a l . , 1977; T a y l o r e t a l . , 1978; M i t c h e l l et a l . , 1982). In r a t s , c i m e t i d i n e 30 sulfoxide, 5-hydroxymethylcimetidine and N-desmethyl-cimetidine have been shown to be generated from cimetidine by r a t hepatic microsomes (Zbaida et al., 1984). 1.2.4 Cimetidine Drug-Drug Interactions Since the f i r s t p u blication of a c o n t r o l l e d c l i n i c a l study of a cimetidine drug i n t e r a c t i o n with warfarin i n 1979 ( S e r l i n et al., 1979), numerous other drug-drug interactions in v o l v i n g cimetidine have been i d e n t i f i e d . In many of these cases, the i n t e r a c t i o n i s due to i n h i b i t i o n of hepatic drug metabolism by cimetidine. The topic of cimetidine drug-drug in t e r a c t i o n s i n man has been reviewed extensively i n the l i t e r a t u r e (e.g. Somogyi and Muirhead, 1987; Smith and Kendall, 1988; Feldman and Burton, 1990a). In man, cimetidine impairs the clearance of drugs that undergo extensive cytochrome P-450-mediated hepatic oxidative metabolism, but does not a f f e c t the clearance of drugs that are mainly eliminated by conjugation reactions such as glucuronidation, sulphation and a c e t y l a t i o n . In animal studies, the in vitro addition of cimetidine to hepatic microsomes has been shown to i n h i b i t the cytochrome P-450-catalyzed oxidation of many substrates (Table 4). Observations from human and animal studies have led to the perception that cimetidine i s a general i n h i b i t o r of cytochrome P-450 ( R e i l l y et a l . , 1988; Leclercq et a l . , 1989). However, a d e t a i l e d analysis of the l i t e r a t u r e 31 TABLE 4 INHIBITION OF HEPATIC MICROSOMAL CYTOCHROME P-450 ENZYME ACTIVITIES BY IN VITRO CIMETIDINE IN RATS Enzyme A c t i v i t y Type o f I n h i b i t i o n K i (mM) R e f e r e n c e a m i n o p y r i n e N-demethylase n o n - c o m p e t i t i v e N.D. P e l k o n e n and Puurunen, 1980 a m i n o p y r i n e N-demethylase n o n - c o m p e t i t i v e N.D. Tanaka e t al., 1985 a m i n o p y r i n e N-demethylase mixed 0.13 Speeg e t al., 1982 a m i n o p y r i n e N-demethylase mixed 0.7 Imai e t al., 1986 b e n z o [ a ] p y r e n e h y d r o x y l a s e n o n - c o m p e t i t i v e N.D. P e l k o n e n and Puurunen, 1980 7-ethoxycoumarin O - d e e t h y l a s e c o m p e t i t i v e 0.18 0.3 Re n d i c e t al., 1979 7-ethoxycoumarin 0 - d e e t h y l a s e c o m p e t i t i v e 0.8 J e n s e n and G u g l e r , 1985 m e p e r i d i n e N-demethylase c o m p e t i t i v e 0.45 K n o d e l l e t al., 1982 m e p e r i d i n e N-demethylase n o n - c o m p e t i t i v e N.D. Dawson and V e s t a l , 1984 m e t o p r o l o l a - h y d r o x y l a s e c o m p e t i t i v e 0.009 Lennard e t al., 1986 m e t o p r o l o l a - h y d r o x y l a s e c o m p e t i t i v e 0.019 W r i g h t e t al., 1991 m e t o p r o l o l O-desmethylase c o m p e t i t i v e 0.038 Le n n a r d e t a l . , 1986 morphine N-demethylase c o m p e t i t i v e 0.068 R e i l l y and Winzor, 1984 p e n t o b a r b i t a l h y d r o x y l a s e c o m p e t i t i v e 0.13 K n o d e l l e t al., 1982 t r i m e t h a d i o n e N-demethylase n o n - c o m p e t i t i v e N.D. Tanaka e t a l . , 1985 N.D. = not determined 32 i n d i c a t e d s u b s t a n t i a l , but i n d i r e c t , evidence t h a t c e r t a i n cytochrome P-450 enzymes may be l e s s s u s c e p t i b l e or even r e f r a c t o r y t o the i n h i b i t o r y a c t i o n o f c i m e t i d i n e . 1.2.5 D i f f e r e n t i a l I n h i b i t i o n of Cytochrome P-450-Mediated H e p a t i c Drug Metabolism by In Vivo C i m e t i d i n e Treatment 1.2.5.1 C l i n i c a l S t u d i e s I t has been shown i n c l i n i c a l s t u d i e s t h a t c i m e t i d i n e does not a f f e c t the c l e a r a n c e o r s t e a d y - s t a t e serum c o n c e n t r a t i o n s o f s e v e r a l drugs t h a t are m e t a b o l i z e d mainly by cytochrome P-450 enzymes. The r e s u l t s of these s t u d i e s are summarized i n Table 5. A few of t h e s e w i l l be e x p l a i n e d i n d e t a i l . Tolbutamide i s metabolized i n man t o hydroxy-t o l b u t a m i d e , which i s then p a r t i a l l y c o n v e r t e d t o c a r b o x y t o l b u t a m i d e (Thomas and Ikeda, 1966). V a r i o u s drugs have been shown t o a f f e c t the c l e a r a n c e of tolbutamide, i n c l u d i n g sulphaphenazole (Hansen and C h r i s t e n s e n , 1977). The a d m i n i s t r a t i o n of c i m e t i d i n e t o h e a l t h y human v o l u n t e e r s has no e f f e c t on the t o t a l body c l e a r a n c e of tolbutamide (Dey e t a l . , 1983; S t o c k l e y e t a l . , 1986; Adebayo and Coker, 1988) o r the f o r m a t i o n c l e a r a n c e of the hydroxytolbutamide m e t a b o l i t e ( S t o c k l e y et al., 1986). These o b s e r v a t i o n s suggest t h a t the cytochrome P-450 enzyme(s) i n v o l v e d i n the h y d r o x y l a t i o n o f tolbutamide i s ( a r e ) not s u b j e c t t o 33 TABLE 5 CLINICAL STUDIES WITH CIMETIDINE: LACK OF A DRUG-DRUG INTERACTION Drug V a r i a b l e Not N Reference A f f e c t e d by Cimetidine c a r b a m a z e p i n e s t e a d y - s t a t e s e r u m c o n c e n t r a t i o n * 7 11 8 Sonne e t a l . , 1983 L e v i n e e t a l . , 1985 D a l t o n e t al., 1986 c y c l o s p o r i n e t o t a l body c l e a r a n c e 2 J a r e w e n k o e t a l . , 1986 d e s i p r a m i n e t o t a l body c l e a r a n c e " 4 S t e i n e r and S p i n a , 1987 f o r m a t i o n c l e a r a n c e o f 2 - h y d r o x y -d e s i p r a m i n e " 4 S t e i n e r and S p i n a , 1987 e s t r a d i o l 1 6 a - h y d r o x y l a t i o n o f e s t r a d i o l i n s e r u m 9 G a l b r a i t h and M i c h n o v i c z , 1989 u r i n a r y e x c r e t i o n o f 1 6 a - h y d r o x y -e s t r o n e 9 G a l b r a i t h and M i c h n o v i c z , 1989 m e x i l e t i n e t o t a l body c l e a r a n c e 6 6 K l e i n e t al., 1985 B r o c k m e y e r e t a l . , 1989 f o r m a t i o n c l e a r a n c e o f 4 - h y d r o x y m e t h y l -m e x i l e t i n e 6 B r o c k m e y e r e t a l . , 1989 f o r m a t i o n c l e a r a n c e o f p a r a -h y d r o x y m e t h y -m e x i l e t i n e 6 B r o c k m e y e r e t al., 1989 m i s o n i d a z o l e t o t a l body c l e a r a n c e 6 Begg e t al., 1983 AUC f o r O - d e s m e t h y l -m i s o n i d a z o l e 6 Begg e t a l . , 1983 t o l b u t a m i d e t o t a l body c l e a r a n c e 10 7 8 7 Dey et al., 1983 S t o c k l e y e t a l . , 1986 A d e b a y o e t al., 1988 S t o c k e l y e t al., 1986 f o r m a t i o n c l e a r a n c e o f h y d r o x y t o l b u t a m i d e N = number o f s u b j e c t s ; AUC = area under the serum-c o n c e n t r a t i o n curve * In p a t i e n t s on c h r o n i c carbamazapine therapy ** In slow m e t a b o l i z e r s o f de b r i s o q u i n e 34 inhibition by cimetidine. Recently, i t was reported that at least three human cytochrome P-450 enzymes are involved in the hydroxylation of tolbutamide: cytochrome P450IIC8, cytochrome P450IIC9 and cytochrome P450IIC10 (Brian et a l . , 1989; Relling et al., 1990). The enzyme involved in the hydroxylation of debrisoquine to 4-hydroxydebrisoquine exhibits genetic polymorphism and two distinct phenotypes have been noted: extensive and poor metabolizers (Mahgoub et a l . , 1977). The enzyme which hydroxylates debrisoquine in man has been purified (Gut et al., 1984; Distlerath et al., 1985) and i s referred to as cytochrome P450IID6 (Nebert et al., 1989). The oxidative metabolism of some drugs, including desipramine, has been associated with the polymorphism of debrisoquine hydroxylation (Bertilsson and Aberg-Wistedt, 1983). The concurrent administration of cimetidine and desipramine to healthy human volunteers results in a decrease in the total body clearance of desipramine and a decrease in the formation clearance of the 2-hydroxy-desipramine metabolite in extensive metabolizers of debrisoquine (Steiner and Spina, 1987). In contrast, in poor metabolizers of debrisoquine, cimetidine does not affect the total body clearance of desipramine or the formation clearance of the 2-hydroxydesipramine metabolite (Steiner and Spina, 1987). It has been shown that there i s no immunodetectable cytochrome P450IID6 in hepatic 35 microsomes from poor metabolizers of debrisoquine (Gonzalez et al., 1988; Zanger et al., 1988). In poor metabolizers, other cytochrome P-450 enzyme(s) are l i k e l y to be involved in the oxidation of debrisoquine and other drugs associated with the polymorphism of debrisoquine hydroxylation. The results from the cimetidine-desipramine drug interaction study therefore suggest that cimetidine inhibits cytochrome P450IID6, but not the cytochrome P-450 enzyme(s) involved in the hydroxylation of desipramine in poor metabolizers. Carbamazepine is eliminated mainly by hepatic metabolism (Lertratanangkoon and Horning, 1982). Cimetidine decreases the total body clearance of carbamazepine in healthy human volunteers administered a single dose of carbamazepine (Webster et al., 1984; Dalton et al., 1985). In contrast, in epileptic patients on chronic carbamazepine therapy (Sonne et al., 1983; Levine et al., 1985) and in healthy human volunteers pretreated with multiple doses of carbamazepine (Dalton et al., 1986), cimetidine has no effect on the steady-state serum concentration of carbamazepine. Carbamazepine i s an inducer of cytochrome P-450 and i t induces i t s own metabolism (Eichelbaum et al., 1975, 1985). It is therefore possible that cimetidine inhibits the enzyme(s) involved in carbamazepine metabolism in uninduced subjects, but not the enzyme(s) involved in the metabolism of this drug in subjects undergoing chronic carbamazepine therapy. 36 Antipyrine is used frequently as an in vivo marker for cytochrome P-450-mediated hepatic drug metabolism in man (Vesell, 1979). Feely et al. (1984) demonstrated that the percent decrease in antipyrine clearance by cimetidine was almost two-fold greater in subjects pretreated with rifampin than in subjects who were not pretreated. Rifampin i s an efficacious inducer of human cytochrome P450IIIA enzymes (Combalbert et al., 1989). It i s possible that the cytochrome P-450 enzyme(s) responsible for antipyrine oxidation in the rifampin-induced subjects is(are) more susceptible to inhibition by cimetidine than those involved in the metabolism of antipyrine in uninduced subjects. 1.2.5.2 Animal Studies Antipyrine i s eliminated in rats by hepatic metabolism to y i e l d three major metabolites: 3-hydroxymethylantipyrine, 4-antipyrine and norantipyrine (Danhof et al., 1979). Multiple cytochrome P-450 enzymes are thought to be involved in the formation of the metabolites of antipyrine. The in vivo administration of cimetidine to adult male rats d i f f e r e n t i a l l y inhibits the in vivo formation of the major metabolites of antipyrine (Adedoyin et al., 1987b). Cimetidine i s approximately 50 times more potent in inhibiting the formation of 3-hydroxymethylantipyrine than in inhibiting the formation of 4-hydroxyantipyrine (Adedoyin et al., 1987b). This suggests that the formation of the 37 metabolites of antipyrine is catalyzed by different cytochrome P-450 enzymes and that these enzymes have diff e r e n t i a l susceptibility to inhibition by cimetidine. Drew et a l . (1981) reported that a single intraperitoneal administration of cimetidine (150 mg/kg) to adult male rats pretreated with 3-methylcholanthrene resulted in 89% inhibition of hepatic microsomal benzo[a]pyrene hydroxylase activity, but had no effect on 7-ethoxycoumarin O-deethylase, biphenyl 4-hydroxylase, zoxazolamine hydroxylase or aniline hydroxylase activity. This suggests that certain cytochrome P-450 enzymes are not susceptible to inhibition by in vivo cimetidine treatment in adult male rats. Recently, Galbraith and Jellinck (1989) demonstrated that multiple intraperitoneal injections of cimetidine (173 mg/kg every 12 hours for 5 doses) to adult male rats resulted in a decrease in hepatic microsomal estradiol 2-hydroxylase, estradiol 16a-hydroxylase, ethylmorphine N-demethylase, aniline hydroxylase and benzo[a]pyrene hydroxylase a c t i v i t i e s , but had no effect on 7-ethoxycoumarin O-deethylase activity. In contrast, none of these acti v i t i e s were affected in hepatic microsomes from adult female rats subjected to the same cimetidine treatment protocol. These results are d i f f i c u l t to interpret since cimetidine was administered as multiple injections over several days rather than as a single dose. It has been 38 shown that treatment of adult male rats with multiple injections of cimetidine modestly induces hepatic cytochromes P450IA1/2 and cytochromes P450IIB1/2 (Ioannides et al., 1989). After repeated administration of cimetidine over several days, the relative proportions of various cytochrome P-450 enzymes in the hepatic microsomes may have been changed, due to induction of some enzymes and suppression of others. Thus, a decrease in an enzyme activi t y under these circumstances may be due not only to inhibition, but also to suppression. However, i f the observations by Galbraith and Jellinck (1989) were mainly a result of inhibition, then they would suggest that certain cytochrome P-450 enzymes in adult male and female rats are not susceptible to the inhibitory action of cimetidine. Taken together, one interpretation from the human and animal studies with cimetidine described above is that not a l l cytochrome P-450 enzymes are inhibited following in vivo cimetidine treatment. To date, systematic studies have not been performed to determine whether cimetidine selectively inhibits cytochrome P-450 enzymes. The information generated from such studies would be important for several reasons. F i r s t , in order to develop a detailed understanding of the inhibition of hepatic drug metabolism by cimetidine, i t is necessary to i n i t i a l l y identify the specific cytochrome P-450 enzymes inhibited by the compound before any mechanistic studies can be performed. Second, in 39 certain instances, the information generated may help to predict potential drug-drug interactions with cimetidine. Third, knowledge of the specific enzyme(s) inhibited by cimetidine would allow investigators to use this compound as a pharmacological probe in cytochrome P-450 research; for example, to study the function of a particular enzyme. 1.2.6 Inhibition of Cytochrome P-450 by In Vitro or In Vivo Cimetidine Treatment Inhibition of cytochrome P-450 enzyme ac t i v i t i e s by the in vitro addition of cimetidine to microsomes has been well documented. Cimetidine interacts with rat and human microsomal cytochrome P-450 in vitro by the binding of a ligand nitrogen atom to the heme iron of the hemoprotein at the sixth coordination position, resulting in a characteristic Type II difference spectrum with the peak and trough at 420-432 nm and 390-397 nm, respectively (Rendic et a l . , 1979, 1983, 1984; Pelkonen and Puurunen, 1980; Speeg et a l . , 1982; Knodell et a l . , 1982; Bast et a l . , 1989). At present, the mechanism by which cytochrome P-450 enzymes are inhibited following in vivo cimetidine administration i s s t i l l not known. However, i t has become apparent that the observed inhibition of cytochrome P-450 by in vitro cimetidine i s not equivalent to the effect observed following the in vivo administration of the drug to intact animals or to humans (Somogyi and Muirhead, 1987). 40 The concentration of cimetidine required for the in vitro inhibition of a microsomal cytochrome P-450-mediated enzyme activity is typically 100-1000 times greater than the serum concentration associated with inhibition of drug metabolism in vivo. In in vitro enzyme inhibition studies, the IC S 0 is the concentration of the inhibitor required to reduce an enzyme activity by 50%. In rat hepatic microsomes, the usual IC 5 0 for inhibition of various enzyme act i v i t i e s by in vitro cimetidine is in the range of 1-10 mM (Pelkonen and Puurunen, 1980; Speeg et al., 1982; Dawson and Vestal, 1984; Mosca et al., 1985; Imai et al., 1986; Yee and Shargel, 1986; Wang et a l . , 1988; Bast et al., 1989; Vyas et al., 1990). In contrast, at 1 h after a single intraperitoneal dose (120 mg/kg) of cimetidine to rats, substantial inhibition of aminopyrine elimination i s observed, whereas the serum drug concentration is only approximately 0.008 mM (Speeg et al., 1982). In human hepatic microsomes, the IC S 0 for inhibition of cytochrome P-450 enzyme activity by in vitro cimetidine is also in the range of 1-10 mM (Puurunen et al., 1980, Rendic et al., 1984; Hoensch et al., 1985; Imai et al., 1986; Pasanen et al., 1988; Vyas et al., 1990). During chronic administration of cimetidine at therapeutic doses in man, the serum concentration of the drug i s below 0.006 mM for most of the dosage interval (Somogyi and Gugler, 1983) and 41 inhibition of drug metabolism is known to occur at these low serum concentrations (Cohen et al., 1985). To reconcile the differences between the inhibition of cytochrome P-450 by in vitro and in vivo cimetidine, several interpretations have been made by various investigators. Based on data from spectral binding studies, i t has been suggested that at least two cimetidine-binding sites exist in rat hepatic microsomes, with spectral dissociation constants (Ks) of 0.008 - 0.072 mM and 0.10 - 0.33 mM (Rendic et al., 1979, 1983; Speeg et al., 1982; Reilly et al., 1983; Rekka et al., 1988). To account for the discrepancy between the concentrations required for inhibition by in vivo and in vitro cimetidine, Reilly et al. (1983) proposed that the inhibition observed with in vivo cimetidine i s due to i t s interaction with the higher a f f i n i t y binding site. A major problem with this proposal is that the spectral binding studies with cimetidine were a l l performed with microsomes and not with a purified cytochrome P-450. The observation of biphasic binding may simply reflect the different a f f i n i t i e s of the various cytochrome P-450 enzymes in the microsomes for cimetidine. The IC 5 0 for in vitro inhibition i s dependent on the type of inhibition. In the case of competitive inhibition, IC 5 0 =. Ki (1 + [S] / Km), where Ki i s the inhibitory constant, [S] i s the substrate concentration and Km is the Michaelis constant. Reilly and Winzor (1984) suggested that 42 the high IC 5 0 values observed in various in vitro cimetidine studies may simply be due to the high substrate concentrations usually used in the assays. The value of IC 5 0 is dependent on the ratio of [S] / Km i f the inhibition is competitive. It has been documented that the inhibition of cytochrome P-450 enzyme activities by in vitro cimetidine is not always competitive (Table 4). Moreover, substantial inhibition of microsomal cytochrome P-450-mediated enzyme act i v i t i e s i s observed 2 h after a single intraperitoneal injection of cimetidine to rats (Drew et al., 1981). The standard method for performing in vitro inhibition studies involves the addition of the putative inhibitor to the incubation mixture immediately prior to the i n i t i a t i o n of substrate oxidation. However, this method does not allow for the catalysis-dependent formation of reactive intermediates. Jensen and Gugler (1985) therefore used a preincubation protocol in an attempt to further investigate the inhibition of cytochrome P-450 by cimetidine in rats. They observed that a 10 min preincubation of microsomes with cimetidine (0.25 mM) and NADPH prior to the addition of the substrate increased the inhibition of 7-ethoxycoumarin 0-deethylase activity compared to "control" samples not subjected to preincubation. Based on this observation, these investigators proposed that the inhibition of cytochrome P-450 by cimetidine in vivo involves the formation of a metabolite-intermediate or an activated 43 complex. However, the increase in the inhibition of the enzyme activity reported by Jensen and Gugler (1985) following preincubation may have been due to the presence of NADPH in the preincubation mixture. It has been shown that l i p i d peroxidation by rat hepatic microsomes is NADPH-dependent and that this results in the breakdown of heme from the holoenzyme (Levin et al., 1973). Ioannoni et a l . (1986) claimed that when the effect by NADPH was taken into account, preincubation had no effect on the inhibition of microsomal morphine N-demethylation by 0.5 mM cimetidine. According to Rekka et al. (1988), preincubation of hepatic microsomes with cimetidine (0.1 mM or 0.25 mM) did not increase the inhibition of the microsomal oxidation of tofenacine or 7-ethoxyresorufin. However, they failed to specify whether the control samples were preincubated with NADPH. It i s possible that cimetidine forms a metabolite-intermediate complex only with certain cytochrome P-450 enzymes. To date, i t has not been shown conclusively whether metabolite-intermediate complexation is a mechanism involved in the inhibition of cytochrome P-450 enzymes by cimetidine. It i s apparent that the standard method for conducting inhibition studies with cimetidine added in vitro to microsomes does not necessarily provide results that are equivalent to those that occur following the in vivo administration of the drug to an intact animal. In light of 44 this discrepancy, and in the absence of a suitable in vitro method, cimetidine inhibition studies should be conducted with the drug administered in vivo in order to avoid making erroneous conclusions regarding the enzyme selectively or the mechanism of inhibition by the compound. However, there are limitations in performing inhibition studies with a putative inhibitor administered in vivo to an intact animal. Therefore, there is an obvious need to find a suitable in vitro method that w i l l adequately model the inhibition that occurs following the in vivo administration of an inhibitor. 45 1.3 OBJECTIVES The overall goal of this investigation was to provide a better understanding of the inhibition of hepatic cytochrome P-450 by in vivo and in vitro cimetidine treatment. The following were the objectives: A. to determine whether cimetidine, when administered in vivo to adult male rats, d i f f e r e n t i a l l y inhibits hepatic microsomal cytochrome P-450-mediated enzyme act i v i t i e s , B. to determine whether the differ e n t i a l inhibition of cytochrome P-450-mediated enzyme a c t i v i t i e s by in vivo cimetidine, i f present, occurs when cimetidine i s added to hepatic microsomes in vitro, and C. to determine the effect of preincubation on the inhibition of cytochrome P-450 enzyme act i v i t i e s by in vitro cimetidine treatment. 46 MATERIALS AND METHODS 2.1 CHEMICALS Cimetidine hydrochloride was a g i f t from Smith Kline & French Canada Ltd. (Montreal, Que.). Aminopyrine, erythromycin base, dexamethasone, fi-naphthof lavone, semicarbazide hydrochloride, ammonium acetate, acetylacetone, testosterone, androstenedione and 16-keto-testosterone were obtained from Sigma Chemical Company (St. Louis, MO). 60-, 7a-, 110-, 16a, and 160-hydroxytesto-sterone were bought from Steraloids, Inc. (Wilton, NH). 2a-and 20-hydroxytestosterone were provided by Professor D.N. Kirk, MRC Steroid Reference Collection, Queen Mary's College, London, United Kingdom. Ethoxyresorufin (7-ethoxyphenoxazone) and pentoxyresorufin (7-pentoxyphenoxazone) were supplied by Molecular Probes, Inc. (Eugene, OR). Resorufin (phenoxazone) was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI). Phenobarbital sodium and NADPH were purchased from British Drug House (Toronto, Ont.) and Boehringer Mannheim Canada Ltd. (Dorval, Que.), respectively. Formaldehyde was obtained from Fisher Scientific Company (Fair Lawn, NJ). Bovine serum albumin was purchased as part of the Bio-Rad Protein Assay K i t R (Bio-Rad Laboratories, Mississauga, Ont.). Monospecific polyclonal rabbit-anti-rat cytochrome 47 P450IIC11 antibody was generously provided by Dr. S. M. Bandiera of the Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada. Control rabbit IgG (ChromPureR) was supplied by Jackson Immunoresearch Lab. Inc. (West Grove, PA). A l l other chemicals were reagent grade. 2.2 ANIMALS Adult male Wistar rats (51-55 days old, weighing 250-300 g) were obtained from Canadian Breeding Farms (Montreal, Que.) and were allowed to acclimatize in our animal care f a c i l i t y for at least seven days prior to i n i t i a t i o n of treatment. The temperature of the animal room was maintained at 22°C and fluorescent lighting in the room was controlled by an automatic timer (0800 h on, 2200 h o f f ) . The animals were housed on LobundR corncob bedding (Paxton Processing Ltd., Paxton Processing Ltd., Paxton, IL) and were provided with Rodent Laboratory Chow #5001R (Ralston Purina Canada Inc., Longueuil, Que.) and tap water ad libitum up to the time of sacrifice. 2.3 TREATMENT Phenobarbital sodium and cimetidine hydrochloride were dissolved in d i s t i l l e d water. Dexamethasone was suspended in 2% w/v Tween 80 (e.g. Schuetz and Guzelian, 1984; Wrighton et a l . , 1985a), whereas fJ-naphthof lavone was 48 suspended in corn o i l (e.g. Guengerich et al., 1982b; Thomas et al., 1983; Waxman, 1984). A l l injections were by the intraperitoneal route. 2.3.1 Induction Protocol (Pretreatment) Rats were pretreated with a compound known to preferentially induce the cytochrome P-450 enzyme(s) of interest. To induce cytochrome P450IIB1 and cytochrome P450IIB2, rats were pretreated with phenobarbital sodium, 80 mg/kg once daily for four days. Rats pretreated in this manner w i l l be referred to as "phenobarbital-induced rats". Control rats received 0.9% saline. To induce cytochrome P450IIIA1, rats were pretreated with dexamethasone, 100 mg/kg once daily for 3 days. Rats pretreated in this manner w i l l be referred to as "dexamethasone-induced rats". Control rats received the vehicle, 2% w/v Tween 80. To induce cytochrome P450IA1, rats were pretreated with fi-naphthoflavone, 40 mg/kg once daily for three days. Rats pretreated in this manner w i l l be referred to as "f3-naphthoflavone-induced rats". Control rats received the vehicle, corn o i l . These are standard injection protocols for maximally inducing the cytochrome P-450 enzymes indicated above (e.g. Guengerich et al., 1982b; Waxman, 1984; Halpert et al., 1985a; Dutton and Parkinson, 1989). The saline- or vehicle-treated rats (four rats per group) were included in each induction/inhibition study as an 49 internal control group. To study the cytochrome P-450 enzymes in the uninduced state, the rats were not pretreated (e.g. Guengerich et a l . , 1982b; Waxman et a l . , 1985; Wrighton et al., 1985a). The term "uninduced rats" w i l l be used to refer to those animals not subjected to any injections during the pretreatment phase. 2.3.2 Inhibition Protocol (Treatment) In the in vivo inhibition experiments, a single dose of cimetidine hydrochloride (150 mg/kg) or 0.9% saline (uninhibited control) was administered to uninduced and induced rats 24 h after the last pretreatment dose. In cases where the drug was administered in this manner, the term "in vivo cimetidine" w i l l be used. This dosage of cimetidine has been used by other investigators (Drew et a l . , 1981; Reichen et a l . , 1986). In a preliminary experiment, a dose of 150 mg/kg intraperitoneally yielded maximal inhibition of aminopyrine N-demethylase activity when rats were sacrificed 90 min after the cimetidine injection. In the in vitro inhibition experiments, cimetidine hydrochloride, which was dissolved in d i s t i l l e d water, was added directly to the incubation mixture. In cases where the drug was added in this manner, the term "in vitro cimetidine" w i l l be used. 50 2.4 TIME OF SACRIFICE In the in vivo cimetidine experiments, rats were sacrificed 90 min after the single injection of cimetidine or saline. In the previous studies reported by other investigators, the time of sacrifice varied between one to two hours (Drew et al. , 1981, Mosca et al., 1985; Yee and Shargel, 1986). In a preliminary experiment, maximal inhibition of aminopyrine N-demethylase activity was observed when rats were sacrificed 90 min after a single injection of cimetidine. In the other experiments, the animals were sacrificed 24 h after the last dose of the inducer or vehicle (e.g. Gontovnick and Bellward, 1980). 2 .5 PREPARATION OF HEPATIC MICROSOMES Hepatic microsomes were prepared by a standard method (Lu and Levin, 1972). Each rat was stunned, decapitated and exsanguinated. Immediately after the abdominal cavity was opened, the l i v e r was excised and immersed in 20 mL of 50 mM TRIS / 1.15% potassium chloride (pH 7.5) at 4 °C. A l l subsequent procedures were performed at 4 °C. Depending on the particular experiment, either individual or pooled livers were homogenized in the 50 mM TRIS / 1.15% potassium chloride buffer using a Potter-Elvejhem homogenizer with a motor-driven teflon pestle. The homogenate was centrifuged at 10,000 x g for 20 min. Following centrifugation, the 51 supernatant was f i l t e r e d through f o u r l a y e r s o f c h e e s e c l o t h t o remove the s u r f a c e l i p i d and was then s u b j e c t e d t o u l t r a c e n t r i f u g a t i o n a t 100,000 x g f o r 60 min. The p e l l e t was suspended i n 20 mL of the 10 mM EDTA / 1.15% potassium c h l o r i d e (pH. 7.4) b u f f e r and r e c e n t r i f u g e d a t 100,000 x g f o r 60 min. The microsomal p e l l e t was resuspended i n 4 mL of 0.25 M s u c r o s e . A l i q u o t s (0.5 - 1 mL) of each microsomal p r e p a r a t i o n were p l a c e d i n cryotubes and s t o r e d a t -80 °C u n t i l use. When prepared by t h i s method, the microsomes were found t o be s t a b l e f o r a t l e a s t two years (Thomas et al., 1983). 2.6 DETERMINATION OF TOTAL CYTOCHROME P-450 CONTENT T o t a l microsomal cytochrome P-450 con t e n t was determined from the sodium d i t h i o n i t e - r e d u c e d carbon monoxide d i f f e r e n c e spectrum (Omura and Sato, 1964) u s i n g a molar e x t i n c t i o n c o e f f i c i e n t o f 91 cm - 1 mM"1 between 450 nm and 490 nm. Microsomal suspensions were d i l u t e d i n a b u f f e r c o n t a i n i n g 100 mM potassium phosphate (pH 7.4), 20% v/v g l y c e r o l and 0.1 mM EDTA (Thomas e t al., 1983). Microsomes from uninduced r a t s were d i l u t e d t o 1:20 v/v, w h i l e those from induced r a t s were d i l u t e d t o 1:50 v/v. A few m i l l i g r a m s o f sodium d i t h i o n i t e were p l a c e d i n both the sample and r e f e r e n c e c u v e t t e s c o n t a i n i n g the d i l u t e d microsomes. The contents o f the sample c u v e t t e were then g e n t l y s a t u r a t e d w i t h carbon monoxide f o r 60 seconds a t 52 a p p r o x i m a t e l y 1 bubble per second. A f t e r a few minutes the spectrum was recorded from 325 nm t o 625 nm a t room temperature. The scanning was repeated u n t i l no f u r t h e r i n c r e a s e i n the absorbance a t 450 nm was observed. A l l d e t e r m i n a t i o n s were performed i n d u p l i c a t e u s i n g a SLM-Aminco DW-2 scanning spectrophotometer. 2.7 MICROSOMAL PROTEIN ASSAY Microsomal p r o t e i n c o n c e n t r a t i o n was determined by the method o f B r a d f o r d (1976) w i t h the Bio-Rad P r o t e i n Assay K i t R . Absorbance was measured at 595 nm u s i n g a Hewlett-Packard Model 8452A diode a r r a y spectrophotometer. The c o n c e n t r a t i o n o f the unknown sample was determined from a st a n d a r d curve o f absorbance versus the c o n c e n t r a t i o n o f bovine serum albumin. A l l d e t e r m i n a t i o n s were performed i n d u p l i c a t e . 2.8 ENZYME ASSAYS 2.8.1 Enzyme Assay C o n d i t i o n s P r e l i m i n a r y experiments were performed f o r each enzyme assay w i t h microsomes from uninduced and induced r a t s t o ensure t h a t the amount o f product formed was l i n e a r w i t h r e s p e c t t o i n c u b a t i o n time and microsomal p r o t e i n c o n c e n t r a t i o n . The c o n c e n t r a t i o n o f the s u b s t r a t e and the 53 concentration of NADPH used in each assay were shown to yield maximal product formation. 2.8.2 Aminopyrine N-Demethylase Assay The N-demethylation of aminopyrine was estimated spectrophotometrically by a standard method (Gontovnick and Bellward, 1980) with modifications. The production of formaldehyde was measured by the method of Nash (1953). The reaction mixture, in a fi n a l volume of 1.5 mL, included: 1 mL of 100 mM potassium phosphate (pH 7.4); 0.1 mL of 62.5 mM semicarbazide hydrochloride; 0.1 mL of 111.5 mM magnesium chloride; 0.1 mL of 15 mM aminopyrine dissolved in d i s t i l l e d water; and 0.1 mL of microsomes diluted in 0.25 M sucrose. The fi n a l substrate concentration was 1 mM. The fi n a l protein concentrations in the reaction mixture were 0.5 mg/mL and 0.15 mg/mL for microsomes from uninduced and induced rats, respectively. After the mixture was preincubated for 75 seconds at 37 °C in a shaking water bath, the reaction was initiated by the addition of 0.1 mL of 15 mM NADPH (dissolved in 100 mM potassium phosphate, pH 7.4). The reaction was allowed to proceed for 10 min at 37 °C before being terminated by the addition of 0.5 mL of ice-cold 20% w/v trichloroacetic acid. After mixing on a vortex, each test tube was placed on ice. Substrate and microsomes were then added to the blank tubes that were preincubated without the substrate and microsomes, 54 respectively. A l l tubes were centrifuged for 15 min at 1,000 x g. The supernatant (1.5 mL) was transferred to a new test tube followed by the addition of 0.5 mL of Nash reagent (15 g of ammonium acetate, 0.2 mL of acetylacetone in 50 mL of d i s t i l l e d water). The tubes were incubated at 60 °C for 15 min in a shaking water bath. After the samples were allowed to cool to room temperature under the fume hood, the intensity of the yellow colour produced was measured on a Hewlett-Packard Model 8452A diode array spectrophotometer. The amount of formaldehyde produced was determined from a standard curve of absorbance at 412 nm versus formaldehyde concentration. The standard samples contained inactivated microsomes (inactivated in a 60 °C water bath). A l l determinations were performed in duplicate. The mean absorbance of the blank samples was subtracted from the mean absorbance of the experimental samples. Aminopyrine N-demethylase activity is expressed as nanomoles of formaldehyde formed per min per mg of microsomal protein. 2.8.3 Pentoxyresorufin O-Dealkylase Assay The 0-dealkylation of pentoxyresorufin was determined fluorometrically by the formation of resorufin (Lubet et a l . , 1985). The reaction mixture, in a f i n a l volume of 2 mL, included: 1.93 mL of 100 mM HEPES / 5 mM magnesium chloride (pH 7.8), 10 u.L of 1 mM pentoxyresorufin dissolved 55 in dimethylsulf oxide and 50 u.L of microsomes diluted in 0.25 M sucrose. The f i n a l substrate concentration was 5 uM. The f i n a l protein concentrations in the reaction mixture were 150 u.g/mL and 50 u.g/mL for microsomes from uninduced and induced rats, respectively. The reaction was carried out in an optical glass fluorescence c e l l (1 cm path length) at 37 °C and initiated by the addition of 10 u,L of 50 mM NADPH (dissolved in 100 mM HEPES / 5 mM magnesium chloride, pH 7.8). The fluorescence reading was recorded after a 5 min reaction period. The quantity of resorufin formed was determined from a standard curve of fluorescence versus resorufin concentration. The standard samples contained inactivated microsomes. The fluorescence of the blank sample (without NADPH) was subtracted from the experimental sample. Both pentoxyresorufin and resorufin were dissolved in dimethylsulf oxide (Burke et al. 1985) and stored in the dark. The experimental procedures were performed under subdued lighting with the overhead light off. A l l determinations were performed in duplicate using a Shimadzu RF-540 spectrophotofluorometer interfaced with a Shimadzu DR-3 data recorder. The excitation wavelength was set at 530 nm ( s l i t width, 5 nm) and the emission wavelength was set at 582 nm ( s l i t width, 5 nm). Pentoxyresorufin 0-dealkylase activity is expressed as nanomoles of resorufin formed per min per mg of microsomal protein. 56 2.8.4 Erythromycin N-Demethylase Assay The N-demethylation of erythromycin was estimated spectrophotometrically (Arlotto et al., 1987). The production of formaldehyde was measured by the method of Nash (1953). The reaction mixture, in a fi n a l volume of 1.5 mL, included: 1 mL of 100 mM potassium phosphate (pH 7.4); 0.1 mL of 75 mM semicarbazide hydrochloride; 0.1 mL of 45 mM magnesium chloride; 0.1 mL of 6 mM erythromycin base dissolved in 30% v/v ethanol; and 0.1 mL of microsomes diluted in 0.25 M sucrose. The fin a l substrate concentration was 0.4 mM. The fi n a l protein concentrations in the reaction mixture were 0.6 mg/mL and 0.15 mg/mL for microsomes from uninduced and induced rats, respectively. The other steps were the same as those described for the aminopyrine N-demethylase assay. Erythromycin N-demethylase activity i s expressed as nanomoles of formaldehyde formed per min per mg of microsomal protein. 2.8.5 E t h o x y r e s o r u f i n O-Deethylase Assay The 0-deethylation of ethoxyresorufin was determined fluorometrically by resorufin formation (Burke and Mayer, 1974). This assay was performed using the same experimental procedures and conditions as the pentoxyresorufin 0-dealkylase assay except that ethoxyresorufin was the substrate. Ethoxyresorufin 0-deethylase activity is 57 expressed as nanomoles of resorufin formed per min per mg of microsomal protein. 2.8.6 Testosterone Oxidase Assay The microsomal hydroxylation and oxidation of testosterone was determined by the method of Wood et a l . (1983). The reaction mixture, i n a f i n a l volume of 1 mL, included: 0.5 mL of 100 mM potassium phosphate (pH 7.4); 0.1 mL of 30 mM magnesium chloride; 0.2 mL of 0.25 M sucrose; 20 uL of 12.5 mM testosterone dissolved i n methanol; and 80 uX. of microsomes d i l u t e d i n 0.25 M sucrose. The f i n a l substrate concentration was 0.25 mM. The f i n a l protein concentrations i n the reaction mixture were 0.5 mg/mL and 0.15 mg/mL for samples from uninduced and induced r a t s , r e s p e c t i v e l y . A f t e r the mixture was preincubated for 75 seconds at 37 °C i n a shaking water bath, the reaction was i n i t i a t e d by adding 0.1 mL of 10 mM NADPH dissolved i n 50 mM potassium phosphate (pH 7.4) to each incubation tube. The reaction was allowed to proceed f o r 5 min at 37 °C before i t was terminated by the addition of 6 mL of methylene c h l o r i d e . Subsequently, 0.1 mL of i n t e r n a l standard (16-keto-testosterone or 11 p*-hydroxy testosterone, 3 nmol per tube) was added and the incubation contents were mixed on a vortex f o r 30 seconds. The samples were centrifuged for 2 min at 800 x g. The aqueous phase (upper layer) was aspirated and 4 mL of the organic phase was transferred to a 58 test tube and evaporated under a stream of nitrogen at 35 °C. The residue was dissolved in 0.2 mL of methanol. The sample was f i l t e r e d through a 0.45 um Type HV f i l t e r (Millipore Ltd., Mississauga, Ont.). A volume of 10 u.L was used for high performance liquid chromatographic analysis. Formation of microsomal testosterone oxidation products was quantitated by a high performance liquid chromatographic method based on that described by Wood et a l . (1983). The system consisted of two Waters Model 501 pumps, an automatic sample injector (Waters Model 712 WISPR) and a Waters Model 484 ultraviolet-visible absorbance detector. A software program, BASELINE 810 Chromatography Workstation11, was used to control the operation of these devices as well as for data analysis. A l l samples were analyzed using a reverse phase column preceded by a Pelliguard R LC-18, 2 cm guard column (Supelco, Inc., Beliefonte, PA). In samples from uninduced and dexamethasone-treated rats, the separation of 2a-, 2 0 - , 16a-and 160-hydroxytestosterone as well as androstenedione was performed using a 5 urn octyldecylsilane, 4.6 x 150 mm inner diameter, reverse phase column (Supelco, Inc., Bellefonte, PA). A concave gradient (Option No.7 in the BASELINE 810 software program) from 100% Solvent A to 100% Solvent B was used over a 25 min period at a flow rate of 1.5 mL/min. Solvent A contained 465 mL of methanol, 530 mL of d i s t i l l e d water and 11 mL of acetonitrile. Solvent B contained 760 mL 59 of methanol, 220 mL of d i s t i l l e d water and 11 mL of acetonitrile. The same conditions were used for the separation of these metabolites in samples from phenobarbital-induced rats except that the composition of Solvent A was 430 mL of methanol, 600 mL of d i s t i l l e d water and 11 mL of acetonitrile and the gradient time was 33 min. Methanol and acetonitrile were HPLC grade. Water was d i s t i l l e d and further purified with a Millipore Milli-Q apparatus (Millipore Ltd., Mississauga, Ont.). For the separation of 6(3- and 7a-hydroxytestosterone in microsomes from a l l groups, different chromatographic conditions were used to improve the resolution of these two metabolites. The main column was a ZorbaxR 5 um octyldecylsilane, 4.6 x 150 mm diameter, reverse phase column (Dupont Canada Inc., Mississauga, Ont.). The mobile phase was 14% tetrahydrofuran and 86% d i s t i l l e d water for the f i r s t 18 min followed by a mobile phase of 69% tetrahydrofuran and 31% d i s t i l l e d water for an additional 5 min. The flow rate was 1.5 mL/min. An unidentified enzymatic product co-eluted with 16-keto-hydroxytestosterone in samples from phenobarbital-induced rats. Consequently, the internal standard used in these samples was lip-hydroxytestosterone. In a l l other cases, 16-keto-testosterone was used as the internal standard. 60 A l l chromatographic separations were performed at room temperature. The absorbance of the column ef f l u e n t s was monitored at 254 nm. Metabolites were i d e n t i f i e d by comparing retention times to those of authentic standards. The amount of each metabolite formed was determined by l i n e a r regression analysis of a standard curve of peak height r a t i o ( a n a l y t e / i n t e r n a l standard) versus concentration r a t i o (analyte/internal standard). Peak height was calculated by the BASELINE 810 software program. Inactivated microsomes were used i n the standard samples. A l l determinations were performed i n duplicate. A c t i v i t y i s expressed as nanomoles of testosterone metabolite formed per min per mg of microsomal protein. 2.9 IMMUNOINHIBITION STUDIES The e f f e c t of a preparation of monospecific polyclonal anti-cytochrome P450IIC11 antibody on aminopyrine N-demethylase, pentoxyresorufin 0-dealkylase, erythromycin N-demethylase and testosterone oxidase a c t i v i t i e s was determined by a standard method (Thomas et a l . , 1981). Hepatic microsomes (0.3 nmol of t o t a l cytochrome P-450) from uninduced, phenobarbital- or dexamethasone-induced rats were preincubated with anti-cytochrome P450IIC11 antibody or con t r o l rabbit IgG i n phosphate-buffered s a l i n e (pH 7.4) f o r 10 min at room temperature. (Phosphate-buffered s a l i n e contained 137 mM NaCl, 3 mM KCl, 8 mM Na2HP04, 1 mM KH2P04 61 and 0.2 mM EDTA). Each te s t tube contained 0.25, 0.5, 1, 2.5, 5 or 10 mg of anti-cytochrome P450IIC11 antibody or co n t r o l rabbit IgG per nmol of t o t a l cytochrome P-450. Substrate oxidation was i n i t i a t e d as described above (see Sections 2.8.2, 2.8.3, 2.84 and 2.86). Microsomes prepared from pooled l i v e r s were used. A l l determinations were performed i n duplicate. Results are expressed as a percent of c o n t r o l a c t i v i t y . 2.10 STATISTICAL ANALYSES In experiments where microsomal samples i s o l a t e d from i n d i v i d u a l l i v e r s were used, the data were subjected to formal s t a t i s t i c a l analysis using the UBC SPSS-X computer program ( L a i , 1986). The s i g n i f i c a n c e of the difference between the means of two treatment groups was evaluated by the two-tailed independent Student's t - t e s t . The a priori l e v e l of s i g n i f i c a n c e was set at p < 0.05. S t a t i s t i c a l analyses were not performed on the data obtained from experiments where the microsomal samples were prepared from pooled l i v e r s . In these cases, the sample s i z e was unity. 62 RESULTS 3.1 STUDIES WITH IN VIVO CIMETIDINE To determine whether hepatic cytochrome P-450 enzymes are i n h i b i t e d s e l e c t i v e l y by in vivo cimetidine treatment i n adult male r a t s , the animals were pretreated as described i n "Materials and Methods" (Section 2.3.1). The treatment phase f o r studying i n h i b i t i o n consisted of a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n of cimetidine (150 mg/kg) or s a l i n e (uninhibited control) as described i n "Materials and Methods" (Section 2.3.2). In cases where the purpose was to examine the e f f e c t of cimetidine on a p a r t i c u l a r cytochrome P-450 i n microsomes, an enzyme-specific a c t i v i t y was determined. I t i s important to note that i n the d i f f e r e n t enzyme assays, aliquots of the same microsomal suspension were used. 3.1.1 Total Cytochrome P-450 Content Phenobarbital, dexamethasone and fi-naphthof lavone increased the t o t a l microsomal cytochrome P-450 content by 1.9-, 1.5- and 1.5-fold, respectively (Table 6). Jn vivo cimetidine treatment did not change the t o t a l cytochrome P-450 content i n microsomes from uninduced, phenobarbital-, dexamethasone- or {J-naphthoflavdne-induced rats (Figure 3). 63 TABLE 6 EFFECT OF PHENOBARBITAL, DEXAMETHASONE AND (3-NAPHTHOFLAVONE ON TOTAL MICROSOMAL CYTOCHROME P-450 CONTENT Pretreatment N Total Cytochrome P-450 (nmol/mg protein) Saline 4 1.54 + 0.05 Phenobarbital 8 2.98 + 0.27* 2% Tween 80 4 1.51 + 0.08 Dexamethasone 8 2.29 + 0.07* Corn O i l 4 1.14 + 0.07 P-naphthoflavone 8 1.73 + 0.09* Results are expressed as the mean ± SEM f o r the number (N) of rats indicated. *p < 0.05, compared to the corresponding control group. Effect of in vivo cimetidine on total microsomal cytochrome P-450 content. Results are expressed as the mean ± SEM for 8 rats per group. SALINE 1 I ^ CIMETIDINE UNINDUCED PB INDUCED DEX INDUCED BNF INDUCED 65 3.1.2 Aminopyrine N-Demethylase Activity Aminopyrine i s a substrate which is known to be demethylated by many non-inducible and inducible cytochrome P-450 enzymes (Guengerich et al., 1982a). Since the inducing agents used in this study were known to induce different cytochrome P-450 enzymes, aminopyrine N-demethylase was used i n i t i a l l y as a non-selective marker to probe for d i f f e r e n t i a l inhibition by cimetidine. Phenobarbital and dexamethasone pretreatment increased aminopyrine N-demethylase activity by 2.6- and 2.2-fold, respectively, whereas 6-naphthoflavone pretreatment decreased this activity by 0.46-fold (Table 7). In vivo cimetidine inhibited aminopyrine N-demethylase activity by 62% in microsomes from uninduced rats, whereas i t inhibited this activity by only 33%, 20% and 28% in microsomes from rats induced with phenobarbital, dexamethasone and P-naphthoflavone, respectively (Figure 4). The apparent increased inhibition of aminopyrine N-demethylase activity by in vivo cimetidine in the uninduced rats suggested that at least one of the cytochrome P-450 enzymes present in these animals is more susceptible to inhibition by cimetidine than are the inducible cytochrome P-450 enzymes. Based on this observation, subsequent experiments were designed to study the effects of cimetidine on substrates known to be specifically metabolized by particular 66 TABLE 7 EFFECT OF PHENOBARBITAL, DEXAMETHASONE AND 0-NAPHTHOFLAVONE ON AMINOPYRINE N-DEMETHYLASE ACTIVITY Pretreatment N Activity (nmol/min/mg protein) Saline Phenobarbital 4 5.84 ± 0.61 8 15.20 ± 0.84* 2% Tween 80 4 6.34 ± 0.68 Dexamethasone 8 13.96 ± 0.70* Corn O i l 4 6.36+0.74 P-naphthoflavone 8 2.90 ± 0.26* Results are expressed as the mean ± SEM for the number (N) of rats indicated. *p < 0.001, compared to the corresponding control group. 67 F i g u r e 4 E f f e c t o f in vivo c i m e t i d i n e on a m i n o p y r i n e N-demethylase a c t i v i t y . R e s u l t s a r e e x p r e s s e d as the mean ± SEM f o r 8 r a t s p e r g r o u p . *p < 0 .02 , # p < 0 .001 , compared t o the c o r r e s p o n d i n g s a l i n e -t r e a t e d g r o u p . UNINDUCED PB DEX BNF INDUCED INDUCED INDUCED 68 cytochrome P-450 enzymes under defined pretreatment conditions. 3.1.3 Pentoxyresorufin O-Dealkylase Activity Jn vivo cimetidine inhibited pentoxyresorufin 0-dealkylase activity by 38% in microsomes from uninduced rats (Figure 5A). Phenobarbital and dexamethasone pretreatment increased pentoxyresorufin 0-dealkylase activity by 108- and 13-fold, respectively (Table 8). Jn vivo cimetidine had no effect on this activity in microsomes from phenobarbital- or dexamethasone-induced rats (Figures 5A and 5B). Cytochromes P450IIB1/2 account for more than 90% of the pentoxyresorufin 0-dealkylase activity i n hepatic microsomes from phenobarbital-induced rats (Lubet et al., 1985; Waxman et al., 1987; Dutton and Parkinson, 1989). Hepatic microsomes from uninduced rats contain low levels of cytochromes P450IIB1/2 (Guengerich et al., 1982a; Thomas et al., 1983; Waxman et al., 1985). The major cytochrome P-450 enzymes responsible for pentoxyresorufin 0-dealkylase activity in microsomes from uninduced rats have not been identified, but i t has been reported that cytochrome P450IIB1/2 do not contribute to this activity in these microsomes (Waxman et al., 1987). Although dexamethasone pretreatment induces cytochrome P450IIB1/2 (Yamazoe et al., 1987), i t has not yet been determined which cytochrome P-450 69 F i g u r e 5 E f f e c t o f in vivo c i m e t i d i n e on p e n t o x y r e s o r u f i n O-dealkylase a c t i v i t y . Microsomes were i s o l a t e d from (A) uninduced, (B) p h e n o b a r b i t a l - i n d u c e d and (C) dexamethasone-induced r a t s . R e s u l t s are expressed as the mean ± SEM f o r 8 r a t s per group. *p < 0.02, compared t o the co r r e s p o n d i n g s a l i n e -t r e a t e d group. O) 0.03 | c "o Q. O) E 0.02 \ c E \ o J 0.01 >-> o < 0.00 • SALINE ESS CIMETIDINE UNINDUCED DEX INDUCED TABLE 8 EFFECT OF PHENOBARBITAL AND DEXAMETHASONE ON PENTOXYRESORUFIN O-DEALKYLASE ACTIVITY Pretreatment N Activity (nmol/min/mg protein) Saline 4 0.05 ± 0.01 Phenobarbital 8 5.42 ± 0.30* 2% Tween 80 4 0.03 ± 0.01 Dexamethasone 8 0.40 ± 0.03* Results are expressed as the mean ± SEM for the number (N) of rats indicated. *p < 0.001, compared to the corresponding control group. 71 enzymes contribute to pentoxyresorufin 0-dealkylase acti v i t y following dexamethasone pretreatment. 3.1.4 Erythromycin N-Demethylase A c t i v i t y Jn vivo cimetidine inhibited erythromycin N-demethylase activity by 40% in microsomes from uninduced rats (Figure 6A). Phenobarbital and dexamethasone pretreatment increased this activity by 2.8- and 9.8-fold, respectively (Table 9). Jn vivo cimetidine did not affect erythromycin N-demethylase activity in microsomes from either phenobarbital- or dexamethasone-induced rats (Figures 6B and 6C). Cytochromes P4 50IIIA1/2 account for a majority of the erythromycin N-demethylase activity i n hepatic microsomes from dexamethasone-induced rats (Wrighton et al., 1985a). Both cytochrome P450IIIA1 mRNA and cytochrome P450IIIA2 mRNA are present in livers of adult male rats treated with phenobarbital (Gonzalez et al., 1986), whereas cytochrome P450IIIA2, but not cytochrome P450IIIA1, i s expressed in livers of uninduced adult male rats (Cooper et al., 1990). It has not yet been reported which cytochrome P-450 enzymes contribute to erythromycin N-demethylase activity in these microsomes from uninduced or phenobarbital-induced rats. 72 Figure 6 Effect of in vivo cimetidine on erythromycin N-demethylase activity. Microsomes were isolated from (A) uninduced, (B) phenobarbital-induced and (C) dexamethasone-induced rats. Results are expressed as the mean ± SEM for 8 rats per group. *p < 0.005, compared to the corresponding saline-treated group. > o < (A) 1.5 1.0 0.5 0.0 • SALINE ESS CIMETIDINE UNINDUCED (B) C T ! SALINE ESS CIMETIDINE PB INDUCED (C) C D SALINE CIMETIDINE DEX INDUCED 73 TABLE 9 EFFECT OF PHENOBARBITAL AND DEXAMETHASONE ON ERYTHROMYCIN N-DEMETHYLASE ACTIVITY Pretreatment N Activity (nmol/min/mg protein) Saline 4 1.15 ± 0.12 Phenobarbital 8 3.25 ± 0.31* 2% Tween 80 4 1.06 ± 0.12 Dexamethasone 8 10.44 ± 0.27" Results are expressed as the mean ± SEM for the number (N) of rats indicated. *p < 0.001, compared to the corresponding control group. 74 3.1.5 Ethoxyresorufin O-Deethylase Activity In vivo cimetidine inhibited ethoxyresorufin O-deethylase activity by 84% in microsomes from uninduced rats (Figure 7A). p-Naphthoflavone pretreatment increased this activity by 23-fold (Table 10). In vivo cimetidine did not affect ethoxyresorufin O-deethylase activity in microsomes from p-naphthoflavone-induced rats (Figure 7B). It has been reported that, in hepatic microsomes from uninduced rats, cytochrome P450IA2 accounts for approximately 80% of the ethoxyresorufin O-deethylase activity and that cytochrome P450IA1 accounts for the remainder of the activity (Kelley et al., 1987). Recently, Nakajima et al. (1990) showed that cytochrome P450IIC11 also contributed to this enzyme activity. Therefore, ethoxyresorufin O-deethylase activity in hepatic microsomes from uninduced rats may not be a specific marker for cytochrome P450IA2. Although fi-naphthoflavone pretreatment induces both cytochrome P450IA1 and cytochrome P450IA2 (Guengerich et al., 1982a; Waxman et al., 1985), i t has yet to be reported whether these enzymes contribute to ethoxyresorufin O-deethylase activity in microsome from fJ-naphthoflavone-induced rats. 75 F i g u r e 7 E f f e c t o f in vivo c i m e t i d i n e on e t h o x y r e s o r u f i n O-deethylase a c t i v i t y . Microsomes were i s o l a t e d from (A) uninduced and (B) p-naphthoflavone-induced r a t s . R e s u l t s are expressed as the mean ± SEM f o r 8 r a t s per group. *p < 0.02, compared t o the c o r r e s p o n d i n g s a l i n e - t r e a t e d group. c 'OJ o C L cn E \ c E \ o E c O < 0.5 0.4 0.3 0.2 t 0.1 0.0 (A) • SALINE K53 CIMETIDINE UNINDUCED (B) c '«> "o a. u> E \ c "E \ o E c >-> I— o < 15 10 CZ1 SALINE ESS CIMETIDINE BNF INDUCED TABLE 10 EFFECT OF p* -NAPHTHOFLAVONE ON ETHOXYRESORUFIN O-DEETHYLASE ACTIVITY Pretreatment N Activity (nmol/min/mg protein) Saline 4 0.52 ± 0.06 P-Naphthoflavone 8 11.95 ± 0.65* Results are expressed as the mean ± SEM for the number (N) of rats indicated. *p < 0.001, compared to the corresponding control group. 77 3.1.6 T e s t o s t e r o n e Oxidase A c t i v i t i e s The results from the preceding sections indicated that several cytochrome P-450 enzyme activities (aminopyrine N-demethylase, pentoxyresorufin 0-dealkylase, erythromycin N-demethylase and ethoxyresorufin O-deethylase) in microsomes from uninduced adult male rats were inhibited by in vivo cimetidine (Figures 8 and 9). In addition, some of these enzyme activ i t i e s known to be specific for particular cytochrome P-450 enzymes in induced rats were not affected by cimetidine, indicating that these enzymes may not be subject to inhibition by this drug. To further explore this apparent di f f e r e n t i a l inhibition of cytochrome P-450 by cimetidine, microsomal testosterone oxidation was determined under defined pretreatment conditions. This was done because of the known spec i f i c i t y of several of the testosterone hydroxylase ac t i v i t i e s for particular cytochrome P-450 enzymes. For example, testosterone 2a- and 16a-hydroxylase ac t i v i t i e s in hepatic microsomes from uninduced adult male rats are specific for cytochrome P450IIC11 (Waxman, 1984; Waxman et a l . , 1987). Since cytochrome P450IIC11 was known to be a major enzyme in livers of uninduced adult male rats (Guengerich et a l . , 1982a; Dannan et a l . , 1983; Waxman et a l . , 1985)., this enzyme was a candidate for inhibition by cimetidine in these animals. 78 Figure 8 Summary of the e f f e c t s of in vivo cimetidine on aminopyrine N-demethylase, pentoxyresorufin O-dealkylase and erythromycin N-demethylase a c t i v i t i e s i n microsomes from uninduced, phenobarbital-induced and dexamethasone-induced ra t s . Results are based on the data from Figures 4-6. #p < 0.001, sp < 0.005, *p < 0.02 compared to the saline-treated control group. 60 # o CQ Ld O CC LU Q_ 40 20 0 # ! APND 1 UNINDUCED PB-INDUCED DEX—INDUCED f 1 PROD EMND 79 Figure 9 Summary of the e f f e c t s of in vivo cimetidine on aminopyrine N-demethylase and ethoxyresorufin O-deethylase a c t i v i t i e s i n microsomes from uninduced and ft-naphthoflavone-induced r a t s . Results are based on the data from Figure 4 and Figure 7 . #p < 0 . 0 0 1 , *p < 0 . 0 2 compared to the saline-treated control group. 100 80 UNINDUCED ESS BNF-INDUCED m rr. S 60 o UJ Q L 40 20 0 APND EROD 80 3.1.6.1 T e s t o s t e r o n e 2cc-Hydroxylase A c t i v i t y In vivo cimetidine inhibited testosterone 2a-hydroxylase activity by 65% in microsomes from uninduced rats (Figure 10). It has been shown that cytochrome P450IIC11 accounts for more than 85% of this activity in hepatic microsomes from uninduced adult male rats (Waxman, 1984; Waxman et al., 1987). Phenobarbital pretreatment decreased testosterone 2a-hydroxylase activity by 51% (Table 11). In vivo cimetidine inhibited the remaining activity by 73% in microsomes from phenobarbital-induced rats (Figure 10). It is not known whether cytochrome P450IIC11 is the major enzyme responsible for this activity in microsomes from phenobarbital-induced adult male rats. Dexamethasone pretreatment decreased testosterone 2a-hydroxylase activity by 58% (Table 11). In vivo cimetidine inhibited 4 6% of the remaining activity in microsomes from dexamethasone-induced rats (Figure 10). It is not known whether cytochrome P450IIC11 is the major enzyme responsible for this activity in microsomes from dexamethasone-induced adult male rats. 81 F i g u r e 10 E f f e c t o f i n vivo c i m e t i d i n e on t e s t o s t e r o n e 2a-hydroxyiase a c t i v i t y . Microsomes were i s o l a t e d from uninduced, p h e n o b a r b i t a l - i n d u c e d and dexamethasone-induced r a t s . R e s u l t s are expressed as the mean ± SEM. The sample s i z e (N) was 8 r a t s per group, except f o r the uninduced, c i m e t i d i n e -t r e a t e d group (N = 7) and the dexamethasone-induced, c i m e t i d i n e - t r e a t e d group (N = 5 ) . *p < 0.001, compared t o the c o r r e s p o n d i n g s a l i n e -t r e a t e d c o n t r o l group. c ~o >_ Q. CO E \ C ' £ \ o E >~ > I— o < 2.5 2.0 1.5 1.0 0.5 0.0 SALINE ESS CIMETIDINE 1 UNINDUCED PB DEX INDUCED INDUCED 82 TABLE 11 EFFECT OF PHENOBARBITAL AND DEXAMETHASONE ON TESTOSTERONE 2a-HYDROXYLASE ACTIVITY Pretreatment N A c t i v i t y (nmol/min/mg p r o t e i n ) S a l i n e 4 2.38 ± 0.32 P h e n o b a r b i t a l 8 1.17 ± 0.13* 2% Tween 80 4 1.93 ± 0.34 Dexamethasone 8 0.81 ± 0.11* R e s u l t s are expressed as the mean ± SEM f o r the number (N) o f r a t s i n d i c a t e d . *p < 0.001, compared t o the c o r r e s p o n d i n g c o n t r o l group. 83 3.1.6.2 Testosterone 20- and 60-Hydroxylase Activities Phenobarbital pretreatment increased testosterone 20-and 60-hydroxylase acti v i t i e s by 6.1- and 3.7-fold, respectively (Tables 12 and 13). Dexamethasone pretreatment also increased these two a c t i v i t i e s , and the magnitude of the increase was 12-fold for testosterone 20-hydroxylase activity and 5.5-fold for testosterone 60-hydroxylase activity (Tables 12 and 13). Jn vivo cimetidine did not inhibit either testosterone 20- or 60-hydroxylase activity in microsomes from uninduced, phenobarbital- or dexamethasone-induced rats (Figures 11 and 12). It has been reported that cytochromes P450IIIA1/2 account for more that 85% of the testosterone 20- or 60-hydroxylase activity in hepatic microsomes from uninduced, phenobarbital- or dexamethasone-induced adult male rats (Halvorson et a l . , 1990). The results from the cimetidine experiments on testosterone 20- and 60-hydroxylase a c t i v i t i e s are consistent with the lack of inhibition of erythromycin N-demethylase activity in microsomes from dexamethasone-induced rats (Figure 6) since, in the latter case, cytochromes P450IIIA1/2 are also the major contributors (Wrighton et al., 1985a). 84 TABLE 12 EFFECT OF PHENOBARBITAL AND DEXAMETHASONE ON TESTOSTERONE 20-HYDROXYLASE ACTIVITY Pretreatment N A c t i v i t y (nmol/min/mg p r o t e i n ) S a l i n e 4 0.07 ± 0.01 P h e n o b a r b i t a l 8 0.43 ± 0.03* 2% Tween 80 4 0.07+0.02 Dexamethasone 8 0.84 ± 0.05* R e s u l t s are expressed as the mean ± SEM f o r the number (N) o f r a t s i n d i c a t e d . *p < 0.001, compared t o the c o r r e s p o n d i n g c o n t r o l group. 85 TABLE 13 EFFECT OF PHENOBARBITAL AND DEXAMETHASONE ON TESTOSTERONE 6p*-HYDROXYLASE ACTIVITY Pretreatment N Activity (nmol/min/mg protein) Saline 4 2.36 ± 0.19 Phenobarbital 8 8.77 ± 0.88* 2% Tween 80 4 - 2.46 ± 0.37 Dexamethasone 8 13.53 ± 0.81# Results are expressed as the mean ± SEM for the number (N) of rats indicated. *p < 0.002, compared to the corresponding control group. #p < 0.001, compared to the corresponding control group. 86 Figure 11 Effect of in vivo cimetidine on testosterone 2p-hydroxylase activity. Microsomes were isolated from uninduced, phenobarbital-induced and dexamethasone-induced rats. Results are expressed as the mean ± SEM. The sample size (N) was 8 rats per group, except for the uninduced, cimetidine-treated group (N = 7) and the dexamethasone-induced, cimetidine-treated group (N = 5 ) . c 'co ~*— o l_ C L a> E \ c £ \ o E c >-> < 1.0 0.8 0.6 0.4 5= 0.2 0.0 SALINE ESS CIMETIDINE UNINDUCED I PB INDUCED 1 DEX INDUCED 87 Figure 12 E f f e c t of in vivo cimetidine on testosterone 6f3-hydroxylase a c t i v i t y . Microsomes were i s o l a t e d from uninduced, phenobarbital-induced and dexamethasone-induced r a t s . Results are expressed as the mean ± SEM. The sample si z e (N) was 8 rats per group, except f o r the uninduced, cimetidine-treated group (N = 7) and the dexamethasone-induced, cimetidine-treated group (N = 5 ) . 88 3.1.6.3 T e s t o s t e r o n e 7a-Hydroxylase A c t i v i t y In vivo cimetidine did not inhibit testosterone 7a-hydroxylase activity in microsomes from uninduced rats (Figure 13). Neither phenobarbital nor dexamethasone had any effect on testosterone 7cc-hydroxylase activity (Table 14). In vivo cimetidine did not inhibit this a c t i v i t y in microsomes from phenobarbital- or dexamethasone-induced rats (Figure 13). It has been shown that cytochrome P450IIA1 accounts for 80-96% of the testosterone 7a-hydroxylase activity in hepatic microsomes from uninduced, phenobarbital- or dexamethasone-induced rats (Levin et a l . , 1987; Waxman et a l . , 1988b; Arlotto and Parkinson, 1989). 89 F i g u r e 13 E f f e c t o f in vivo c i m e t i d i n e on t e s t o s t e r o n e 7 a-hydroxylase a c t i v i t y . Microsomes were i s o l a t e d from uninduced, p h e n o b a r b i t a l - i n d u c e d and dexamethasone-induced r a t s . R e s u l t s are expressed as the mean ± SEM. The sample s i z e (N) was 8 r a t s per group, except f o r the uninduced, c i m e t i d i n e -t r e a t e d group (N = 7) and the dexamethasone-induced, c i m e t i d i n e - t r e a t e d group (N = 5 ) . c CD *5 C L D ) E \ c "E \ o E c >-> r — o < 1.0 0.5 0.0 SALINE ESS CIMETIDINE UNINDUCED 1 i PB INDUCED 1 DEX INDUCED 90 TABLE 14 EFFECT OF PHENOBARBITAL AND DEXAMETHASONE ON TESTOSTERONE 7a-HYDROXYLASE ACTIVITY Pretreatment N A c t i v i t y (nmol/min/mg p r o t e i n ) S a l i n e 4 0.45 + 0.08 P h e n o b a r b i t a l 8 0 . 8 3 ± 0 . 1 8 2% Tween 80 4 0.35 ± 0.08 Dexamethasone 8 0 . 3 1 1 0 . 0 4 R e s u l t s are expressed as the mean ± SEM f o r the number (N) o f r a t s i n d i c a t e d . 91 3.1.6.4 T e s t o s t e r o n e 16a-Hydroxylase A c t i v i t y In vivo cimetidine inhibited testosterone 16a-hydroxylase activity by 60% in microsomes from uninduced rats (Figure 14). This activity was increased 2.1-fold by phenobarbital, but was decreased 4 0% by dexamethasone (Table 15). In vivo cimetidine did not affect this activity in microsomes from either phenobarbital- or dexamethasone-induced rats (Figure 14). Cytochrome P450IIC11 accounts for more than 85% of the testosterone 16a-hydroxylase activity i n hepatic microsomes from uninduced adult male rats (Waxman, 1984; Waxman et a l . , 1987). This enzyme i s suppressed in livers of rats pretreated with phenobarbital or dexamethasone. It has been shown that cytochrome P450IIC11 i s not the major enzyme responsible for testosterone 16a-hydroxylase activity in hepatic microsomes from phenobarbital-induced adult male rats (Waxman, 1984; Waxman et a l . , 1987). In this case, the major contributors are cytochromes P450IIB1/2 (Thomas et a l . , 1981; Reik et a l . , 1985; Waxman et a l . , 1987). However, i t has not yet been determined which cytochrome P-450 enzymes contribute to this activity in microsomes from dexamethasone-induced male rats. 92 Figure 14 Effect of in vivo cimetidine on testosterone 16cc-hydroxylase activity. Microsomes were isolated from uninduced, phenobarbital-induced and dexamethasone-induced rats. Results are expressed as the mean ± SEM. The sample size (N) was 8 rats per group, except for the uninduced, cimetidine-treated group (N =7) and the dexamethasone-induced, cimetidine-treated group (N = 5). *p < 0.001, compared to the saline-treated group. c 'a> ~o i _ Q. cn E \ c £ \ o £ c >-> I— O < 0 SALINE CSS CIMETIDINE UNINDUCED 1. PB INDUCED 1 DEX INDUCED 93 TABLE 15 EFFECT OF PHENOBARBITAL AND DEXAMETHASONE ON TESTOSTERONE 16<x-HYDROXYLASE ACTIVITY Pretreatment N Activity (nmol/min/mg protein) Saline 4 3.72 ± 0.53 Phenobarbital 8 7.78 ± 0.79# 2% Tween 80 4 3.04 ± 0.50 Dexamethasone 8 1.81+0.21* Results are expressed as the (N) of rats indicated. *p corresponding control group, corresponding control group. mean ± SEM for the number < 0.025, compared to the #p < 0.01, compared to the 94 3.1.6.5 Testosterone 160-Hydroxylase Activity Only trace levels (less than 0.1 nmol/min/mg protein) of the 160-hydroxytestosterone metabolite were formed by microsomes from uninduced rats (Table 16) and accurate quantitation of this metabolite was not possible. As a result, the effect of in vivo cimetidine on this activity in microsomes from uninduced rats was not determined. Phenobarbital pretreatment increased testosterone 160-hydroxylase activity by at least 48-fold (Table 16). In vivo cimetidine did not inhibit this activity in microsomes from phenobarbital-induced rats (Figure 15). It has been shown that cytochromes P450IIB1/2 account for more than 90% of the testosterone 160-hydroxylase activity in hepatic microsomes from phenobarbital-induced rats (Reik et al., 1985; Waxman et al., 1987). A possible explanation for the lack of effect of cimetidine on testosterone 160-hydroxylase activity could be that the inhibition by cimetidine was competitive and the substrate concentration used in the assay was too high for inhibition to be detected. Therefore, the effect of in vivo cimetidine on testosterone 160-hydroxylase activity was determined at lower substrate concentrations. However, even at l/25th of the usual substrate concentration, in vivo cimetidine did not inhibit testosterone 160-hydroxylase 95 TABLE 16 EFFECT OF PHENOBARBITAL AND DEXAMETHASONE ON TESTOSTERONE 160-HYDROXYLASE ACTIVITY Pretreatment N A c t i v i t y (nmol/min/mg p r o t e i n ) S a l i n e 4 < 0.1 P h e n o b a r b i t a l 8 4 . 8 2 ± 0 . 4 5 2% Tween 80 4 < 0.1 Dexamethasone 8 0.53 ± 0.04 R e s u l t s are expressed as the mean ± SEM f o r the number (N) o f r a t s i n d i c a t e d . S t a t i s t i c a l a n a l y s i s was not performed on these d a t a . 96 Figure 15 Effect of in vivo cimetidine on testosterone 16|3-hydroxylase activity. Microsomes were isolate d from phenobarbital-induced and dexamethasone-induced rats. Results are expressed as the mean ± SEM. The sample size (N) was 8 rats per group, except for the uninduced, cimetidine-treated group (N = 7) and the dexamethasone-induced, cimetidine-treated group (N = 5). PB INDUCED DEX INDUCED 97 activity in microsomes from phenobarbital-induced rats (Figure 16). Dexamethasone pretreatment increased testosterone 16|5-hydroxylase activity by at least 5-fold (Table 16). In vivo cimetidine also did not inhibit this activity in microsomes from dexamethasone-induced rats (Figure 15). It is not known which cytochrome P-450 enzymes contribute to testosterone 16(3-hydroxylase activity in microsomes from dexamethasone-induced rats. 3.1.6.6 Andrbstenedione Formation In vivo cimetidine inhibited androstenedione formation by 31% in microsomes from uninduced rats (Figure 17). The activity was increased 2.8-fold by phenobarbital pretreatment, but was decreased 40% following dexamethasone pretreatment (Table 17). Jn vivo cimetidine did not affect androstenedione formation in microsomes from phenobarbital-or dexamethasone-induced rats (Figure 17). Cytochromes P450IIB1/2 account for 60-70% of the formation of androstenedione in hepatic microsomes from phenobarbital-treated adult male rats (Reik et al., 1985). It i s not known which enzymes are responsible for the remainder of the activity in microsomes from this group of rats. As well, i t has yet to be shown which cytochrome P-450 enzymes are involved in androstenedione formation in 98 F i g u r e 16 Lineweaver-Burk p l o t f o r the e f f e c t o f i n vivo c i m e t i d i n e on t e s t o s t e r o n e 16p~hydroxylase a c t i v i t y . P h e n o b a r b i t a l - i n d u c e d r a t s were s a c r i f i c e d 90 min a f t e r a s i n g l e i n j e c t i o n o f c i m e t i d i n e HCl (150 mg/kg) o r s a l i n e ( c o n t r o l ) . Microsomes were prepared from a p o o l o f f o u r l i v e r s i n each group. The enzyme a c t i v i t y (V) was d e t e r m i n e d a t v a r i o u s s u b s t r a t e [S] c o n c e n t r a t i o n s . The symbols i n d i c a t e v a l u e s o f the transformed d a t a and the l i n e s were generated by l i n e a r r e g r e s s i o n a n a l y s i s . 99 F i g u r e 17 E f f e c t o f in vivo c i m e t i d i n e on androstenedione f o r m a t i o n . Microsomes were i s o l a t e d from u n i n d u c e d , p h e n o b a r b i t a l - i n d u c e d a n d dexamethasone-induced r a t s . R e s u l t s are expressed as the mean ± SEM. The sample s i z e (N) was 8 r a t s per group, except f o r the uninduced, c i m e t i d i n e -t r e a t e d group (N = 7) and the dexamethasone-induced, c i m e t i d i n e - t r e a t e d group (N = 5 ) . *p < 0.001, compared t o the c o r r e s p o n d i n g s a l i n e -t r e a t e d group. 8 c O Q_ £ \ c £ o E c >-> o < 0 CZD SALINE ES3 CIMETIDINE UNINDUCED PB DEX INDUCED INDUCED 100 TABLE 17 EFFECT OF PHENOBARBITAL AND DEXAMETHASONE ON ANDROSTENEDIONE FORMATION Pretreatment N Activity (nmol/min/mg protein) Saline 4 2.45 ± 0.10 Phenobarbital 8 6.97 ± 0.39* 2% Tween 80 4 1.88 ± 0.29 Dexamethasone 8 1.04 ± 0.10# Results are expressed as the mean ± SEM for the number (N) of rats indicated. #p < 0.005, compared to the corresponding control group. *p < 0.001, compared to the corresponding control group. 101 microsomes from uninduced or dexamethasone-induced adult male rats. 3.1.6.7 Summary The effects of in vivo cimetidine on testosterone oxidation by microsomes from uninduced, phenobarbital-induced and dexamethasone-induced rats are summarized in Figure 18. Testosterone 2a- and 16a-hydroxylase acti v i t i e s in microsomes from the uninduced rats were inhibited by in vivo cimetidine. The inhibition observed in these two cases, in conjunction with the known enzyme-specificity of these two a c t i v i t i e s , indicated that in vivo cimetidine has an inhibitory effect on cytochrome P450IIC11. In the other cases where inhibition of testosterone oxidation by cimetidine was observed, the enzyme-specificity of the act i v i t i e s was unknown. Furthermore, the lack of an effect of in vivo cimetidine on testosterone 20, 60, 7a or 160-hydroxylase activity suggests that cytochromes P450IIIA1/2, cytochrome P450IIA1 and cytochromes P450IIB1/2 are not inhibited by cimetidine. 102 18 Summary o f the e f f e c t s o f in vivo c i m e t i d i n e on t e s t o s t e r o n e o x i d a t i o n . R e s u l t s are based on the da t a from F i g u r e s 10-15 and 17. p < O.uUl, compared t o the s a l i n e - t r e a t e d c o n t r o l group. UNINDUCED R \ \ \ \ \ \ \ . s • N \ \ \ \ \ \ \ \ \ \ \ \ \ . M l K S 3 PB-INDUCED MM DEX-INDUCED 2a 2/3 6/3 7a 16a 16/3 TESTOSTERONE METABOLITES 103 3.2 IMMUNOINHIBITION STUDIES WITH MONOSPECIFIC ANTI-CYTOCHROME P450IIC11 ANTIBODY The major purpose of the immunoinhibition studies was to determine the role of cytochrome P450IIC11 i n the microsomal enzyme activities that were inhibited by in vivo cimetidine; that i s , aminopyrine N-demethylase, pentoxyresorufin O-dealkylase, erythromycin N-demethylase, testosterone 2a-hydroxylase, testosterone 16a-hydroxylase and androstenedione formation. Testosterone 2$-, 6^ -, 7a-and 16p~hydroxylase activities were also determined in the presence or absence of the antibody. .3.2.1 Aminopyrine N-Demethylase A c t i v i t y At saturating concentrations, the antibody inhibited aminopyrine N-demethylase activity by approximately 35% in microsomes from uninduced adult male rats (Figure 19). In contrast, there was l i t t l e or no inhibitory effect of the antibody on this activity in microsomes from phenobarbital-induced rats. 3.2.2 Pentoxyresorufin O-Dealkylase A c t i v i t y At the highest concentration used, the antibody inhibited pentoxyresorufin O-dealkylase activity by more than 90% in microsomes from uninduced rats (Figure 20). In contrast, there was l i t t l e or no inhibitory effect of the 104 F i g u r e 19 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 antibody on aminopyrine N-demethylase a c t i v i t y . Microsomes were i s o l a t e d from a p o o l o f l i v e r s from e i t h e r f o u r uninduced o r p h e n o b a r b i t a l -induced r a t s . Microsomes (0.3 nmol o f t o t a l cytochrome P-450) were p r e i n c u b a t e d f o r 10 min a t room temperature w i t h 0.25, 0.5, 1, 2.5, 5 o r 10 mg of anti-cytochrome P450IIC11 a n t i b o d y o r c o n t r o l IgG per nmol o f t o t a l cytochrome P-450. Enzyme a c t i v i t y was determined as d e s c r i b e d under " M a t e r i a l s and Methods". R e s u l t s are expressed as a p e r c e n t of c o n t r o l a c t i v i t y . >-> i— o < o CC o o o 100 80 60 40 o oc D_ 20 - - B UNINDUCED - A PB-INDUCED 0 0.1 1.0 10.0 ANTI-P450IIC1 1 IgG ( m g / n m o l P450) 105 Figure 20 Effect of monospecific anti-cytochrome P450IIC11 antibody on pentoxyresorufin 0-dealkylase activity. Microsomes were isolated from a pool of livers from either four uninduced or phenobarbital-induced rats. Microsomes (0.3 nmol of total cytochrome P-450) were preincubated for 10 min at room temperature with 0.25, 0.5, 1, 2.5, 5 or 10 mg of anti-cytochrome P450IIC11 antibody or control IgG per nmol of total cytochrome P-450. Enzyme activity was determined as described under "Materials and Methods". Results are expressed as a percent of control activity. 0 1 — • — . . — 0.1 1.0 10.0 ANTI-P450IIC1 1 IgG (mg/nmol P450) 106 antibody on this activity in microsomes from phenobarbital-induced rats. 3.2.3 Erythromycin N-Demethylase Activity At the concentrations used, the antibody had l i t t l e or no inhibitory effect on erythromycin N-demethylase activity in microsomes from uninduced rats (Figure 21). 3.2.4 Testosterone 2a-Hydroxylase Activity At saturating concentrations, the antibody completely inhibited testosterone 2a-hydroxylase activity in microsomes from uninduced adult male rats (Figure 22). This observation i s consistent with published data (Waxman, 1984; Waxman et a l . , 1987) and confirms that cytochrome P450IIC11 is the enzyme responsible for testosterone 2a-hydroxylase activity in microsomes from uninduced adult male rats. At saturating concentrations, the antibody also completely inhibited this activity in microsomes from phenobarbital-induced and dexamethasone-induced adult male rats (Figure 22). These results indicate that cytochrome P450IIC11 remains the enzyme responsible for microsomal testosterone 2oc-hydroxylase activity in these groups of rats. 107 Figure 21 Effect of monospecific anti-cytochrome P450IIC11 antibody on erythromycin N-demethylase activity. Microsomes were isolated from a pool of livers from four uninduced rats. Microsomes (0.3 nmol of total cytochrome P-450) were preincubated for 10 min at room temperature with 0.25, 0.5, 1, 2.5, 5 or 10 mg of anti-cytochrome P450IIC11 antibody or control IgG per nmol of total cytochrome P-450. Enzyme activity was determined as described under "Materials and Methods". Results are expressed as a percent of control activity. >-> I— o < _ J o cc I— z o o U . o o DC U l Q _ 1.0 10.0 ANTI-P450IIC1 1 IgG ( m g / n m o l P450) 108 Figure 22 Effect of monospecific anti-cytochrome P450IIC11 antibody on testosterone 2a-hydroxylase ac t i v i t y . Microsomes were isolated from a pool of livers from four uninduced, phenobarbital-induced or dexamethasone-induced rats. Microsomes (0.3 nmol of total cytochrome P-450) were preincubated for 10 min at room temperature with 0.25, 0.5, 1, 2.5, 5 or 10 mg of anti-cytochrome P450IIC11 antibody or control IgG per nmol of total cytochrome P-450. Enzyme activity was determined as described under "Materials and Methods". Results are expressed as a percent of control activity. > i— < 100 80 • • U N I N D U C E D A • PB-IN0UCED • 0 D E X — I N D U C E D O CC 60 o o Lu o (— LU o cc LU CL 40 20 0 0.1 1.0 10.0 ANTI-P450IIC1 1 IgG ( m g / n m o l P450 ) 109 3.2.5 T e s t o s t e r o n e 16a-Hydroxylase A c t i v i t y At saturating concentrations, the antibody inhibited testosterone 16a-hydroxylase activity by approximately 95% in microsomes from uninduced adult male rats (Figure 23). This i s consistent with published data (Waxman, 1984; Waxman et a l . , 1987) and confirms that cytochrome P450IIC11 i s the enzyme responsible for microsomal testosterone 16a-hydroxylase activity in this group of rats. As shown in Figure 23, at saturating concentrations, the antibody had l i t t l e or no inhibitory effect on this activity in microsomes from phenobarbital-induced adult male rats. It has been shown that cytochrome P450IIC11 i s not the major contributor to testosterone 16a-hydroxylase activity in microsomes from phenobarbital-induced adult male rats (Waxman, 1984; Waxman et a l . , 1987). At saturating concentrations, the antibody inhibited testosterone 16a-hydroxylase activity by approximately 65% in microsomes from dexamethasone-treated rats (Figure 23), indicating that cytochrome P450IIC11 partially contributes to microsomal testosterone 16a-hydroxylase activity in this group of rats. 3.2.6 Androstenedione Formation At saturating concentrations, the antibody inhibited androstenedione formation by approximately 60% in microsomes from uninduced adult male rats (Figure 24). In contrast, i t 110 Figure 23 Effect of monospecific anti-cytochrome P450IIC11 antibody on testosterone 16a-hydroxylase a c t i v i t y . Microsomes were isolated from a pool of livers from four uninduced, phenobarbital-induced or dexamethasone-induced rats. Microsomes (0.3 nmol of total cytochrome P-450) were preincubated for 10 min at room temperature with 0.25, 0.5, 1, 2.5, 5 or 10 mg of anti-cytochrome P450IIC11 antibody or control IgG per nmol of total cytochrome P-450. Enzyme activity was determined as described under "Materials and Methods". Results are expressed as a percent of control activity. 0 1 . . — • • — 0.1 1.0 10.0 ANTI-P450IIC1 1 IgG (mg/nmol P450) I l l F i g u r e 24 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 a n t i b o d y on androstenedione f o r m a t i o n . Microsomes were i s o l a t e d from a p o o l o f l i v e r s from f o u r uninduced, p h e n o b a r b i t a l - i n d u c e d o r dexamethasone-induced r a t s . Microsomes (0.3 nmol o f t o t a l cytochrome P-450) were p r e i n c u b a t e d f o r 10 min a t room temperature with 0.25, 0.5, 1, 2.5, 5 o r 10 mg o f anti-cytochrome P450IIC11 a n t i b o d y o r c o n t r o l IgG per nmol of t o t a l cytochrome P-450. Enzyme a c t i v i t y was determined as d e s c r i b e d under " M a t e r i a l s and Methods". R e s u l t s are expressed as a p e r c e n t o f c o n t r o l a c t i v i t y . >-> h-o < o CC \— o o U. o o CC Ul CL. 100 80 60 40 20 0 • UNINDUCED •A PB-INDUCED O DEX—INDUCED 0.1 1.0 10.0 ANTI-P450IIC11 IgG (mg /nmo l P450) 112 had l i t t l e or no inhibitory effect on this reaction in microsomes from phenobarbital-induced adult male rats. In microsomes from dexamethasone-induced adult male rats, the antibody inhibited this reaction by approximately 40%. 3.2.7 T e s t o s t e r o n e 26-, 60-, 7a- and 160-Hydroxylase A c t i v i t i e s The antibody did not inhibit the cytochrome P450IIIAl/2-mediated testosterone 20- and 60-hydroxylase act i v i t i e s or the cytochrome P450IIAl-mediated testosterone 7a-hydroxylase activity in microsomes from uninduced, phenobarbital- or dexamethasone-induced rats (Figures 25-27). It also did not inhibit the cytochrome P450IIB1/2-mediated testosterone 160-hydroxylase activity in microsomes from phenobarbital-induced rats (Figure 28). 113 F i g u r e 25 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 antibody on t e s t o s t e r o n e 2f3-hydroxylase a c t i v i t y . Microsomes were i s o l a t e d from a p o o l o f l i v e r s from f o u r uninduced, p h e n o b a r b i t a l - i n d u c e d o r dexamethasone-induced r a t s . Microsomes (0.3 nmol of t o t a l cytochrome P-450) were p r e i n c u b a t e d f o r 10 min a t room temperature w i t h 0.25, 0.5, 1, 2.5, 5 o r 10 mg of anti-cytochrome P450IIC11 an t i b o d y o r c o n t r o l IgG per nmol o f t o t a l cytochrome P-450. Enzyme a c t i v i t y was determined as d e s c r i b e d under " M a t e r i a l s and Methods". R e s u l t s are expressed as a p e r c e n t o f c o n t r o l a c t i v i t y . >-> o < _ J o oc o o 200 150 100 UNINDUCED PB-INDUCED DEX-INDUCED Ul o DC 50 0 0.1 1.0 10.0 ANTI-P450IIC1 1 IgG (mg/nmol P450) 114 F i g u r e 26 E f f e c t o f m o n o s p e c i f i c anti-cytochrome P450IIC11 ant i b o d y on t e s t o s t e r o n e 66-hydroxylase a c t i v i t y . Microsomes were i s o l a t e d from a p o o l o f l i v e r s from f o u r uninduced, p h e n o b a r b i t a l - i n d u c e d o r dexamethasone-induced r a t s . Microsomes (0.3 nmol o f t o t a l cytochrome P-450) were p r e i n c u b a t e d f o r 10 min a t room temperature w i t h 0.25, 0.5, 1, 2.5, 5 o r 10 mg of anti-cytochrome P450IIC11 a n t i b o d y o r c o n t r o l IgG per nmol o f t o t a l cytochrome P-450. Enzyme a c t i v i t y was determined as d e s c r i b e d under " M a t e r i a l s and Methods". R e s u l t s are expressed as a p e r c e n t o f c o n t r o l a c t i v i t y . >-> I— o < _ I o O o 200 150 100 - • U N I N D U C E D - • P B - I N D U C E D • D E X - I N D U C E D : LoJ O cn U l C L . 50 0 0.1 1.0 10.0 ANTI-P450IIC1 1 IgG ( m g / n m o l P450) 115 Figure 27 Effect of monospecific anti-cytochrome P450IIC11 antibody on testosterone 7a-hydroxylase a c t i v i t y . Microsomes were isolated from a pool of livers from four uninduced, phenobarbital-induced or dexamethasone-induced rats. Microsomes (0.3 nmol of total cytochrome P-450) were preincubated for 10 min at room temperature with 0.25, 0.5, 1, 2.5, 5 or 10 mg of anti-cytochrome P450IIC11 antibody or control IgG per nmol of total cytochrome P-450. Enzyme activ i t y was determined as described under "Materials and Methods". Results are expressed as a percent of control activity. >-> o < _ j o cc o o 200 150 100 UNINDUCED PB-INDUCED DEX—INDUCED z L d o cc 50 0 0.1 1.0 10.0 ANTI-P450IIC1 1 IgG (mg/nmol P450) 116 Figure 28 Effect of monospecific anti-cytochrome P450IIC11 antibody on testosterone 16p~hydroxylase activity. Microsomes were isolated from a pool of livers from four phenobarbital-induced rats. Microsomes (0.3 nmol of total cytochrome P-4 50) were preincubated for 10 min at room temperature with 0.25, 0.5, 1, 2.5, 5 or 10 mg of anti-cytochrome P450IIC11 antibody or control IgG per nmol of total cytochrome P-450. Enzyme activity was determined as described under "Materials and Methods". Results are expressed as a percent of control activity. >-> r-o < o cc t— O o L i . o Ld o cc 200 1 5 0 100 50 0 0.1 1.0 10.0 ANTI-P450IIC1 1 IgG (mg/nmol P450) 117 3.3 STUDIES WITH IN VITRO CIMETIDINE The r e s u l t s presented i n the f o r e g o i n g s e c t i o n s i n d i c a t e d t h a t in vivo c i m e t i d i n e i n h i b i t s h e p a t i c cytochrome P450IIC11, but a p p a r e n t l y not cytochrome P450IIA1, cytochromes P450IIB1/2 or cytochromes P450IIIA1/2 i n the a d u l t male r a t . Experiments were performed t o determine whether the same r e s u l t s would be o b t a i n e d w i t h in vitro c i m e t i d i n e , u s i n g the same enzyme a c t i v i t i e s as i n the s t u d i e s w i t h in vivo c i m e t i d i n e . 3.3.1 P e n t o x y r e s o r u f i n O-Dealkylase A c t i v i t y Jn vitro c i m e t i d i n e i n h i b i t e d p e n t o x y r e s o r u f i n 0 - d e a l k y l a s e a c t i v i t y i n microsomes from p h e n o b a r b i t a l -induced r a t s ( F i g u r e 29). The I C S 0 v a l u e was 1.0 mM (Table 18). T h i s i n d i c a t e s t h a t i n v i t r o c i m e t i d i n e i n h i b i t s cytochromes P450IIB1/2 and i s i n c o n t r a s t t o the apparent l a c k of i n h i b i t i o n of p e n t o x y r e s o r u f i n 0 - d e a l k y l a s e a c t i v i t y i n microsomes from p h e n o b a r b i t a l - i n d u c e d r a t s by in vivo c i m e t i d i n e ( S e c t i o n 3.1.3). 3.3.2 E r y t h r o m y c i n N-Demethylase A c t i v i t y Jn vitro c i m e t i d i n e i n h i b i t e d e r y t h r o m y c i n N-demethylase a c t i v i t y i n microsomes from dexamethasone-induced r a t s ( F i g u r e 30). The I C 5 0 v a l u e was 2.8 mM (Table 18). T h i s i n d i c a t e s t h a t in vitro c i m e t i d i n e 118 Figure 29 Effect of in vitro cimetidine on pentoxyresorufin 0-dealkylase activity. Livers from four phenobarbital-induced rats were pooled and microsomes were prepared. Cimetidine hydrochloride (0.15 - 10 mM) or d i s t i l l e d water (control) was added in vitro. Results are expressed as a percent of control activity. The activity of the control sample was 5.03 nmol/min/mg protein. 100 >-t: > h-o < _J o ce o o u. o UJ o CC Ul Q_ CIMETIDINE HCl CONCENTRATION (mM) 119 TABLE 18 I C 5 0 VALUES FOR THE INHIBITION OF PENTOXYRESORUFIN O-DEALKYLASE, ERYTHROMYCIN N-DEMETHYLASE AND ETHOXYRESORUFIN O-DEETHYLASE ACTIVITIES BY CIMETIDINE IN VITRO Enzyme Activity Inducer Substrate IC 5 0 Concentration (mM) (mM) Pentoxyresorufin O-Dealkylase Erythromycin N-Demethylase Ethoxyresorufin O-Deethylase Phenobarbital Dexamethasone None 0.005 0.4 0.005 1.0 2.8 0.3 Ethoxyresorufin 0-Deethylase p-Naphtho-flavone 0.005 7.1 IC 5 0 values were determined graphically based on the data from Figures 29-31. 120 Figure 30 Effect of in vitro cimetidine on erythromycin N-demethylase activity. Livers from four dexamethasone-induced rats were pooled and microsomes were prepared. Cimetidine hydrochloride (0.625 - 10 mM) or d i s t i l l e d water (control) was added in vitro. Results are expressed as a percent of control activity. The activity of the control sample was 10.41 nmol/min/mg protein. 100 >-0 '——•—• • • • . . . . .—. . . • 0.5 1.0 10.0 CIMETIDINE HCl CONCENTRATION (mM) inhibits cytochromes P450IIIA1/2 and i s in contrast to the apparent lack of inhibition of erythromycin N-demethylase activity in microsomes from dexamethasone-induced rats by in vivo cimetidine (Section 3.1.4). 3.3.3 Ethoxyresorufin O-Deethylase Activity In vitro cimetidine inhibited ethoxyresorufin O-deethylase activity in microsomes from uninduced rats (Figure 31). The IC 5 0 value was 0.3 mM (Table 18). This indicates that in vitro cimetidine inhibits cytochrome P450IA2 and is consistent with the observed inhibition of ethoxyresorufin O-deethylase activity in microsomes from uninduced rats by in vivo cimetidine (Section 3.1.5). In vitro cimetidine also inhibited ethoxyresorufin O-deethylase activity in microsomes from (3-naphthoflavone-induced rats (Figure 31). The IC 5 0 value was 7.1 mM (Table 18). This is in contrast to the apparent lack of inhibition of ethoxyresorufin O-deethylase activity in microsomes from p-naphthoflavone-induced rats by in vivo cimetidine (Section 3.1.5). 3.3.4 Testosterone 2a-Hydroxylase Activity Jn vitro cimetidine inhibited testosterone 2a-hydroxylase activity in microsomes from uninduced rats (Figure 32). The IC 5 0 value was 7.4 mM (Table 19). In vitro cimetidine also inhibited this activity in microsomes 122 F i g u r e 31 E f f e c t o f i n vitro c i m e t i d i n e on e t h o x y r e s o r u f i n O-deethylase a c t i v i t y . Microsomes were i s o l a t e d from a po o l o f l i v e r s from e i t h e r f o u r uninduced or 6-naphthoflavone-induced r a t s . C i m e t i d i n e h y d r o c h l o r i d e (0.02 - 10 mM) o r d i s t i l l e d water ( c o n t r o l ) was added in vitro. R e s u l t s are expressed as a perce n t o f c o n t r o l a c t i v i t y . C o n t r o l a c t i v i t y i n nmol/min/mg p r o t e i n : uninduced, 0.42; 6-naphthoflavone-induced, 11.71. 0 1 • — • • — < . — . . i 0.01 0.10 1.00 10.00 CIMETIDINE HCl CONCENTRATION (mM) 123 Figure 32 Effect of in vitro cimetidine on testosterone 2a-hydroxylase activity. Microsomes were isolated from a pool of livers from either four uninduced or phenobarbital-induced rats. Cimetidine hydrochloride (0.02 - 10 mM) or d i s t i l l e d water (control) was added in vitro. Results are expressed as a percent of control activity. Control activity in nmol/min/mg protein: uninduced, 1.55; phenobarbital-induced, 0.92. >-> r— O < _ J o cc o o L i _ o Ld O CC Ld Q_ 100 80 60 40 20 • UNINDUCED •A PB-INDUCED 0.01 0.10 1.00 10.00 CIMETIDINE HCI CONCENTRATION (mM) 124 TABLE 19 IC S 0 VALUES FOR THE INHIBITION OF TESTOSTERONE OXIDATION BY CIMETIDINE IN VITRO T e s t o s t e r o n e Uninduced P h e n o b a r b i t a l -M e t a b o l i t e induced 2a 26 66 7a 16a 166 A 7.4 1.7 1.7 N.I. 6.4 N.D. > 5 3.7 1.6 2.2 N.I. 4.2 3.3 5.5 The s u b s t r a t e c o n c e n t r a t i o n was 0.25 mM. I C 5 0 v a l u e s (mM) were determined g r a p h i c a l l y based on the da t a from F i g u r e s 32-38. A b b r e v i a t i o n s : N.I., no i n h i b i t i o n w i t h c i m e t i d i n e c o n c e n t r a t i o n s o f up t o 10 mM; N.D., not determined; A, androstenedione. 125 from phenobarbital-induced rats (Figure 32) and the IC S 0 value in that case was 3.7 mM (Table 19). These results indicate that in vitro cimetidine inhibits cytochrome P450IIC11 and are consistent with the observed inhibition of testosterone 2a-hydroxylase activity in microsomes from uninduced and phenobarbital-induced rats by in vivo cimetidine (Section 3.1.6.1). 3 . 3 . 5 Testosterone 20- and 60-Hydroxylase Activities In vitro cimetidine inhibited testosterone 20- and 60-hydroxylase activities in microsomes from uninduced rats (Figures 33 and 34). The IC S 0 value was 1.7 mM in both cases (Table 19). In vitro cimetidine also inhibited these two a c t i v i t i e s in microsomes from phenobarbital-induced rats (Figures 33 and 34). The IC 5 0 value was 1.6 mM for the inhibition of testosterone 20-hydroxylase activity and 2.2 mM for the inhibition of testosterone 6p*-hydroxylase acti v i t y (Table 19). These results indicate that in vitro cimetidine inhibits cytochromes P450IIIA1/2 and are in contrast to the apparent lack of inhibition of testosterone 20- and 60-hydroxylase acti v i t i e s in microsomes from uninduced and phenobarbital-induced rats by in vivo cimetidine (Section 3.1.6.2). 126 F i g u r e 33 E f f e c t o f in vitro c i m e t i d i n e on t e s t o s t e r o n e 26-hydroxylase a c t i v i t y . Microsomes were i s o l a t e d from a p o o l o f l i v e r s from e i t h e r f o u r uninduced o r p h e n o b a r b i t a l - i n d u c e d r a t s . C i m e t i d i n e h y d r o c h l o r i d e (0.02 - 10 mM) o r d i s t i l l e d water ( c o n t r o l ) was added in vitro. R e s u l t s are expressed as a p e r c e n t of c o n t r o l a c t i v i t y . C o n t r o l a c t i v i t y i n nmol/min/mg p r o t e i n : uninduced, 0.04; p h e n o b a r b i t a l - i n d u c e d , 0.29. >-> I— O < o cn o o L U o cn 100 80 60 40 20 UNINDUCED PB-INDUCED 0.01 0.10 1.00 10.00 CIMETIDINE HCl CONCENTRATION (mM) 127 F i g u r e 34 E f f e c t o f in vitro c i m e t i d i n e on t e s t o s t e r o n e 6B-hydroxylase a c t i v i t y . Microsomes were i s o l a t e d from a p o o l o f l i v e r s from e i t h e r f o u r uninduced o r p h e n o b a r b i t a l - i n d u c e d r a t s . C i m e t i d i n e h y d r o c h l o r i d e (0.02 - 10 mM) o r d i s t i l l e d water ( c o n t r o l ) was added in vitro. R e s u l t s are expressed as a p e r c e n t o f c o n t r o l a c t i v i t y . C o n t r o l a c t i v i t y i n nmol/min/mg p r o t e i n : uninduced, 1.55; p h e n o b a r b i t a l - i n d u c e d , 7.16. 0.01 0.10 1.00 10.00 CIMETIDINE HCl CONCENTRATION (mM) 128 3.3.6 T e s t o s t e r o n e 7a-Hydroxylase A c t i v i t y In vitro c i m e t i d i n e , a t c o n c e n t r a t i o n s o f up t o 10 mM, d i d not i n h i b i t t e s t o s t e r o n e 7cc-hydroxylase a c t i v i t y i n microsomes from uninduced or p h e n o b a r b i t a l - i n d u c e d r a t s ( F i g u r e 35). These r e s u l t s suggest t h a t in vitro c i m e t i d i n e does not i n h i b i t cytochrome P450IIA1 and are c o n s i s t e n t w i t h the apparent l a c k of i n h i b i t i o n o f t e s t o s t e r o n e 7a-hydroxylase a c t i v i t y i n microsomes from uninduced and p h e n o b a r b i t a l - i n d u c e d r a t s by in vivo c i m e t i d i n e ( S e c t i o n 3.1.6.3). 3.3.7 T e s t o s t e r o n e 16a-Hydroxylase A c t i v i t y In vitro c i m e t i d i n e i n h i b i t e d t e s t o s t e r o n e 16a-hydroxylase a c t i v i t y i n microsomes from uninduced r a t s ( F i g u r e 36). The I C S 0 v a l u e was 6.4 mM (Table 19). T h i s i n d i c a t e s t h a t in vitro c i m e t i d i n e i n h i b i t s cytochrome P450IIC11 and i s c o n s i s t e n t w i t h the i n h i b i t i o n o f t e s t o s t e r o n e 16a-hydroxylase a c t i v i t y i n microsomes from uninduced r a t s by in vivo c i m e t i d i n e ( S e c t i o n 3.1.6.4). In vitro c i m e t i d i n e a l s o i n h i b i t e d t e s t o s t e r o n e 16ct-hydroxylase a c t i v i t y i n microsomes from p h e n o b a r b i t a l -induced r a t s ( F i g u r e 36). The I C 5 0 v a l u e was 4.2 mM (Table 19). T h i s i s i n c o n t r a s t t o the apparent l a c k o f i n h i b i t i o n o f t e s t o s t e r o n e 16a-hydroxylase a c t i v i t y i n microsomes from p h e n o b a r b i t a l - i n d u c e d r a t s by in vivo c i m e t i d i n e ( S e c t i o n 129 Figure 35 Effect of in vitro cimetidine on testosterone 7a-hydroxylase activity. Microsomes were isolated from a pool of livers from either four uninduced or phenobarbital-induced rats. Cimetidine hydrochloride (0.02 - 10 mM) or d i s t i l l e d water (control) was added in vitro. Results are expressed as a percent of control activity. Control activity in nmol/min/mg protein: uninduced, 0.42; phenobarbital-induced, 0.84. > h-O < _ I o cn i— z o o U J o cn U J CL. 120 100 80 60 40 20 • • UNINDUCED A A PB- INDUCED 0.01 0.10 1.00 10.00 CIMETIDINE HCl CONCENTRATION (mM) 130 Figure 36 Effect of in vitro cimetidine on testosterone 16a-hydroxylase activity. Microsomes were isolated from a pool of livers from either four uninduced phenobarbital-induced rats. Cimetidine hydrochloride (0.02 - 10 mM) or d i s t i l l e d water (control) was added in vitro. Results are expressed as a percent of control activity. Control activity in nmol/min/mg protein: uninduced, 2.22; phenobarbital-induced, 6.24. L U 0_ 0 1 1 - • -.I . ^ _ J _ ^ ^ J _ 0.01 0.10 1.00 10.00 CIMETIDINE HCI CONCENTRATION (mM) 131 3.1.6.4). Furthermore, the differential effect of in vivo cimetidine on testosterone 16a-hydroxylase activity in microsomes from uninduced and phenobarbital-induced rats (Section 3.1.6.4) was not observed with in vitro cimetidine. 3 . 3 . 8 Testosterone 168-Hydroxylase Activity Jn vitro cimetidine inhibited testosterone 166-hydroxylase activity in microsomes from phenobarbital-induced rats (Figure 37). The IC 5 0 was 3.3 mM (Table 19). This indicates that in vitro cimetidine inhibits cytochromes P450IIB1/2 and i s in contrast to the apparent lack of inhibition of testosterone 168-hydroxylase activity in microsomes from phenobarbital-induced rats by in vivo cimetidine (Section 3.1.6.5). 3 . 3 . 9 Androstenedione Formation Jn vitro cimetidine inhibited androstenedione formation in microsomes from uninduced rats (Figure 38). The IC 5 0 value was greater than 5 mM (Table 19). This i s consistent with the inhibition of androstenedione formation in microsomes from uninduced rats by in vivo cimetidine (Section 3.1.6.6). Jn vitro cimetidine also inhibited androstenedione formation in microsomes from phenobarbital-induced rats (Figure 38) and the IC 5 0 value was 5.5 mM (Table 19). This i s i n contrast to the apparent lack of inhibition of 132 Figure 37 Effect of in vitro cimetidine on testosterone 160-hydroxylase activity. Microsomes were isolated from a pool of livers from four phenobarbital-induced rats. Cimetidine hydrochloride (0.02 - 10 mM) or d i s t i l l e d water (control) was added in vitro. Results are expressed as a percent of control activity. Control activity in nmol/min/mg protein: 3.74. 0_ 0 1 •—•—• • .—. . ' .—.— 0.01 0.10 1.00 10.00 CIMETIDINE HCI CONCENTRATION (mM) 133 Figure 38 Effect of in vitro cimetidine on androstenedione formation. Microsomes were isolated from a pool of livers from either four uninduced or phenobarbital-induced rats. Cimetidine hydrochloride (0.02 - 10 mM) or d i s t i l l e d water (control) was added in vitro. Results are expressed as a percent of control activity. Control activity in nmol/min/mg protein: uninduced, 1.34; phenobarbital-induced, 5.23. >~ > t— o < _ J o Cd h-z o o Ul o Cd. UI 0_ 100 80 60 40 20 0 • UNINDUCED A A PB- INDUCED 0 0.01 0.10 1.00 1 0.00 CIMETIDINE HCI CONCENTRATION (mM) 134 androstenedione f o r m a t i o n i n microsomes from p h e n o b a r b i t a l -induced r a t s by in vivo c i m e t i d i n e ( S e c t i o n 3.1.6.6). As was the case w i t h t e s t o s t e r o n e 16ct-hydroxylase a c t i v i t y , the d i f f e r e n t i a l e f f e c t o f in vivo c i m e t i d i n e on androstenedione f o r m a t i o n i n the uninduced and p h e n o b a r b i t a l - i n d u c e d groups ( S e c t i o n 3.1.6.6) was not observed w i t h in vitro c i m e t i d i n e . 3.3.10 Enzyme K i n e t i c s o f the I n h i b i t i o n o f T e s t o s t e r o n e 2a-Hydroxylase A c t i v i t y by In Vitro and Jn Vivo C i m e t i d i n e The r e s u l t s p r e s e n t e d i n the f o r e g o i n g s e c t i o n s have p r o v i d e d evidence t h a t c i m e t i d i n e , whether a d m i n i s t e r e d in vitro o r in vivo, i n h i b i t s cytochrome P450IIC11. To g a i n f u r t h e r i n s i g h t i n t o the i n h i b i t o r y e f f e c t o f c i m e t i d i n e on t h i s enzyme, k i n e t i c experiments were performed t o determine the type o f i n h i b i t i o n o f t e s t o s t e r o n e hydroxylase a c t i v i t y by c i m e t i d i n e . Microsomes from uninduced a d u l t male r a t s were used. Based on Lineweaver-Burk p l o t s o f the d a t a , the i n h i b i t i o n o f t e s t o s t e r o n e 2a-hydroxylase a c t i v i t y by in vitro c i m e t i d i n e was c o m p e t i t i v e ( F i g u r e 39), whereas the i n h i b i t i o n o f t h i s a c t i v i t y by in vivo c i m e t i d i n e was non-c o m p e t i t i v e ( F i g u r e 40). 135 Figure 39 Linweaver-Burk plot for the inhibition of testosterone 2a-hydroxylase activity by in vitro cimetidine. Microsomes were prepared from a pool of four livers from uninduced rats. Cimetidine hydrochloride (2 mM, 4 mM) or d i s t i l l e d water (control) was added in vitro. The enzyme acti v i t y (V) was determined at various substrate [S] concentrations. The symbols indicate values of the transformed data and the lines were generated by linear regression analysis. 136 Figure 40 Linweaver-Burk plot for the inhibition of testosterone 2a-hydroxylase activity by in vivo cimetidine. Uninduced rats were sacrificed 90 min after a single injection of cimetidine HCl (150 mg/kg) or saline (control). Microsomes were prepared from a pool of four livers in each group. The enzyme activity (V) was determined at various substrate [S] concentrations. The symbols indicate values of the transformed data and the lines were generated by linear regression analysis. 137 3.4 PREINCUBATION STUDIES WITH IN VITRO CIMETIDINE If cimetidine inhibits cytochrome P-450-mediated hepatic drug metabolism in vivo by a non-competitive mechanism, as suggested by the result shown in Figure 40, then this would explain why relatively low concentrations of cimetidine in man and rats can inhibit drug clearance. However, this does not explain why cimetidine i s not a more potent inhibitor in vitro. Preincubation studies were performed to determine whether cimetidine, at relatively low concentrations, can selectively inhibit microsomal testosterone oxidation in a manner similar to that observed with in vivo cimetidine (Section 3.1.6). 3.4.1 Preliminary Experiments The effect of in vitro cimetidine on microsomal testosterone oxidation with the inclusion of a preincubation step in the assay protocol was investigated. In these experiments, microsomes were f i r s t preincubated with NADPH and cimetidine. Subsequently, testosterone oxidation was init i a t e d with the addition of the substrate and the reaction was allowed to proceed as described in "Materials and Methods" (Section 2.8.6). 138 3.4.1.1 Microsomes from Uninduced Rats The i n i t i a l experiment was performed to determine whether with a preincubation step, a relatively low concentration of cimetidine can selectively inhibit microsomal testosterone oxidation. Microsomes were preincubated with cimetidine (0, 0.025, 0.05, 0.1, 0.2 or 0.4 mM) and 1 mM NADPH for 20 min prior to the i n i t i a t i o n of testosterone oxidation. As shown in Figure 41, cimetidine resulted in a concentration-dependent inhibition of both testosterone 2a-hydroxylase and testosterone 6|3-hydroxylase a c t i v i t i e s . However, in microsomes preincubated with 0.05 mM cimetidine and NADPH for 20 min, testosterone 2a-hydroxylase activity was approximately 3 0% lower compared to those preincubated for the same period of time with NADPH only, whereas testosterone 60-hydroxylase activity was unaffected by cimetidine (Figure 41). To determine whether NADPH in the preincubation medium causes any substantial decrease in testosterone oxidation, microsomes were preincubated with 1 mM NADPH for 0 (control) 5, 10, 15 or 20 min prior to the i n i t i a t i o n of testosterone oxidation. As shown in Figure 42, the preincubation of microsomes with NADPH (in the absence of cimetidine) for the times indicated resulted in l i t t l e or no decrease in each of the a c t i v i t i e s . An experiment was performed to determine whether the observed inhibition of testosterone 2a-hydroxylase activity 139 Figure 41 Testosterone 2a- and 66-hydroxylase a c t i v i t i e s i n microsomes preincubated with NADPH and cimetidine. Livers from four uninduced rats were pooled and microsomes were prepared. Microsomes were preincubated with cimetidine hydrochloride (0.025 - 0.4 mM) or d i s t i l l e d water (control) and NADPH (1 mM) for 20 min. Results are expressed as a percent of control activity. Control acti v i t y in nmol/min/mg protein: 2a, 1.67; 66, 2.01. 0 1 • • • — — — i — • . . — I 0.01 0.10 1.00 CIMETIDINE HCl CONCENTRATION (mM) 140 F i g u r e 42 T e s t o s t e r o n e o x i d a t i o n i n microsomes p r e i n c u b a t e d f o r v a r i o u s times w i t h NADPH i n the absence o f c i m e t i d i n e . L i v e r s from f o u r uninduced r a t s were pooled and microsomes were prepared. Microsomes were p r e i n c u b a t e d w i t h 1 mM NADPH f o r 0 ( c o n t r o l ) , 5, 10, 15 o r 20 min p r i o r t o the i n i t i a t i o n o f t e s t o s t e r o n e o x i d a t i o n . R e s u l t s are expressed as a per c e n t o f c o n t r o l a c t i v i t y . C o n t r o l a c t i v i t y i n nmol/min/mg p r o t e i n : 2a, 1.72; 20, 0.05; 60, 2.11; 7a, 0.25; 16a, 2.20; A (androstenedione), 1.54. >-> r -O < o cc I— z o o L d o cc L d 0_ 100 80 0 60 - o--o 2a A — — A 2/3 40 o--o 6/3 " V — — V 7a ^ •-• 16a 20 - A.— — A A 0 10 15 20 25 PREINCUBATION TIME (MIN) 141 by c i m e t i d i n e a f t e r p r e i n c u b a t i o n i s a NADPH-dependent p r o c e s s . Microsomes were p r e i n c u b a t e d w i t h c i m e t i d i n e (0, 0.025, 0.1 o r 0.4 mM) i n the presence o r absence o f 1 mM NADPH f o r 20 min p r i o r t o the i n i t i a t i o n o f t e s t o s t e r o n e o x i d a t i o n . As shown i n F i g u r e 43, NADPH was r e q u i r e d i n t h e p r e i n c u b a t i o n medium f o r the i n h i b i t i o n o f t e s t o s t e r o n e 2ct-hydroxylase a c t i v i t y by c i m e t i d i n e . A time-course experiment was performed t o determine an o p t i m a l p r e i n c u b a t i o n p e r i o d f o r the s e l e c t i v e i n h i b i t i o n o f t e s t o s t e r o n e 2a-hydroxylase a c t i v i t y . Microsomes were p r e i n c u b a t e d w i t h NADPH and 0.05 mM c i m e t i d i n e o r d i s t i l l e d water ( c o n t r o l ) f o r 0, 10, 15, or 20 min p r i o r t o t h e i n i t i a t i o n o f t e s t o s t e r o n e o x i d a t i o n . As shown i n F i g u r e 44, near maximal i n h i b i t i o n o f t e s t o s t e r o n e 2cc-hydroxylase a c t i v i t y was a t t a i n e d a f t e r a 15 min p r e i n c u b a t i o n p e r i o d a t t h i s c o n c e n t r a t i o n o f c i m e t i d i n e . No i n h i b i t i o n o f t e s t o s t e r o n e 66-hydroxylase a c t i v i t y was observed w i t h a p r e i n c u b a t i o n p e r i o d o f up t o 20 min ( F i g u r e 44). 3.4.1.2 Microsomes from P h e n o b a r b i t a l - i n d u c e d Rats The p r e i n c u b a t i o n step was then used t o determine the e f f e c t o f in vitro c i m e t i d i n e on t e s t o s t e r o n e o x i d a t i o n i n microsomes from p h e n o b a r b i t a l - i n d u c e d r a t s . Microsomes were p r e i n c u b a t e d w i t h 1 mM NADPH and 0, 0.025, 0.05, 0.1 or 0.4 mM c i m e t i d i n e f o r 15 min p r i o r t o the i n i t i a t i o n o f 142 Figure 43 Testosterone 2a-hydroxylase activity in microsomes preincubated with cimetidine in the presence and absence of NADPH. Livers from four uninduced rats were pooled and microsomes were prepared. Microsomes were preincubated with cimetidine hydrochloride (0.025 - 0.4 mM) or d i s t i l l e d water (control) and with or without NADPH (1 mM) for 20 min prior to the i n i t i a t i o n of testosterone oxidation. In those cases where NADPH was absent in the preincubation medium, i t was added just prior to the start of testosterone oxidation. c '<Si ~o CL cn E \ c £ \ o E c >-> o < ESS WITHOUT NADPH 0 i 1 1 1 1 1 1 3 WITH NADPH 0 0 .025 0.1 0.4 CIMETIDINE HCl CONCENTRATION (mM) 143 F i g u r e 44 T e s t o s t e r o n e 2a- and 6B-hydroxylase a c t i v i t i e s i n microsomes p r e i n c u b a t e d f o r v a r i o u s times w i t h 0.05 mM c i m e t i d i n e and NADPH. L i v e r s from f o u r uninduced r a t s were pool e d and microsomes were prepared. Microsomes were p r e i n c u b a t e d w i t h c i m e t i d i n e h y d r o c h l o r i d e (0.05 mM) o r d i s t i l l e d water ( c o n t r o l ) and NADPH (1 mM) f o r 1, 10, 15 o r 20 min. R e s u l t s a re expressed as a percent o f c o n t r o l a c t i v i t y . C o n t r o l a c t i v i t y i n nmol/min/mg p r o t e i n f o r t e s t o s t e r o n e 2a-hydroxylase: 0 min, 1.72; 10 min, 1.69; 15 min, 1.61; 20 min, 1.59. C o n t r o l a c t i v i t y i n nmol/min/mg p r o t e i n f o r t e s t o s t e r o n e 66-hydroxylase: 0 min, 2.11; 10 min, 2.03; 15 min, 1.91; 20 min, 1.82. >-> t— o < o cn o o o o cn ui CL. 100 5 10 15 20 PREINCUBATION PERIOD (min) 25 144 t e s t o s t e r o n e o x i d a t i o n . As shown i n F i g u r e 45, p r e i n c u b a t i o n o f these microsomes w i t h NADPH and 0.025 o r 0.05 mM c i m e t i d i n e f o r 15 min r e s u l t e d i n the i n h i b i t i o n of t e s t o s t e r o n e 2a-hydroxylase a c t i v i t y , but not t e s t o s t e r o n e 60- o r 160-hydroxylase a c t i v i t y ( F i g u r e 45). 3.4.2 Experiments With I n d i v i d u a l Microsomal Samples The p r e l i m i n a r y experiments were performed wi t h microsomes prepared from pooled l i v e r s . To q u a n t i t a t e the e x t e n t o f i n h i b i t i o n o f t e s t o s t e r o n e o x i d a t i o n by i n vitro c i m e t i d i n e and determine the s e l e c t i v i t y o f the i n h i b i t i o n , a d d i t i o n a l experiments were performed w i t h microsomes pre p a r e d from i n d i v i d u a l l i v e r s . The c o n t r o l groups were: 1) no c i m e t i d i n e and no p r e i n c u b a t i o n ; 2) 0.05 mM c i m e t i d i n e and no p r e i n c u b a t i o n ; 3) no c i m e t i d i n e but the microsomes were p r e i n c u b a t e d w i t h 1 mM NADPH f o r 15 min p r i o r t o the i n i t i a t i o n o f t e s t o s t e r o n e o x i d a t i o n . In the exp e r i m e n t a l group, the microsomes were p r e i n c u b a t e d w i t h 0.05 mM c i m e t i d i n e and 1 mM NADPH f o r 15 min p r i o r t o the i n i t i a t i o n o f t e s t o s t e r o n e o x i d a t i o n . The r e s u l t s from these experiments are d e s c r i b e d i n the f o l l o w i n g s e c t i o n s . 3.4.2.1 T e s t o s t e r o n e 2a-Hydroxylase A c t i v i t y In microsomes from uninduced r a t s , t e s t o s t e r o n e 2oc-hydroxylase a c t i v i t y was 25% lower (p=0.011) i n microsomes p r e i n c u b a t e d w i t h c i m e t i d i n e and NADPH than i n 1 4 5 F i g u r e 45 Testosterone 2a-, 60- and 160-hydroxylase ac t i v i t i e s in microsomes preincubated for 15 min with cimetidine and NADPH. Livers from four phenobarbital-induced rats were pooled and microsomes were prepared. Microsomes were preincubated with cimetidine hydrochloride (0.025 - 0.2 mM) or d i s t i l l e d water (control) and NADPH (1 mM) for 15 min prior to the i n i t i a t i o n of testosterone oxidation. Results are expressed as a percent of control activity. Control activity in nmol/min/mg protein: 2a, 0.78; 60, 5.97; 160, 3.62. CIMETIDINE HCI CONCENTRATION (mM) 146 those preincubated with NADPH only (Figure 46A). In microsomes from phenobarbital-induced rats, this activity was 32% lower (p = 0.023) in microsomes preincubated with cimetidine and NADPH than in those preincubated with NADPH only (Figure 46B). These results are consistent with the inhibition of testosterone 2a-hydroxylase activity by in vivo cimetidine (Section 3.1.6.1). Without the preincubation step, cimetidine did not inhibit testosterone 2a-hydroxylase activity (Figures 46A and 46B). 3.4.2.2 Testosterone 20- and 60-Hydroxylase Activities With or without the preincubation step, cimetidine did not inhibit the cytochromes P450IIIAl/2-mediated-testosterone 20- or 60-hydroxylase activity in microsomes from uninduced or phenobarbital-induced rats (Figures 47A, 47B, 48A and 48B) . 3.4.2.3 Testosterone 7cx-Hydroxylase Activity With or without the preincubation step, cimetidine did not inhibit the cytochrome P4 50lIAl-mediated testosterone 7a-hydroxylase activity in microsomes from uninduced or phenobarbital-induced rats (Figures 49A and 49B) . 147 Figure 46 Effect of preincubation of microsomes with cimetidine and NADPH on testosterone 2<x-hydroxylase activity. Microsomes were preincubated with NADPH (1 mM) and cimetidine hydrochloride (0.05 mM) or d i s t i l l e d water for 1 or 15 min prior to the i n i t i a t i o n of testosterone oxidation. Results are expressed as the mean ± SEM activity for four individual microsomal samples per group. Panel A: uninduced rats; Panel B: phenobarbital-induced rats. *p < 0.05, compared to the group preincubated with NADPH for the same period of time in the absence of cimetidine. ( A ) c 5.0 <a "o a. 2.5 £ \ c 2.0 "E \ 1.5 o E c 1.0 >-> 0.5 o < 0.0 rZZl CONTROL ESS CIMETIDINE WITHOUT PREINCUBATION WITH PREINCUBATION (B) c <D O 1_ CL cn E \ c E \ o E c >-> 1— o < 0.5 0.0 CZI CONTROL E S 3 CIMETIDINE WITHOUT PREINCUBATION WITH PREINCUBATION 148 Figure 47 Effect of preincubation of microsomes with cimetidine and NADPH on testosterone 2p*-hydroxylase activity. Microsomes were preincubated with NADPH (1 mM) and cimetidine hydrochloride (0.05 mM) or d i s t i l l e d water for 0 or 15 min prior to the initation of testosterone oxidation. Results are expressed as the mean ± SEM activ i t y for four individual microsomal per group. Panel A: uninduced rats; Panel B: phenobarbital-induced rats. ( A ) £ ~o i_ o_ E \ c 'E \ o £ c >-> I— O < 0.06 0.04 0.02 0.00 an CONTROL ESS CIMETIDINE W I T H O U T P R E I N C U B A T I O N WITH P R E I N C U B A T I O N W I T H O U T WITH P R E I N C U B A T I O N P R E I N C U B A T I O N 149 Figure 48 Effect of preincubation of microsomes with cimetidine and NADPH on testosterone 66-hydroxylase activity. Microsomes were preincubated with NADPH (1 mM) and cimetidine hydrochloride (0.05 mM) or d i s t i l l e d water for 0 or 15 min prior to the initation of testosterone oxidation. Results are expressed as the mean ± SEM activity for four individual microsomal samples per group. Panel A: uninduced rats; Panel B: phenobarbital-induced rats. (B) W I T H O U T WITH P R E I N C U B A T I O N P R E I N C U B A T I O N 150 Figure 49 Effect of preincubation of microsomes with cimetidine and NADPH on testosterone 7a-hydroxylase activity. Microsomes were preincubated with NADPH (1 mM) and cimetidine hydrochloride (0.05 mM) or d i s t i l l e d water for 0 or 15 min prior to the initation of testosterone oxidation. Results are expressed as the mean ± SEM activity for four individual microsomal samples per group. Panel A: uninduced rats; and Panel B: phenobarbital-induced rats. ( A ) c 15 D . Ui E \ c E \ o E c >-> I— O < 0.4 0.3 0.2 0.1 0.0 HZ] C O N T R O L E S S C I M E T I D I N E W I T H O U T P R E I N C U B A T I O N WITH P R E I N C U B A T I O N (B) c "o t -Q. D > E \ c *E \ o £ c >-> I— o < 0.8 0.6 0.4 0.2 0.0 L ~ j C O N T R O L E S S C I M E T I D I N E W I T H O U T P R E I N C U B A T I O N WITH P R E I N C U B A T I O N 151 3.4.2.4 T e s t o s t e r o n e 16ct-Hydroxylase A c t i v i t y T e s t o s t e r o n e 16a-hydroxylase a c t i v i t y was 27% lower (p=0.009) i n microsomes (from uninduced r a t s ) p r e i n c u b a t e d w i t h c i m e t i d i n e and NADPH than i n those p r e i n c u b a t e d w i t h NADPH o n l y ( F i g u r e 50A). In c o n t r a s t , w i t h p r e i n c u b a t i o n , c i m e t i d i n e d i d not i n h i b i t t h i s a c t i v i t y i n microsomes from p h e n o b a r b i t a l - i n d u c e d r a t s ( F i g u r e 50B). T h i s d i f f e r e n t i a l e f f e c t o f in vitro c i m e t i d i n e on t e s t o s t e r o n e 16a-hydroxylase a c t i v i t y i n the uninduced and p h e n o b a r b i t a l -induced groups was s i m i l a r t o t h a t observed w i t h in vivo c i m e t i d i n e ( S e c t i o n 3.1.6.4). Without the p r e i n c u b a t i o n s t e p , c i m e t i d i n e d i d not i n h i b i t t e s t o s t e r o n e 16a-hydroxylase a c t i v i t y ( F i g u r e s 50A and 50B). 3.4.2.5 T e s t o s t e r o n e 166-Hydroxylase A c t i v i t y With o r wit h o u t the p r e i n c u b a t i o n s t e p , c i m e t i d i n e d i d not i n h i b i t t he cytochromes P450IIBl/2-mediated t e s t o s t e r o n e 16B-hydroxylase a c t i v i t y i n microsomes from p h e n o b a r b i t a l -induced r a t s ( F i g u r e 51). 3.4.2.6 Androstenedione Formation With o r without the p r e i n c u b a t i o n step, c i m e t i d i n e d i d not i n h i b i t androstenedione formation i n microsomes from uninduced o r p h e n o b a r b i t a l - t r e a t e d r a t s ( F i g u r e s 52A and 52B). 152 Figure 50 Effect of preincubation of microsomes with cimetidine and NADPH on testosterone 16a-hydroxylase activity. Microsomes were preincubated with NADPH (1 mM) and cimetidine hydrochloride (0.05 mM) or d i s t i l l e d water for 0 or 15 min prior to the initation of testosterone oxidation. Results are expressed as the mean ± SEM activity for four individual microsomal samples per group. Panel A: uninduced rats; Panel B: phenobarbital-induced rats. *p < 0.01, compared to the group preincubated with NADPH for the same period of time in the absence of cimetidine. (A) o i_ Q . D ) E \ C "E \ o £ c >-> i— o < 1 -C Z D C O N T R O L E S S C I M E T I D I N E W I T H O U T WITH P R E I N C U B A T I O N P R E I N C U B A T I O N (B) c '(D Q . a> E \ c E \ o E c >-> t— o < UZ3 C O N T R O L E S S C I M E T I D I N E W I T H O U T WITH P R E I N C U B A T I O N P R E I N C U B A T I O N 153 Figure 51 E f f e c t of preincubation of microsomes with cimetidine and NADPH on testosterone 160-hydroxylase a c t i v i t y . Microsomes (from phenobarbital-induced rats) were preincubated with NADPH (1 mM) and cimetidine hydrochloride (0.05 mM) or d i s t i l l e d water for 0 or 15 min p r i o r to the i n i t a t i o n of testosterone oxidation. Results are expressed as the mean ± SEM a c t i v i t y f o r four i n d i v i d u a l microsomal samples per group. .E 'CD ~o i _ CL CD £ c £ \ o E c >-> t— o < CONTROL WITHOUT PREINCUBATION ESS CIMETIDINE WITH PREINCUBATION 154 Figure 52 Effect of preincubation of microsomes with cimetidine and NADPH on androstenedione act i v i t y . Microsomes were preincubated with NADPH (1 mM) and cimetidine hydrochloride (0.05 mM) or d i s t i l l e d water for 0 or 15 min prior to the initation of testosterone oxidation. Results are expressed as the mean ± SEM activity for four individual microsomal samples per group. Panel A: uninduced rats; Panel B: phenobarbital-induced rats. (A) £. 2.0 c 'm ~o i. CL D) E \ C o E c >-> I— < 1.5 1.0 0.5 0.0 WITHOUT PREINCUBATION (ZZ1 CONTROL ESS CIMETIDINE WITH PREINCUBATION (B) c °5 ~o i_ a O ) E \ c '£ \ o E c > I— o < 2 -LZZ1 CONTROL ESS CIMETIDINE WITHOUT WITH PREINCUBATION PREINCUBATION 155 3.5 SUMMARY OF THE EFFECTS OF IN VIVO AND IN VITRO CIMETIDINE ON MICROSOMAL TESTOSTERONE OXIDATION In microsomes from uninduced adult male rats, in vivo cimetidine inhibited testosterone 2a- and 16a-hydroxylase ac t i v i t i e s and androstenedione formation, but not testosterone 20, 60- or 7a-hydroxylase activity (Figure 53A). In vitro cimetidine (5 mM) inhibited a l l of these a c t i v i t i e s , except for testosterone 7a-hydroxylase activity (Figure 53B). With the inclusion of the preincubation step, a low concentration (0.05 mM) of in vitro cimetidine inhibited only testosterone 2a- and 16a-hydroxylase act i v i t i e s (Figure 53C). In microsomes from phenobarbital-induced adult male rats, in vivo cimetidine inhibited testosterone 2a-hydroxylase activity, but not testosterone 20-, 60, 7a-, 16a- or 160-hydroxylase activity or androstenedione formation (Figure 54A). In vitro cimetidine (5 mM) inhibited a l l of these a c t i v i t i e s , except for testosterone 7a-hydroxylase activity (Figure 54B). With the inclusion of the preincubation step, in vitro cimetidine (0.05 mM) inhibited only testosterone 2a-hydroxylase activity (Figure 54C). 156 Figure 53 Summary of the effects of in vivo and in vitro cimetidine on testosterone oxidation in microsomes from uninduced rats. (A) In vivo cimetidine; (B) in vitro cimetidine (5 mM); (C) in vitro cimetidine (0.05 mM) with preincubation. Results are based on the data from Figures 18, 32-38 and 46-52. *p < 0.001, 'p < 0.01, *p < 0.05 compared to the respective control group. In Panel B, st a t i s t i c a l analyses were not performed since the microsomes were prepared from a pool of l i v e r s . (A) 1 0 0 | : : . 8 0 e 60 I z 5 40 o UJ a . 20 0 TESTOSTERONE METABOLITES (B) 1 0 0 2/3 6/3 7 a 16a TESTOSTERONE METABOLITES 50 • (C) KS) WITH PREINCUBATION • WITHOUT PREINCUBATION 3 0 2 0 1 0 I 2a 2(3 6/3 - 7a 16a TESTOSTERONE METABOLITES 157 Figure 54 Summary of the effects of in vivo and in vitro cimetidine on testosterone oxidation in microsomes from phenobarbital-induced rats. (A) In vivo cimetidine; (B) in vitro cimetidine (5 mM); (C) in vitro cimetidine (0.05 mM) with preincubation. Results are based on the data from Figures 18, 3 2-38 and 4 6-52. #p < 0.001, *p < 0.005 compared to the respective control group. In Panel B, s t a t i s t i c a l analyses were not performed since the microsomes were prepared from a pool of l i v e r s . (A) 100 2/3 6/3 7o 16a 16/3 TESTOSTERONE META80LITES (B) 100 20 6(3 7a 16a 16/3 TESTOSTERONE METABOLITES (C) 40 30 10 E 3 WITH PREINCUBATION CT! WITHOUT PREINCUBATION 2a 20 6(3 7a 16a 160 TESTOSTERONE METABOLITES 158 DISCUSSION 4.1 DIFFERENTIAL INHIBITION OF CYTOCHROME P-450-MEDIATED ENZYME ACTIVITIES BY IN VIVO CIMETIDINE 4.1.1 I n h i b i t i o n o f Cytochrome P450IIC11 by In Vivo C i m e t i d i n e O b s e r v a t i o n s from human and animal s t u d i e s have l e d t o the p e r c e p t i o n t h a t c i m e t i d i n e i s a g e n e r a l i n h i b i t o r o f h e p a t i c cytochrome P-450 enzymes ( R e i l l y e t al., 1988; L e c l e r c q e t al., 1989). However, t h e r e i s s u b s t a n t i a l , but i n d i r e c t , evidence t h a t c i m e t i d i n e may d i f f e r e n t i a l l y i n h i b i t h e p a t i c cytochrome P-450 enzymes. An i n i t i a l o b s e r v a t i o n from the prese n t i n v e s t i g a t i o n was t h a t in vivo c i m e t i d i n e i n h i b i t e d aminopyrine N-demethylase a c t i v i t y t o a g r e a t e r e x t e n t i n microsomes from uninduced than i n those from induced a d u l t male r a t s ( F i g u r e s 8 and 9 ) . S l u s h e r et al. (1987) r e p o r t e d t h a t anti-cytochrome P450IA1/2 and a n t i -cytochrome P450IIB1/2 a n t i b o d i e s p a r t i a l l y i n h i b i t e d the f o r m a t i o n o f 4-aminoantipyrine, one o f the N-demethylated m e t a b o l i t e s o f aminopyrine, i n microsomes from a d u l t male r a t s induced w i t h p h e n o b a r b i t a l or 3-methylcholanthrene, but not i n microsomes from uninduced a d u l t male r a t s . T h i s i n d i c a t e s t h a t d i f f e r e n t cytochrome P-450 enzymes are r e s p o n s i b l e f o r aminopyrine N-demethylase a c t i v i t y i n 159 microsomes from uninduced and induced rats. The results shown in Figures 8 and 9, in conjunction with the observations of Slusher et a l . (1987), suggested that at least one of the cytochrome P-450 enzymes present in uninduced rats was more susceptible to inhibition by in vivo cimetidine than those present in induced rats. Therefore, further studies were performed to test the hypothesis that in vivo cimetidine differentially inhibits hepatic cytochrome P-450 enzymes in adult male rats. Cytochrome P450IIC11 is a major hepatic cytochrome P-450 enzyme in uninduced adult male rats (Guengerich et a l . , 1982a; Waxman et a l . , 1985). The results from the present investigation provide the f i r s t evidence that in vivo cimetidine inhibits cytochrome P450IIC11. The evidence for this is derived from the cases in which an enzyme activity has been found to be due entirely to cytochrome P450IIC11. In vivo cimetidine inhibited testosterone 2a-and 16a-hydroxylase activities in microsomes from uninduced rats (Figures 10, 14 and 18). Consistent with published results (Waxman, 1984; Waxman et a l . , 1987), the monospecific anti-cytochrome P450IIC11 completely inhibited testosterone 2a-hydroxylase activity (Figure 22) and inhibited testosterone 16a-hydroxylase activity by 95% (Figure 23) in microsomes from uninduced adult male rats. Since in vivo cimetidine inhibited both of these a c t i v i t i e s by more than 60% in microsomes from uninduced rats (Figures 160 10, 14 and 18), i t is apparent that cytochrome P450IIC11 is subject to inhibition by this drug. The results of antibody inhibition experiments showed that the testosterone 2ct-hydroxylase activity was also entirely due to cytochrome P450IIC11 in microsomes from phenobarbital-induced and dexamethasone-induced rats (Figure 22). This activity was also inhibited by in vivo cimetidine in microsomes from these two pretreatment groups (Figure 10 and 18). In uninduced rats, the antibody was also found to inhibit pentoxyresorufin 0-dealkylase activity by more than 90% (Figure 20), indicating that cytochrome P450IIC11 catalyzed this activity in the absence of induction. In vivo cimetidine inhibited pentoxyresorufin 0-dealkylase activity by 38% in microsomes from uninduced rats (Figure 5). Recently, Nakajima et a l . (1990) reported that an antibody to cytochrome P450IIC11, with cross-reactivity to cytochrome P450IIC6, completely inhibited pentoxyresorufin 0-dealkylase activity in microsomes xfrom uninduced rats. In the present investigation, in each case where cytochrome P450IIC11 was observed to be the major or sole contributor to an enzyme activity, that activity was inhibited by in vivo cimetidine. 161 4.1.2 Lack of Inhibition of Cytochromes P450IIB1/2, Cytochromes P450IIIA1/2 and Cytochrome P450IIA1 by In Vivo Cimetidine Cytochromes P450IIB1/2 are the major cytochrome P-4 50 enzymes inducible by phenobarbital (Guengerich et al., 1982a; Thomas et al., 1983; Waxman et al., 1 9 8 5 ) . Testosterone 166-hydroxylase and pentoxyresorufin 0-dealkylase act i v i t i e s in microsomes from phenobarbital-induced rats are frequently used as markers for cytochromes P450IIB1/2 (Lubet et al., 1985; Reik et al., 1985; Waxman et al., 1985, 1987; Dutton and Parkinson, 1 9 8 9 ) . In vivo cimetidine did not inhibit these two activ i t i e s in microsomes from phenobarbital-induced rats (Figures 5 and 15). If cimetidine is a competitive inhibitor of these two ac t i v i t i e s , then the observed lack of inhibition may have been the result of a relatively high substrate concentration. However, even at lower substrate concentrations, in vivo cimetidine did not affect testosterone 166-hydroxylase activity in microsomes from phenobarbital-induced rats (Figure 16). Alternatively, the apparent lack of inhibition of these two activ i t i e s by cimetidine could be explained by an increase in the clearance of cimetidine from the animals due to the induction of cytochromes P-450 by the phenobarbital pretreatment. This would require, as well, that cimetidine is the active inhibitor and that the inhibition is competitive. However, in the same microsomes in which there 162 was lack of inhibition of testosterone 166-hydroxylase and pentoxyresorufin 0-dealkylase a c t i v i t i e s , in vivo cimetidine inhibited testosterone 2a-hydroxylase activity by 73% (Figures 10 and 18), indicating that the active form of the inhibitor was present in the microsomes. In fact, the extent of inhibition of testosterone 2a-hydroxylase activity by cimetidine in microsomes from phenobarbital-induced rats was similar to that observed in microsomes from uninduced rats (Figure 18). The apparent lack of inhibition of testosterone 16a-hydroxylase activity by in vivo cimetidine in microsomes from phenobarbital-induced rats (Figure 14) i s also consistent with the suggestion that in vivo cimetidine does not inhibit cytochromes P450IIB1/2. It has been shown in immunoinhibition studies that cytochromes P450IIB1/2 account for the majority of the testosterone 16a-hydroxylase activity in microsomes from phenobarbital-induced rats (Thomas et a l . , 1981; Reik et a l . , 1985; Waxman et a l . , 1987). Therefore, the results from the present investigation indicate that in vivo cimetidine administration to adult male rats apparently does not inhibit cytochromes P450IIB1/2. Cytochromes P450IIIA1/2 are the major cytochrome P-450 enzymes inducible by dexamethasone (Heuman et a l . , 1982). Testosterone 26- and 66-hydroxylase acti v i t i e s are markers for cytochromes P450IIIA1/2 in microsomes from uninduced, 163 phenobarbital-induced and dexamethasone-induced rats (Halvorson et al., 1990). Erythromycin N-demethylase activity i s also used as a marker for cytochromes P450IIIA1/2 in microsomes from dexamethasone-induced rats (Wrighton et al., 1985a). In vivo cimetidine did not affect testosterone 20- or 60-hydroxylase activity in microsomes from uninduced, phenobarbital- or dexamethasone-induced rats (Figures 11, 12 and 18). As well, in vivo cimetidine did not inhibit erythromycin N-demethylase activity in microsomes from dexamethasone-induced rats (Figures 6 and 8). However, in the same microsomes from dexamethasone-induced rats, in vivo cimetidine did inhibit testosterone 2a-hydroxylase activity (Figures 10 and 18), and this activity was mediated by cytochrome P450IIC11 (Figure 22). It appears that under the experimental conditions of the present investigation, in vivo cimetidine does not inhibit cytochromes P450IIIA1/2. In a preliminary report, Cooper et al. (1990) claimed that both cytochrome P450IIIA1 and cytochrome P450IIIA2 are present in livers of dexamethasone-treated adult male rats, whereas cytochrome P450IIIA2, but not cytochrome P450IIIA1, is expressed in livers of uninduced adult male rats. Cytochrome P450IIA1 is a female-predominant cytochrome P-450 enzyme (Waxman et al., 1989) and is a minor constituent in livers of uninduced adult male rats (Guengerich et al., 1982a; Waxman et al., 1985). Testosterone 7a-hydroxylase activity is used as a marker for 164 cytochrome P450IIA1 in microsomes from uninduced, phenobarbital-induced and dexamethasone-induced adult male rats (Levin et a l . , 1987; Waxman et a l . , 1988b; Arlotto and Parkinson, 1989). Since in vivo cimetidine did not inhibit this activity in microsomes from these groups of adult male rats (Figures 13 and 18), i t appears that, under the experimental conditions of the present investigation, in vivo cimetidine does not inhibit cytochrome P450IIA1. 4.1.3 Indirect Evidence for the Inhibition of Other Cytochrome P-450 Enzymes by In Vivo Cimetidine There i s evidence from the present investigation that other cytochrome P-450 enzymes in uninduced adult male rats, in addition to cytochrome P450IIC11, are also inhibited by in vivo cimetidine. In vivo cimetidine inhibited erythromycin N-demethylase act i v i t y by 40% in microsomes from uninduced rats (Figures 6 and 8), yet the anti-cytochrome P450IIC11 antibody had l i t t l e or no effect on this activity i n these microsomes (Figure 21). Therefore, the inhibition of erythromycin N-demethylase activity by cimetidine in uninduced rats is l i k e l y to be due to an enzyme(s) other than cytochrome P450IIC11. Based on the observation of a biphasic Eadie-Hofstee plot, i t has been suggested that at least two cytochrome P-450 enzymes are responsible for erythromycin N-demethylase activity in microsomes from uninduced adult male rats (Chang et a l . , 1990), but i t has not been 165 demonstrated in an immunoinhibition experiment which cytochrome P-450 enzyme(s) contribute(s) to this activity in microsomes from this group of rats. However, the enzyme that catalyzes erythromycin N-demethylase in the uninduced state is unlikely to be cytochrome P450IIA1, cytochromes P450IIB1/2 or cytochromes P450IIIA1/2 since in vivo cimetidine did not inhibit the activities that are specific for these enzymes. Aminopyrine N-demethylase activity in microsomes from uninduced rats was inhibited 62% by in vivo cimetidine (Figures 4 and 8). Since cytochrome P450IIC11 accounted for only approximately 35% of the aminopyrine N-demethylase activity in these microsomes (Figure 19), i t i s possible that in vivo cimetidine inhibits another cytochrome P-450 enzyme(s) which contribute(s) to this activity in uninduced rats. 4.1.4 Effect of In Vivo Cimetidine on Cytochrome P450IA1 and Cytochrome P450IA2 Cytochrome P450IA1 is the major cytochrome P-450 enzyme inducible by 6-naphthoflavone and 3-methylcholanthrene (Guengerich et al. , 1982a; Thomas et al. , 1983; Waxman et al., 1985). Purified cytochrome P450IA1 catalyzes ethoxyresorufin O-deethylation (Goldstein et al., 1982; Guengerich et al., 1982a; Astrom and DePierre, 1985). Kelley et al. (1987) reported that an anti-cytochrome P450IA1 antibody inhibited ethoxyresorufin O-deethylase 166 activity in microsomes from 3-methylcholanthrene-induced rats by 82% and an anti-cytochrome P450IA2 antibody inhibited this activity in microsomes from the same group of rats by only 27%. Recently, Nakajima et al. (1990) reported that an antibody to cytochrome P450IA1, with cross-reactivity to cytochrome P450IA2, inhibited ethoxyresorufin O - d e e t h y l a s e a c t i v i t y i n microsomes from 3-methylcholanthrene-induced rats by 79%. However, i t has not been shown in an immunoinhibition experiment whether cytochrome P450IA1 is the major enzyme responsible for ethoxyresorufin O-deethylase activity in microsomes from P-naphthoflavone-induced rats. In the present study, in vivo cimetidine did not inhibit this activity in microsomes from P-naphthoflavone-induced rats (Figures 7 and 9). As an internal control, aminopyrine N-demethylase activity was determined in microsomes from p-naphthoflavone-induced rats. As shown in Figures 4 and 9, in vivo cimetidine did inhibit aminopyrine N-demethylase activity by 28% in these microsomes (Figures 4 and 9), indicating that the inhibitory action of cimetidine was present after p-naphthoflavone pretreatment. If cytochrome P450IA1 is the enzyme responsible for ethoxyresorufin O-deethylase activity in microsomes from p-naphthoflavone-induced rats, then i t would appear that, under the experimental conditions in the present investigation, in vivo cimetidine does not inhibit cytochrome P450IA1. 167 Cytochrome P450IA2 is a major cytochrome P-450 enzyme inducible by isosafrole and i s present in low levels in uninduced rats (Guengerich et al., 1982a; Thomas et al., 1983; Waxman et al., 1985). Kelley et a l . (1987) reported that an anti-cytochrome P450IA2 antibody inhibited ethoxyresorufin O-deethylase activity by 78% in microsomes from uninduced rats. In the present study, this activity was used as a marker for cytochrome P450IA2 in microsomes from uninduced rats. It was found that in vivo cimetidine inhibited ethoxyresorufin O-deethylase activity by 84% in microsomes from this group of rats (Figures 7 and 9). Since the completion of this experiment, Nakajima et al. (1990) reported that an antibody to cytochrome P450IIC11, with cross-reactivity to cytochrome P450IIC6, inhibited ethoxyresorufin O-deethylase activity by 74% in microsomes from uninduced rats. It i s therefore uncertain whether ethoxyresorufin O-deethylase activity i s a reliable marker for cytochrome P450IA2 in microsomes from uninduced rats. It i s possible that both cytochrome P450IA2 and cytochrome P450IIC11 catalyze ethoxyresorufin 0-deethylation in microsomes from uninduced rats. Future experiments should be performed to c l a r i f y the enzyme-specificity of ethoxyresorufin O-deethylase activity in microsomes from uninduced rats and the effect of in vivo cimetidine on cytochrome P450IA2. 168 4.1.5 Effect of Pretreatment on Inhibition of Cytochrome P-450 by Cimetidine In some cases, the extent of inhibition of enzyme act i v i t i e s by in vivo cimetidine depended on prior treatment with a cytochrome P-450 inducer. This appeared to be due to the increasing contribution to such activities by inducible enzymes which were not subject to inhibition by cimetidine. In vivo cimetidine inhibited testosterone 16a-hydroxylase ac t i v i t y in microsomes from uninduced rats, but did not affect this activity in microsomes from phenobarbital-induced rats (Figure 14). The anti-cytochrome P450IIC11 antibody inhibited testosterone 16a-hydroxylase activity i n microsomes from uninduced rats, but had l i t t l e or no effect on this activity in microsomes from phenobarbital-induced rats (Figure 23), consistent with published data (Waxman, 1984; Waxman et a l . , 1987). Cytochromes P450IIB1/2 are the major contributors to testosterone 16a-hydroxylase activity in hepatic microsomes from phenobarbital-induced rats (Thomas et a l . 1981; Reik et a l . , 1985; Waxman et a l . , 1987), but does not contribute to this activity in microsome from uninduced rats (Reik et al., 1985). In vivo cimetidine inhibited pentoxyresorufin O-dealkylase activity i n microsomes from uninduced rats, but did not affect this ac t i v i t y in microsomes from phenobarbital-induced rats (Figure 5). Similarly, the anti-cytochrome P450IIC11 antibody inhibited pentoxyresorufin O-dealkylase activity i n 169 microsomes from uninduced rats, but had l i t t l e or no effect on this activity in microsomes from phenobarbital-induced rats (Figure 20). Cytochromes P450IIB1/2 account for more than 90% of the pentoxyresorufin 0-dealkylase activity in hepatic microsomes from phenobarbital-induced rats (Lubet et a l . , 1985; Waxman et a l . , 1987; Dutton and Parkinson, 1989), but does not contribute to this activity in microsomes from uninduced rats (Waxman et al., 1987). In vivo cimetidine inhibited androstenedione formation in microsomes from uninduced rats, but did not affect this reaction in microsomes from phenobarbital-induced rats (Figure 17). The anti-cytochrome P450IIC11 antibody partially inhibited this reaction in microsomes from uninduced rats, but had l i t t l e or no effect on this reaction in microsomes from phenobarbital-induced rats (Figure 24). Cytochromes P450IIB1/2 account for 60-70% of the formation of androstenedione in hepatic microsomes from phenobarbital-induced rats, but doe^ s/ not contribute to the formation of this metabolite in microsomes from uninduced rats (Reik et al., 1985). In vivo cimetidine inhibited aminopyrine N-demethylase activity to a greater extent in microsomes from uninduced than those from phenobarbital-induced rats (Figure 4). The anti-cytochrome P450IIC11 antibody pa r t i a l l y inhibited this activity in microsomes from uninduced rats, but had l i t t l e or no effect on this activity in microsomes from phenobarbital-induced rats (Figure 19). 170 4.1.6 A P o s s i b l e E f f e c t of S u b s t r a t e on the I n h i b i t i o n of Cytochrome P-450-Mediated Enzyme A c t i v i t i e s by C i m e t i d i n e There has been no s y s t e m a t i c i n v e s t i g a t i o n o f the i n h i b i t o r y e f f e c t of c i m e t i d i n e on s p e c i f i c cytochrome P-450 enzymes. Only one previous study has examined the e f f e c t o f a s i n g l e dose o f c i m e t i d i n e on h e p a t i c microsomal cytochrome P-450-mediated enzyme a c t i v i t i e s i n r a t s (Drew e t al., 1981). In t h a t study, a d u l t male r a t s , f o u r per group, were uninduced or p r e t r e a t e d w i t h e i t h e r p h e n o b a r b i t a l o r 3-methylcholanthrene and s a c r i f i c e d 2 h f o l l o w i n g a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n o f c i m e t i d i n e (150 mg/kg). S e v e r a l h e p a t i c microsomal enzyme a c t i v i t i e s were determined, i n c l u d i n g benzo[a]pyrene h y d r o x y l a s e and 7-ethoxycoumarin O-deethylase a c t i v i t i e s . A c c o r d i n g to t h e s e i n v e s t i g a t o r s , c i m e t i d i n e i n h i b i t e d benzo[a]pyrene h y d r o x y l a s e a c t i v i t y by 89%, but had no e f f e c t on 7-ethoxycoumarin O-deethylase a c t i v i t y o r s e v e r a l n o n - s p e c i f i c enzyme a c t i v i t i e s , i n microsomes from each o f the t h r e e pretreatment groups. The r e s u l t s from the study o f Drew e t al. (1981) are d i f f i c u l t t o i n t e r p r e t . I t has been shown i n i m m u n o i n h i b i t i o n experiments t h a t cytochrome P450IA1 accounts f o r more than 80% o f the benzo[a]pyrene hydroxylase a c t i v i t y i n microsomes from r a t s induced w i t h 3-methylcholanthrene (Ryan et al., 1982b). T h i s would l e a d one t o conclude t h a t c i m e t i d i n e i n h i b i t s the c a t a l y t i c 171 function of cytochrome P450IA1. However, the inhibition of benzo[a]pyrene hydroxylase activity by cimetidine i s inconsistent with the lack of effect of cimetidine on 7-ethoxycoumarin O-deethylase activity. In microsomes from the 3-methylcholanthrene-induced rats, cytochrome P450IA1 accounts for 60-70% of the 7-ethoxycoumarin O-deethylase activity (Park et a l . , 1982; Hietanen et a l . , 1987). Therefore, i f cimetidine inhibits cytochrome P450IA1, inhibition of 7-ethoxycoumarin O-deethylase activity by cimetidine should have been observed in the study conducted by Drew et a l . (1981). As well, i f cytochrome P450IA1 is the enzyme responsible for ethoxyresorufin O-deethylase activity in microsomes from p-naphthoflavone-induced rats, then inhibition of ethoxyresorufin O-deethylase activity by in vivo cimetidine should have been observed in microsomes from p-naphthoflavone-induced rats (Figures 7 and 9). The apparent discrepancy in the effect of in vivo cimetidine on the microsomal metabolism of benzo[a]pyrene, ethoxyresorufin and 7-ethoxycoumarin may be substrate-related. It has been proposed that two substrate-binding sites exist on cytochrome P450IA1 (Phillipson et a l . , 1982; Kao and Wilkinson, 1987) and that benzo[a]pyrene and ethoxyresorufin occupy different binding sites on the enzyme (Kao and Wilkinson, 1987). One could then postulate that cimetidine inhibits the site used by benzo[a]pyrene, but does not inhibit the one used by ethoxyresorufin or 7-ethoxycoumarin. 172 Competition for substrate-binding sites leads to alternate substrate inhibition. However, the inhibition of benzo[a]pyrene hydroxylase activity by in vivo cimetidine i s unlikely to be competitive. Due to the short elimination h a l f - l i f e (30-45 min) of cimetidine in rats, the concentration of cimetidine in the l i v e r at 2 h after a single dose of the drug, as well as the concentration of cimetidine in isolated microsomes, i s l i k e l y to be very much lower than the concentration of the substrate (83 uM) used in the assay. Furthermore, as shown in Figure 31, cimetidine, when administered in vitro, did inhibit ethoxyresorufin O-deethylase activity in microsomes from P-naphthoflavone-induced rats. Future studies are required to re-examine the effect of in vivo cimetidine on cytochrome P450IA1-mediated substrate oxidation. The inconsistency in the effect of in vivo cimetidine on the microsomal metabolism of benzo[a]pyrene, 7-ethoxycoumarin and ethoxyresorufin indicates that the inhibitory effect of cimetidine may be both enzyme- and substrate-related. 4.2 INHIBITION OF CYTOCHROME P-450-MEDIATED ENZYME ACTIVITIES BY IN VITRO CIMETIDINE The results from the studies in the present investigation with in vivo cimetidine indicate that differential inhibition of hepatic microsomal cytochrome P-450-mediated enzyme act i v i t i e s occurs following a single intraperitoneal injection of the drug to adult male rats. 173 It has been shown by other investigators that cimetidine, when added to rat hepatic microsomes in vitro, inhibits numerous enzymes activities (Table 4), many of which are not enzyme-specific. Since the concentration of cimetidine required for the in vitro inhibition of a microsomal cytochrome P-450-mediated enzyme activity i s typically 100-1000 times higher than the serum concentration associated with inhibition of hepatic drug metabolism in vivo (Somoygi and Muirhead, 1987), i t is apparent that the inhibition of cytochrome P-450 by in vitro cimetidine i s not necessarily equivalent to that found following the in vivo administration of the drug to intact animals. Experiments were performed to determine whether in vitro cimetidine would di f f e r e n t i a l l y inhibit hepatic microsomal cytochrome P-450-mediated enzyme ac t i v i t i e s , in a manner similar to that observed following the in vivo administration of the drug to rats. Enzyme kinetic experiments were also performed to compare the type of inhibition of the cytochrome P450lICll-mediated testosterone 2a-hydroxylase activity by in vitro and in vivo cimetidine. Except for testosterone 7a-hydroxylase activity, in vitro cimetidine inhibited a l l the enzyme activities examined and the IC 5 0 values were in the low millimolar range (Figures 29-38, Tables 18 and 19). The results obtained with the enzyme-specific a c t i v i t i e s indicate that in vitro cimetidine inhibits the catalytic function of 174 cytochromes P450IIB1/2, cytochrome P450IIC11 and cytochromes P450IIIA1/2, but not cytochrome P450IIA1. Thus, the pattern of the d i f f e r e n t i a l effect of in vivo cimetidine on cytochrome P-450-mediated enzyme activities was not observed with the in vitro administration of cimetidine. In vitro cimetidine inhibited the cytochrome P450IIC11-mediated testosterone 2cc-hydroxylase activity in microsomes from uninduced and phenobarbital-induced rats (Figure 32), consistent with the inhibition of this activity by in vivo cimetidine (Figures 10 and 18). Thus, cimetidine inhibits cytochrome P450IIC11 whether administered in vitro or in vivo. However, the observed inhibition of cytochrome P450IIC11 by in vitro and in vivo cimetidine may not result from the same inhibitory mechanism. The reason for this is that the IC S 0 values of the inhibition of the cytochrome P450lICll-mediated testosterone 2a-hydroxylase activity by in vitro cimetidine in microsomes from uninduced and phenobarbital-induced rats were in the low millimolar range (Table 19), whereas the serum cimetidine concentration in the rat at 90 min after a single intraperitoneal dose of 150 mg/kg of cimetidine is in the low micromolar range (Reichen et al., 1986). To further explore the inhibitory effects of in vitro and in vivo cimetidine on cytochrome P450IIC11, enzyme kinetic experiments were performed with testosterone 2a-hydroxylase. Based on Lineweaver-Burk plots of the data, the inhibition of testosterone 2a-hydroxylase 175 was competitive by in vitro cimetidine (Figure 39), but non-competitive by in vivo cimetidine (Figure 40), indicating that different mechanisms of inhibition are involved. Since i t has been shown that cimetidine is metabolized to a small extent by hepatic microsomes (Zbaida et al., 1984), there could be competition between cimetidine and testosterone for the substrate-binding site(s) on cytochrome P450IIC11. It is therefore possible that the observed competitive inhibition of testosterone 2ct-hydroxylase by in vitro cimetidine reflects alternate substrate inhibition. However, with in vivo cimetidine, competitive inhibition of testosterone 2ct-hydroxylase was not observed. This is not surprising for two reasons. F i r s t , the concentration of cimetidine in the hepatocytes at 90 min after a single dose of the drug is l i k e l y to be very much lower than the in vitro concentration required for inhibition to occur. Second, the concentration of the freely diffusible cimetidine i s further diluted during the preparation of the microsomes. The fact that the inhibition of testosterone 2ct-hydroxylase by in vivo cimetidine was non-competitive provides further evidence that different mechanisms are involved in the inhibition of cytochrome P-450 by in vitro and in vivo cimetidine. Jn vitro cimetidine, at concentrations of up to 10 mM, did not affect the cytochrome P450IIAl-mediated testosterone 7a-hydroxylase activity in microsomes from uninduced and 176 phenobarbital-induced adult male rats (Figure 35). The lack of inhibition may be the result of a very low a f f i n i t y of cimetidine for cytochrome P450IIA1. If so, at suff i c i e n t l y high concentrations, cimetidine should inhibit this enzyme. Alternatively, cimetidine may not be able to bind to the substrate-binding site(s) on cytochrome P450IIA1. This would then suggest that the substrate-binding site(s) on cytochrome P450IIA1 are somehow different from those on the enzymes that are inhibited by in vitro cimetidine. In summary, the specific pattern of the dif f e r e n t i a l effect of in vivo cimetidine on cytochrome P-450-mediated enzyme activ i t i e s does not occur when cimetidine i s administered in vitro. 4.3 EFFECT OF PREINCUBATION ON THE INHIBITION OF CYTOCHROME P-450-MEDIATED ENZYME ACTIVITIES BY LOW CONCENTRATIONS OF CIMETIDINE If cimetidine inhibits cytochrome P-450-mediated hepatic drug metabolism in vivo by a non-competitive mechanism, as suggested by the result shown in Figure 40, then this would explain why relatively low concentrations of cimetidine in man and in rats can inhibit drug clearance. However, i f cimetidine inhibits cytochrome P-450 only by a non-competitive mechanism, then i t should be a more potent inhibitor of cytochrome P-450-mediated enzyme act i v i t i e s in vitro. In fact, the inhibition of testosterone 2a-hydroxylase and several other cytochrome P-450-mediated 177 enzyme a c t i v i t i e s by in vitro cimetidine i s competitive (Figure 3 9 and Table 4). Thus, i t seems that more than one mechanism i s involved i n the i n h i b i t i o n of cytochrome P-4 5 0 enzymes by cimetidine. It i s well-established that an i n h i b i t o r of cytochrome P-450 can act by more than one mechanism. For example, SKF 525-A i n h i b i t s cytochrome P-450 by alternate substrate i n h i b i t i o n and metabolite-intermediate complexation (Schenkman et a l . , 1972; Buening and Franklin, 1976) and chloramphenicol i n h i b i t s cytochrome P-4 50 by alternate substrate i n h i b i t i o n and covalent binding to the apoprotein of cytochrome P-450 (Grogan et al., 1972; R e i l l y and Ivey, 1979; Halpert, 1981). The involvement of d i f f e r e n t mechanisms i n the i n h i b i t i o n of cytochrome P-4 50 by in vivo and in vitro cimetidine may explain the discrepancy i n the s e l e c t i v i t y of the i n h i b i t i o n by i n vivo and in vitro cimetidine. To gain further i n s i g h t into the i n h i b i t i o n of cytochrome P-450 by in vitro cimetidine, experiments were performed to determine whether the s e l e c t i v e i n h i b i t i o n of microsomal testosterone oxidation by in vivo cimetidine can be observed following the preincubation of hepatic microsomes with low concentrations of cimetidine in vitro p r i o r to the i n i t i a t i o n of substrate oxidation. In microsomes from uninduced r a t s , preincubation with 0.05 mM cimetidine and 1 mM NADPH for 15 min resulted i n a decrease i n testosterone 2a- and 16a-hydroxylase a c t i v i t i e s , 178 but did not af f e c t testosterone 26-, 66- or 7a-hydroxylase a c t i v i t y or androstenedione formation, compared to the microsomes preincubated with NADPH for 15 min i n the absence of cimetidine (Figures 4 6-52 and 53C). This pattern of i n h i b i t i o n was s i m i l a r to that observed with in vivo cimetidine (Figure 53A), except for the lack of i n h i b i t i o n of androstenedione formation. With the preincubation of microsomes with cimetidine and NADPH, androstenedione formation was decreased by 17% compared to the microsomes preincubated with NADPH only (Figures 52 and 53C). However, t h i s decrease was not s t a t i s t i c a l l y s i g n i f i c a n t (p = 0.079). A s t a t i s t i c a l l y s i g n i f i c a n t decrease might well be observed with a larger sample s i z e since cytochrome P450IIC11 accounted f o r approximately 60% of the androstenedione formation i n microsomes from uninduced rats (Figure 24). In the absence of preincubation, in vitro cimetidine, at t h i s low concentration (0.05 mM), did not i n h i b i t any of the testosterone oxidase a c t i v i t i e s (Figures 46-52). In microsomes from phenobarbital-induced r a t s , preincubation with 0.05 mM cimetidine and 1 mM NADPH for 15 min resulted i n a decrease i n testosterone 2a-hydroxylase a c t i v i t y , but did not a f f e c t testosterone 26-, 66-, 7a-, 16a- or 166-hydroxylase a c t i v i t y or androstenedione formation, compared to the microsomes preincubated with NADPH i n the absence of cimetidine f o r the same length of time (Figures 46-52 and 54C). Once again, t h i s pattern of i n h i b i t i o n was 179 s i m i l a r to that observed with in vivo cimetidine (Figure 54A) . In the absence of preincubation, i n vitro cimetidine at t h i s low concentration (0.05 mM), did not i n h i b i t any of the a c t i v i t i e s (Figures 46-52). Thus, with preincubation, in vitro cimetidine (0.05 mM) i n h i b i t s cytochrome P450IIC11, but not cytochrome P450IIA1, cytochromes P450IIB1/2 or cytochromes P450IIIA1/2. These re s u l t s are consistent with the pattern of i n h i b i t i o n obtained with in vivo cimetidine. Several important points are evident from the preincubation studies with in vitro cimetidine. F i r s t , the d i f f e r e n t i a l i n h i b i t i o n of microsomal testosterone oxidation by cimetidine was observed only with r e l a t i v e l y low concentrations (0.05 mM or less) of cimetidine (Figure 41). At higher concentrations, the d i f f e r e n t i a l e f f e c t was not apparent. This i l l u s t r a t e s the importance of performing the i n i t i a l experiment to determine the concentration-response r e l a t i o n s h i p (Figure 41). Second, the i n h i b i t i o n of testosterone 2a-hydroxylase a c t i v i t y by in vitro cimetidine (0.05 mM) required the presence of NADPH i n the preincubation medium (Figure 43). When NADPH was absent from the preincubation medium but added just p r i o r to the i n i t i a t i o n of substrate oxidation, i n h i b i t i o n of testosterone 2a-hydroxylase a c t i v i t y at the low cimetidine concentration (0.05 mM) was not observed (Figure 43). The requirement f o r NADPH i n the preincubation medium suggests that the observed i n h i b i t i o n i s the r e s u l t of a c a t a l y s i s -180 dependent process and not simply due to a time-dependent binding of cimetidine to an enzyme. Third, the preincubation of microsomes with only NADPH ( i n the absence of cimetidine) f o r 15 min followed by the usual 5 min of substrate oxidation did not re s u l t i n a substantial reduction i n any of the enzyme a c t i v i t i e s (Figure 42). For t h i s reason, a d d i t i o n a l NADPH was not added just p r i o r to the i n i t i a t i o n of substrate oxidation. I t could be argued that the observed decrease i n the cytochrome P450IIC11-mediated testosterone 2oc-hydroxylase a c t i v i t y i n microsomes from uninduced and phenobarbital-induced rats and testosterone 16a-hydroxylase a c t i v i t y i n microsomes from uninduced rats (Figures 46 and 50) was due to competition f o r the av a i l a b l e NADPH among the enzymes that metabolize testosterone and possibly those that metabolize cimetidine. Cimetidine i s metabolized, although to a small extent, by rat hepatic microsomes (Zbaida et a l . , 1984). However, a saturating concentration of NADPH was used i n the assay. Furthermore, i f NADPH does become r a t e - l i m i t i n g , then one would l i k e l y observe a decrease i n a l l the a c t i v i t i e s and th i s d i d not occur i n the present experiments. The e f f e c t of preincubation of hepatic microsomes with cimetidine and NADPH on cytochrome P-450 enzyme a c t i v i t i e s was investigated i n three previous studies (Jensen and Gugler, 1985; Ioannoni et a l . , 1986; Rekka et a l . , 1988). Jensen and Gugler (1985) determined the e f f e c t of 181 preincubation on the inhibition of 7-ethoxycoumarin O-deethylase activity by cimetidine in microsomes from uninduced adult male rats. In a time-course experiment, these investigators demonstrated an increase in the inhibition of this enzyme activity in microsomes preincubated for 10 min with cimetidine (0.25 mM) and a NADPH-generating system. A 20 min preincubation period did not result in any further increase in the extent of inhibition. The authors hypothesized that either cytochrome P-450 or cimetidine is activated during the preincubation of microsomes, resulting in ligand binding between cimetidine and cytochrome P-450 and the observed inhibition of the enzyme activity. However, the result from this experiment is d i f f i c u l t to interpret due to the lack of an appropriate control group. Since the samples from the "control" group in the study by Jensen and Gugler (1985) were not preincubated, the observed decrease in ethoxycoumarin O-deethylase activity after preincubation may have been due to a breakdown of heme from the holoenzyme as a result of l i p i d peroxidation in the presence of NADPH (Levin et a l . , 1973). In the present investigation, the decrease in the enzyme activities after preincubation was not due to an effect of NADPH on the holoenzyme since in the control group, the microsomes were preincubated with NADPH (Figures 46 and 50). Ioannoni et a l . (1986) examined the effect of preincubation on the inhibition of morphine N-demethylase 182 a c t i v i t y by cimetidine with the purpose of determining whether cimetidine, or a metabolite, causes i r r e v e r s i b l e modification of substrate-binding s i t e s on cytochrome P-450. Microsomes from adult male rats were preincubated with or without 0.5 mM cimetidine i n the presence and absence of a NADPH-generating system for up to 15 min p r i o r to the i n i t i a t i o n of substrate oxidation. Morphine N-demethylase a c t i v i t y was decreased when the microsomes were preincubated with both cimetidine and the NADPH-generating system. However, a s i m i l a r decrease i n t h i s a c t i v i t y occurred when the microsomes were preincubated with the NADPH-generating system i n the absence of cimetidine. Therefore, under t h e i r conditions, preincubation did not enhance the i n h i b i t i o n of morphine N-demethylase a c t i v i t y by cimetidine. While Rekka et a l . (1988) claimed that preincubation had no e f f e c t on the i n h i b i t i o n of the microsomal oxidation of ethoxyresorufin or tofenacine by cimetidine, t h e i r conclusion i s questionable since the experimental protocol was not explained i n s u f f i c i e n t d e t a i l and the data were not shown. As shown i n Figures 53C and 54C, preincubation af f e c t e d the enzyme a c t i v i t i e s s p e c i f i c f o r cytochrome P450IIC11, but not those s p e c i f i c f o r cytochrome P450IIA1, cytochromes P450IIB1/2 and cytochromes P450IIIA1/2. I t i s therefore possible that only c e r t a i n cytochrome P-450 enzymes form a complex with a metabolite of cimetidine. 183 Jensen and Gugler (1985) reported that in rats treated with multiple doses of cimetidine (75 mg/kg intraperitoneally, four times daily for four days), there was a decrease in both the total cytochrome P-450 content and 7-ethoxycoumarin O-deethylase activity compared to saline-treated rats. However, these decreases were not apparent after washing the microsomes from the cimetidine-treated rats with potassium ferricyanide. It has been shown with nitrogen-containing metabolite-intermediate forming agents such as SKF 525-A and triacetyloleandomycin that the oxidation of the ferrous heme iron by potassium ferricyanide dissociates the metabolite-intermediate complex, rendering the enzyme active again (Franklin, 1977; Mansuy, 1987). However, Jensen and Gugler (1985) did not monitor the time-dependent formation of a spectral peak in microsomes incubated with cimetidine and NADPH, which would be indicative of the formation of a metabolite-intermediate complex. Although cimetidine is c l a s s i f i e d as a compound that inhibits cytochrome P-450 by metabolite-intermediate complexation in two books (Gibson and Skett, 1986; Alvares and Pratt, 1990), there i s as yet no definitive evidence that cimetidine, either in vivo or in vitro, forms a metabolite-intermediate complex with cytochrome P-450 enzymes. Further studies are needed to elucidate the mechanisms of inhibition of hepatic cytochrome P-450 enzymes by in vivo and in vitro cimetidine. 184 IMPLICATIONS A novel finding from the present i n v e s t i g a t i o n i s that, i n vivo, cimetidine i s an e f f i c a c i o u s i n h i b i t o r of rat hepatic cytochrome P450IIC11, but that i t does not i n h i b i t several inducible cytochrome P-450 enzymes. When microsomes are preincubated with cimetidine and NADPH, the pattern of i n h i b i t i o n i s the same as that observed following the i n vivo administration of cimetidine. Investigators w i l l be able to use t h i s drug as an experimental agent i n the f i e l d of cytochrome P-4 50 research. For example, to f a c i l i t a t e the understanding of the mechanism of the bi o a c t i v a t i o n of a drug, i t may be necessary to i n h i b i t cytochrome P450IIC11. Cimetidine can be used for t h i s purpose. As well, analogs of cimetidine could be designed and synthesized with the ultimate goal of creating a s p e c i f i c i n h i b i t o r ; that i s , a compound which i n h i b i t s only one cytochrome P-450 enzyme. Such an agent would be useful i n i d e n t i f y i n g the p a r t i c u l a r enzyme involved i n the oxidation of a given drug or substrate. I t would also be p a r t i c u l a r l y valuable i n studies i n which the aim i s to examine the function of a p a r t i c u l a r cytochrome P-450 enzyme i n the metabolism of a given drug i n an i n t a c t animal. The mechanisms of i n h i b i t i o n of cytochrome P-450 by cimetidine are not f u l l y understood. Up to now, the i n h i b i t o r y action of cimetidine has been studied with 185 i s o l a t e d microsomes and with substrates that are not enzyme-s p e c i f i c . In most of the i n vitro microsomal studies with c i m e t i d i n e / the compound has been added immediately p r i o r to the i n i t i a t i o n of substrate oxidation. In the absence of a preincubation step, any i n h i b i t i o n that i s due to a catalysis-dependent process may not be able to occur and most of the observed i n h i b i t i o n may be due to another mechanism that i s less important in vivo. With the finding that a p a r t i c u l a r cytochrome P-450 enzyme ( i . e . cytochrome P450IIC11) i s i n h i b i t e d by cimetidine, investigators may now be able to use an enzyme-specific substrate and the p u r i f i e d enzyme to elucidate the mechanisms of i n h i b i t i o n by cimetidine. Since cimetidine d i f f e r e n t i a l l y i n h i b i t s r a t hepatic cytochrome P-450 enzymes, i t i s also possible that human hepatic cytochrome P-450 enzymes have d i f f e r e n t s u s c e p t i b i l i t i e s to i n h i b i t i o n by t h i s drug. Once the human hepatic cytochrome P-450 enzymes i n h i b i t e d by cimetidine are documented, t h i s compound may also be used as a probe for these enzymes i n c l i n i c a l drug metabolism studies. In addition, i f preincubation of microsomes with cimetidine and NADPH in vitro can be shown to model the i n h i b i t i o n observed in vivo, then s i m i l a r studies with human hepatic microsomes may make metabolic drug-drug interactions more predictable. 186 FUTURE STUDIES 6.1 STUDIES WITH RAT HEPATIC MICROSOMES There i s no d e f i n i t i v e evidence that cimetidine, e i t h e r in vivo or in vitro, forms a metabolite-intermediate complex with cytochrome P-450 enzymes. To determine whether cimetidine i n h i b i t s r at hepatic cytochrome P-450 by metabolite-intermediate complexation, the i n i t i a l experiment would be to incubate microsomes (from uninduced adult male rats) with cimetidine and NADPH and to determine whether there i s time-dependent formation of a s p e c t r a l peak at approximately 448-456 nm, as occurs with other compounds that form metabolite-intermediate complexes (Pershing and Fr a n k l i n , 1982). If the r e s u l t from t h i s experiment i s p o s i t i v e , then one should proceed to determine whether d i s s o c i a t i o n or displacement of the metabolite-intermediate-cytochrome P-450 complex with a compound such as potassium f e r r i c y a n i d e or a displacer would render the enzyme c a t a l y t i c a l l y active again (see Introduction, Section 1.1.5.2). For t h i s purpose, testosterone 2a-hydroxylase a c t i v i t y can be used since the c a t a l y t i c function of cytochrome P450IIC11 has been shown to be i n h i b i t e d by cimetidine. In t h i s experiment, cimetidine may be administered e i t h e r i n vivo or in vitro. I f cimetidine i s given as a sing l e dose in vivo, the i s o l a t e d microsomes 187 should be washed with potassium ferricyanide (or a displacer) prior to conducting the enzyme assay. If cimetidine is added in vitro, then i t s concentration and the time-course of the reaction must be optimized and a preincubation step incorporated to allow for the formation of the metabolite-intermediate-enzyme complex. Potassium ferricyanide (or a displacer) would then be added to the preincubation mixture for an appropriate period of time prior to the i n i t i a t i o n of substrate oxidation. If the inhibition of testosterone 2a-hydroxylase activity by cimetidine i s due to metabolite-intermediate complexation, then this activity should be higher in the potassium ferricyanide-treated group when compared to the appropriate control group. A working hypothesis would be that rat hepatic cytochrome P450IIC11, but not cytochrome P450IIA1, cytochromes P450IIB1/2 or cytochromes P450IIIA1/2, forms a complex with a metabolite of cimetidine. If the results from the above experiments are positive, then a follow-up study would be to determine the chemical basis for the formation of the metabolite-intermediate-enzyme complex. Cimetidine has an amine group in i t s side chain. Other compounds with an amine group such as amphetamine and SKF 525-A have been shown to inhibit cytochrome P-450 by metabolite-intermediate complexatibn (Franklin, 1977). In these cases, the metabolite that complexes the enzyme is the nitrosoalkane derivative, which 188 i s formed by demethylation of the amine group (Mansuy, 1987). Therefore, an i n i t i a l experiment would be to determine whether the N-desmethylcimetidine metabolite has an i n h i b i t o r y e f f e c t on cytochrome P-450; more s p e c i f i c a l l y , the cytochrome P 4 5 0 I I C l l - m e d i a t e d t e s t o s t e r o n e 2a-hydroxylase a c t i v i t y . Induction, suppression and i n h i b i t i o n of cytochrome P-450 enzymes can a l l occur following the chronic administration of a xenobiotic to r a t s . Cytochromes P450IA1/2 and cytochromes P450IIB1/2 are modestly induced i n rats i n j e c t e d with multiple doses of cimetidine over several days (Ioannides et al., 1989). I t i s s t i l l not known whether suppression of cytochrome P-450 contributes to the decrease i n an enzyme a c t i v i t y following chronic cimetidine administration to rats or to man. The e f f e c t of the chronic administration of cimetidine to rats on cytochrome P-450 can be explored further. For example, i n rats treated with cimetidine f o r several days and s a c r i f i c e d 24 h a f t e r the l a s t dose, i s there a decrease i n an enzyme a c t i v i t y such as testosterone 2a-hydroxylase a c t i v i t y ? I f so, i s i t due to suppression and/pr i n h i b i t i o n of cytochrome P450IIC11? To determine whether a suppressive e f f e c t e x i s t s , the l e v e l of hepatic cytochrome P450IIC11 can be determined by immunoquantitation and compared to that from saline-treated c o n t r o l r a t s . To determine whether there i s i n h i b i t i o n of cytochrome P-450 as a r e s u l t of metabolite-intermediate 189 complexation, the microsomes from the cimetidine-treated rats should be washed with potassium f e r r i c y a n i d e and testosterone 2a-hydroxylase a c t i v i t y determined. I f the a c t i v i t y i s higher i n the potassium ferricyanide-washed microsomes compared to the appropriate control group, then t h i s would be an i n d i c a t i o n that an i n h i b i t o r y e f f e c t also e x i s t s . An e a r l i e r study has indicated that cytochrome P450IA2 contributes to most of the ethoxyresorufin O-deethylase a c t i v i t y i n microsomes from uninduced rats (Kelley et al., 1987). Recently, i t has been shown that cytochrome P450IIC11 also contributes to t h i s a c t i v i t y i n microsomes from uninduced rats (Nakajima et a l . , 1990). Therefore, i t i s uncertain whether the i n h i b i t i o n of ethoxyresorufin O-deethylase a c t i v i t y by cimetidine i s due to i n h i b i t i o n of the c a t a l y t i c function of cytochrome P450IA2 or cytochrome P450IIC11 or both. The h i g h - a f f i n i t y phenacetin 0-deethylase a c t i v i t y i n microsomes from uninduced, 3-methyl-cholanthrene-induced or isosafrole-induced rats i s s p e c i f i c f o r cytochrome P450IA2 (Sesardic et a l . , 1990b). Therefore, to examine the e f f e c t of cimetidine on cytochrome P450IA2, one can determine whether the drug i n h i b i t s phenacetin O-deethylase a c t i v i t y i n microsomes from uninduced and isosafrole-induced r a t s . The demonstration that only c e r t a i n hepatic cytochrome P-450 enzymes i n rats are i n h i b i t e d by cimetidine w i l l allow 190 investigators i n the f i e l d of cytochrome P-450 to use t h i s drug to study the c a t a l y t i c function of these enzymes. Analogs of cimetidine could be synthesized as a means to develop s p e c i f i c i n h i b i t o r s of cytochrome P-450 enzymes. As well, i n conjunction with s i t e - d i r e c t e d mutagenesis, one could use cimetidine to study the regions of the r a t hepatic cytochrome P450IIC11 that are c r i t i c a l for enzyme i n h i b i t i o n . The p o t e n t i a l i n h i b i t o r y e f f e c t of a compound on cytochrome P-450 enzymes i s often "screened" i n i n vitro microsomal experiments, using a substrate known to be catalyzed by these enzymes. The experimental conditions of the assays are such that any observable i n h i b i t i o n of the enzyme a c t i v i t y by the t e s t compound i s often associated only with a competitive mechanism of i n h i b i t i o n . As demonstrated i n the present i n v e s t i g a t i o n , the observed i n h i b i t i o n of an enzyme a c t i v i t y by the addition of an i n h i b i t o r to microsomes in vitro does not necessarily r e f l e c t that which occurs following the in vivo administration of the compound to an i n t a c t animal. Therefore, for i n h i b i t o r s which appear more potent in vivo than they do in vitro, i t may be necessary to re-examine t h e i r i n h i b i t o r y e f f e c t s i n an attempt to elucidate t h e i r mechanisms of i n h i b i t i o n in vivo. Results from the present in v e s t i g a t i o n indicate that cytochrome P450IIC11 i s the major or sole c a t a l y s t involved 191 i n the oxidative metabolic pathway of c e r t a i n substrates; fo r example, pentoxyresorufin O-dealkylation i n microsomes from uninduced adult male r a t s . Cytochrome P450IIC11 i s not expressed i n the l i v e r s of female rats (Kamataki et a l . , 1985; Waxman et a l . , 1985). Yet, such substrates are oxidized by hepatic cytochrome P-450 enzymes i n uninduced female r a t s . A question to be answered i s which enzymes i n l i v e r s of female rats are responsible f o r the oxidation of these substrates. Cytochrome P450IIC11 i s developmentally regulated. Its l e v e l i n l i v e r s of male rats i s low p r i o r to puberty, increases a f t e r puberty and becomes n e g l i g i b l e i n o l d age (Waxman, 1984; Kamataki et a l . , 1985). In l i v e r s of 24 month-old male ra t s , the l e v e l of cytochrome P450IIC11 i s n e g l i g i b l e , whereas cytochrome P450IIC12, which i s a "female-specific" enzyme, i s expressed (Kamataki et a l . , 1985). For substrates (drugs) that are oxidized by cytochrome P450IIC11, i s t h i s enzyme s t i l l involved i n the oxidation of these substrates (drugs) i n l i v e r s of old male rats? I f not, does cytochrome P450IIC12 become the major c a t a l y s t i n these cases? To answer these questions, immunoinhibition studies should be performed with the appropriate monospecific antibody (e.g. anti-cytochrome P450IIC11 and anti-cytochrome P450IIC12 antibodies) to determine the contribution of the enzymes to the oxidation of the substrate (drug). 192 6.2 STUDIES WITH HUMAN HEPATIC MICROSOMES Based on the d i f f e r e n t i a l i n h i b i t i o n o f r a t h e p a t i c cytochrome P-450 enzymes by c i m e t i d i n e observed i n the p r e s e n t i n v e s t i g a t i o n , i t i s p o s s i b l e t h a t human h e p a t i c cytochrome P-450 enzymes have d i f f e r e n t s u s c e p t i b i l i t i e s t o the i n h i b i t o r y e f f e c t o f c i m e t i d i n e . To i n v e s t i g a t e the e f f e c t o f c i m e t i d i n e on s p e c i f i c human h e p a t i c cytochrome P-450 enzymes, an approach would be t o p r e i n c u b a t e human h e p a t i c microsomes w i t h low c o n c e n t r a t i o n s o f c i m e t i d i n e i n the presence o f NADPH p r i o r t o the i n i t i a t i o n o f s u b s t r a t e o x i d a t i o n . I n i t i a l l y , an i n t e r n a l c o n t r o l experiment s h o u l d be conducted w i t h a s u b s t r a t e , the o x i d a t i v e metabolism of which i s known t o be i n h i b i t e d by c i m e t i d i n e i n human h e p a t i c microsomes. The next step would be t o s e l e c t a drug, the c l e a r a n c e o f which i s impaired by c i m e t i d i n e i n man, and t o determine whether p r e i n c u b a t i o n o f human h e p a t i c microsomes w i t h a low c o n c e n t r a t i o n o f c i m e t i d i n e and NADPH has an e f f e c t on the i n h i b i t i o n o f the o x i d a t i o n o f t h i s drug by c i m e t i d i n e . In subsequent experiments, enzyme-s p e c i f i c a c t i v i t i e s w i l l be used. These would i n c l u d e : a) p h e n a c e t i n O-deethylase a c t i v i t y f o r human cytochrome P450IA2 ( S e s a r d i c e t a l . , 1988); b) N-nitrosodimethylamine N-demethylase ( I s h i z a k i e t a l . , 1991) o r chlor z o x a z o n e 6-hy d r o x y l a s e a c t i v i t y (Guengerich et a l . , 1991) f o r human cytochrome P450IIE1; c) t e s t o s t e r o n e 66-hydroxylase a c t i v i t y f o r human cytochrome P450IIIA enzymes (Waxman e t a l . , 193 1988a); d) debrisoquine 4-hydroxylase a c t i v i t y for human cytochrome P450IID6 ( D i s t l e r a t h et al., 1985). In each case where one of the above enzyme a c t i v i t i e s i s i n h i b i t e d by cimetidine, the mechanism(s) of i n h i b i t i o n should be investigated. Experiments can be performed to determine whether a metabolite-intermediate i s involved. This can be done by incubating human hepatic microsomes with cimetidine and NADPH and recording the time-dependent formation of a s p e c t r a l peak at approximately 448-456 nm as well as performing the enzyme assays i n the presence and absence of potassium f e r r i c y a n i d e . For these experiments, i t i s important to use l i v e r t i s s u e from s u r g i c a l patients who are not taking drugs that are known to r e s u l t i n the formation of metabolite-intermediate complexes; f o r example, erythromycin, amiodarone and amphetamine. An important study would be to evaluate whether the r e s u l t obtained from a preincubation experiment with human hepatic microsomes and low concentrations of cimetidine can be used to model the e f f e c t of cimetidine on hepatic drug clearance i n man. The approach to t h i s series of experiments would be to use the drugs as the substrates i n the i n vitro enzyme assays. Human hepatic microsomes would be preincubated with low concentrations of cimetidine i n the presence of NADPH p r i o r to the i n i t i a t i o n of drug oxidation. One group of drugs to be used would be those i n which the clearance of the drug and the major metabolites have been 194 shown t o be a f f e c t e d by c i m e t i d i n e ; f o r example, t h e o p h y l l i n e ( G r y g i e l et a l . , 1984; Cusack e t a l . , 1985; V e s t a l e t a l . , 1987). Another group o f drugs t o be used would be those i n which these v a r i a b l e s have been shown not t o be a f f e c t e d by c i m e t i d i n e ; f o r example, t o l b u t a m i d e (Dey et al., 1983; S t o c k l e y e t al., 1986; Adebayo e t al., 1988). 195 SUMMARY AND CONCLUSIONS 7.1 STUDIES WITH CIMETIDINE 1. The in vivo administration of a single dose of cimetidine (150 mg/kg) to adult male rats, which were sacrificed 90 min after the injection, d i f f e r e n t i a l l y affected the hepatic microsomal cytochrome P-450-mediated enzyme a c t i v i t i e s . A) In vivo cimetidine inhibited the enzyme act i v i t i e s specific for cytochrome P450IIC11. Evidence was also obtained suggesting that cimetidine inhibited cytochrome P450IA2; however, this requires further investigation. B) Indirect evidence also indicated that unidentified enzymes other than cytochrome P450IIC11 were inhibited by in vivo cimetidine in microsomes from uninduced adult male rats. C) In contrast, in vivo cimetidine treatment in adult male rats apparently did not affect the enzyme acti v i t i e s specific for cytochrome P450IIA1, cytochromes P450IIB1/2 or cytochromes P450IIIA1/2. It remains possible that the lack of inhibition of these ac t i v i t i e s is substrate-related. D) In some cases, the extent of inhibition by in vivo cimetidine depended on prior treatment with an 196 enzyme inducer. This can be explained by the increasing contribution to such a c t i v i t i e s by inducible enzymes which were not subject to i n h i b i t i o n by cimetidine. 2. The d i f f e r e n t i a l e f f e c t of i n vivo cimetidine on cytochrome P-450-mediated enzyme a c t i v i t i e s was not observed when cimetidine was added to hepatic microsomes in vitro immediately p r i o r to the i n i t i a t i o n of substrate oxidation. A) Jn v i t r o , cimetidine, at concentrations of up to 10 mM, did not a f f e c t an enzyme a c t i v i t y s p e c i f i c f o r cytochrome P450IIA1. This i s the f i r s t case of a cytochrome P-450 enzyme not i n h i b i t e d by cimetidine e i t h e r i n vivo or i n vitro. In contrast, i n v i t r o cimetidine did i n h i b i t the enzyme a c t i v i t i e s s p e c i f i c for cytochromes P450IIB1/2, cytochrome P450IIC11, cytochromes P450IIIA1/2 and possibly cytochrome P450IA2. The IC 5 0 values f o r the various enzyme a c t i v i t i e s were i n the range of 1.0 - 7.4 mM. B) In microsomes from uninduced r a t s , the cytochrome P450IICll-mediated testosterone 2oc-hydroxylase a c t i v i t y was i n h i b i t e d competitively by i n v i t r o cimetidine, but non-competitively by i n vivo cimetidine. 197 3. P r e i n c u b a t i o n o f h e p a t i c microsomes w i t h c i m e t i d i n e and NADPH r e s u l t e d i n i n h i b i t i o n o f cytochrome P-450 t h a t was more c h a r a c t e r i s t i c o f t h a t observed w i t h in vivo c i m e t i d i n e . A) P r e i n c u b a t i o n o f microsomes w i t h 0.05 mM c i m e t i d i n e i n the presence o f NADPH f o r 15 min r e s u l t e d i n the i n h i b i t i o n o f the enzyme a c t i v i t i e s s p e c i f i c f o r cytochrome P450IIC11, but not those f o r cytochrome P450IIA1, cytochromes P450IIB1/2 o r cytochromes P450IIIA1/2, s i m i l a r t o the p a t t e r n o f i n h i b i t i o n observed w i t h in vivo c i m e t i d i n e . T h i s d i f f e r e n t i a l e f f e c t w i t h i n vitro c i m e t i d i n e (0.05 mM) d i d not oc c u r i n the absence o f NADPH i n the p r e i n c u b a t i o n medium o r without the p r e i n c u b a t i o n s t e p . 4. The r e s u l t s from the pr e s e n t i n v e s t i g a t i o n suggest t h a t in vivo, c i m e t i d i n e i n h i b i t s cytochrome P450IIC11 by a c a t a l y s i s - d e p e n d e n t process i n a d u l t male r a t s . T h i s i s a p p a r e n t l y by a mechanism d i f f e r e n t from the i n h i b i t i o n o f cytochrome P-450 by c i m e t i d i n e in vitro i n the absence o f a p r e i n c u b a t i o n s t e p . The p r e c i s e mechanism o f i n h i b i t i o n o f cytochrome P-450 by c i m e t i d i n e should be e l u c i d a t e d i n f u t u r e s t u d i e s . 198 7.2 STUDIES WITH MONOSPECIFIC ANTI-CYTOCHROME P450IIC11 ANTIBODY 1. Testosterone 2a-hydroxylase activity was not only a good marker for cytochrome P450IIC11 in hepatic microsomes from uninduced adult male rats, but was also found to be a good marker for this activity in those microsomes from adult male rats treated with phenobarbital or dexamethasone. 2. Testosterone 16a-hydroxylase activity was a good marker for cytochrome P450IIC11 in hepatic microsomes from uninduced adult male rats. However, in those microsomes from adult male rats treated with phenobarbital or dexamethasone, cytochrome P450IIC11 was only a minor contributor to testosterone 16a-hydroxylase activity. 3. Cytochrome P450IIC11 accounted par t i a l l y for the oxidation of testosterone to androstenedione in hepatic microsomes from uninduced and dexamethasone-induced, but not phenobarbital-induced, adult male rats. 4. Cytochrome P450IIC11 accounted for almost a l l of the pentoxyresorufin 0-dealkylase activity in hepatic microsomes from uninduced adult male rats. However, this enzyme did not appear to contribute to this activity in microsomes from adult male rats treated with phenobarbital. 199 5. Cytochrome P450IIC11 accounted p a r t i a l l y for the aminopyrine N-demethylase a c t i v i t y i n hepatic microsomes from uninduced adult male ra t s , but i t did not appear to contribute to t h i s a c t i v i t y i n microsomes from rats treated with phenobarbital. 6. Cytochrome P450IIC11 did not appear to play a major r o l e i n the N-demethylation of erythromycin i n hepatic microsomes from uninduced adult male r a t s . 200 REFERENCES Adebayo GI, Coker HAB. Lack of e f f i c a c y of cimetidine and r a n i t i d i n e as i n h i b i t o r s of tolbutamide metabolism. Eur. J . C l i n . Pharmacol. 34: 653-565, 1988. Adedoyin A, Aarons L, Houston JB. Dose-dependent pharmaco-k i n e t i c s of cimetidine i n the r a t . Xenobiotica 17: 595-604, 1987a. Adedoyin A, Aarons L, Houston JB. Plasma concentration-response r e l a t i o n s h i p f o r cimetidine i n h i b i t i o n of drug metabolism i n the r a t . Drug Metab. Dispos. 15: 127-132, 1987b. Adesnick M, Atchison M. Genes for cytochrome P-450 and t h e i r regulation. C r i t . Rev. Biochem. 19: 247-305, 1986. Alvares AP, Pratt WB. Pathways of drug metabolism. In: Pratt WB, Taylor P (eds.). P r i n c i p l e s of Drug Action. The Basis of Pharmacology. 3rd Ed. New York: C h u r c h i l l Livingstone, 365-422, 1990. Alvares AP, S c h i l l i n g G, Levin W, Kuntzman R. Studies on the i n d i c a t i o n of CO-binding pigments i n l i v e r microsomes by phenobarbital and 3-methylcholanthrene. Biochem. Biophys. Res. Commun. 29: 521-526, 1967. A r l o t t o MP, Parkinson A. I d e n t i f i c a t i o n of cytochrome P450a (P450IIA1) as the p r i n c i p a l testosterone 7a-hydroxylase i n r a t l i v e r microsomes and i t s regulation by thyroid hormones. Arch. Biochem. Biophys. 270: 458-471, 1989. A r l o t t o MP, Sonderfan AJ, Klassen CD, Parkinson A. Studies on the pregnenolone-16-alpha-carbonitrile-inducible form of r a t l i v e r microsomal cytochrome P-450 and UDP-glucuronosyl transferase. Biochem. Pharmacol. 36: 3859-3866, 1987. Astrom A, DePierre JW. Metabolism of 2-acetylaminofluorene by eight d i f f e r e n t forms of cytochrome P-450 i s o l a t e d from r a t l i v e r . Carcinogenesis 6: 113-120, 1985. Astrom A, DePierre JW. Rat l i v e r microsomal cytochrome P-450 p u r i f i c a t i o n , characterization, m u l t i p l i c i t y and induction. Biochem. Biophys. Acta 853: 1-127, 1986. 201 Backes WL, Jansson I, Mole JE, Gibson GG, Schenkman, JB. I s o l a t i o n and comparison of f o u r cytochrome P-4 5 0 enzymes from p h e n o b a r b i t a l - i n d u c e d r a t l i v e r : t h r e e forms p o s s e s s i n g i d e n t i c a l NH 2-terminal sequences. Pharmacology 31: 155-169, 1985. Ban d i e r a S, Ryan DE, L e v i n W, Thomas PE. Age- and sex-r e l a t e d e x p r e s s i o n o f cytochromes P450f and P450g i n r a t l i v e r . A r c h . Biochem. Biophys. 248: 658-676, 1986. Bast A, Smid K, Timmerman H. The e f f e c t s o f c i m e t i d i n e , r a n i t i d i n e and fa m o t i d i n e on r a t h e p a t i c microsomal cytochrome P-4 50 a c t i v i t i e s . Agents A c t i o n 27: 188-191, 1989. Begg E J , W i l l i a m s KM, Wade DN, O'Shea KF. No s i g n i f i c a n t e f f e c t o f c i m e t i d i n e on the pharmacokinetics o f m i s o n i d a z o l e i n man. Br. J . C l i n . Pharmacol. 15: 575-576, 1983. B e l l w a r d GD, Chang T, Rodrigues B, M c N e i l l JH, Maines S, Ryan DE, L e v i n W, Thomas PE. Hepatic cytochrome P -450J i n d u c t i o n i n the spontaneously d i a b e t i c BB r a t . Mol. Pharmacol. 33: 140-143, 1988. B e r t i l s s o n L, Aberg-Wistedt A. The d e b r i s o q u i n e h y d r o x y l a t i o n t e s t p r e d i c t s s t e a d y - s t a t e plasma l e v e l s o f desipr a m i n e . Br. J . C l i n . Pharmacol. 15: 388-390, 1983. B l a c k JW, Duncan WAM, Durant CJ, G a n e l l i n CR, Parsons ME. D e f i n i t i o n and antagonism o f histamine H 2 - r e c e p t o r s . Nature 236: 385-390, 1972. B r a d f i e l d CA, G l o v e r E, Poland A. P u r i f i c a t i o n and N-t e r m i n a l amino a c i d sequence o f the Ah r e c e p t o r from the C57BL/6J mouse. Mol. Pharmacol. 39: 13-19, 1991. B r a d f o r d MM. A r a p i d and s e n s i t i v e method f o r the q u a n t i t a t i o n o f microgram q u a n t i t i e s o f p r o t e i n u t i l i z i n g the p r i n c i p l e o f p r o t e i n - d y e b i n d i n g . A n a l . Biochem. 72: 248-254, 1976. B r i a n WR, S r i v a s t a v a PK, Umbenhauer DR, L l o y d RS, Guengerich FP. E x p r e s s i o n o f a human l i v e r cytochrome P-450 p r o t e i n w i t h t o l b u t a m i d e hydroxylase a c t i v i t y i n Saccharomyces cerevisiae. B i o c h e m i s t r y 28: 4993-4999, 1989. Brimblecombe RW, Duncan WAM, Durant GJ, Emmett JC, G a n e l l i n CR, Parsons ME. C i m e t i d i n e - a nonthiourea H 2 - r e c e p t o r a n t a g o n i s t . J . I n t . Med. Res. 3: 86-92, 1975. 202 Brockmeyer NH, Breithaupt H, Ferdinand W, von Hattingberg M, Ohnhaus EE. Kinetics of o r a l and intravenous mexiletine: lack of e f f e c t of cimetidine and r a n i t i d i n e . Eur. J . C l i n . Pharmacol. 36: 375-378, 1989. Buening MK, Franklin MR. SKF 525-A i n h i b i t i o n , induction and 452-nm complex formation. Drug Metab. Dispos. 4: 244-255, 1976. Burke MD, Mayer RT. Ethoxyresorufin: d i r e c t f l u o r i m e t r i c assay of a microsomal 0-dealkylation which i s p r e f e r e n t i a l l y inducible by 3-methylcholanthrene. Drug Metab. Dispos. 2: 583-588, 1974. Burke MD, Thompson S, Elcombe CR, Halpert J, Haaparanta T, Mayer RT. Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to d i s t i n g u i s h between d i f f e r e n t induced cytochromes P-450. Biochem. Pharmacol. 34: 3337-3345, 1985. Burland WL, Duncan WAM, Hesselbo T, M i l l s JG, Sharpe PC, Haggie SJ, Wyllie JH. Pharmacological evaluation of cimetidine, a new histamine H 2-receptor antagonist, i n healthy man. Br. J . C l i n . Pharmacol. 2: 481-486, 1975. Chang T, Levine M, Bellward GD. Influence of sex and inducer treatment on the high- and l o w - a f f i n i t y forms of hepatic microsomal erythromycin N-demethylase i n r a t s . Can. J . Phys i o l . Pharmacol. 68: 1510-1513, 1990. Cheng KC, Schenkman JB. P u r i f i c a t i o n and characterization of two c o n s t i t u t i v e forms of rat l i v e r microsomal cytochrome P-450. J . B i o l . Chem. 257: 2378-2385, 1982. Cohen IA, Johnson CE, Berardi RR, Hyneck ML, Achem SR. Cimetidine-theophylline i n t e r a c t i o n : e f f e c t s of age and cimetidine dose. Ther. Drug Monit. 7: 426-434, 1985. Combalbert J, Fabre I, Fabre G, Dalet I, Derancourt J, Cano JP, Maurel P. Metabolism of cyclosporin A. IV. P u r i f i c a t i o n and i d e n t i f i c a t i o n of the rif a m p i c i n -inducible human l i v e r cytochrome P-450 (cyclosporine A oxidase) as a product of P450IIIA gene subfamily. Drug Metab. Dispos. 17: 197-207, 1989. Conney AH. Induction of microsomal enzymes by foreign chemicals and carcinogenesis by p o l y c y c l i c aromatic hydrocarbons: GHA Clowes memorial lecture. Cancer Res. 42: 4875-4917, 1982. 203 Conney AH, G i l l e t t e JR, Inscoe JK, Trams ER, Posner HS. Induced synthesis of l i v e r microsomal enzymes which metabolize foreign compounds. Science 130: 1478-1479, 1959. Cooper KO, Reik LM, Bandiera S, Kelley M, Ryan DE, Daniel R, McCluskey SA, Levin W, Thomas PE. Monoclonal antibodies d i s t i n g u i s h between two members of a steroid-inducible cytochrome P-450 subfamily i n r a t s . FASEB J . 4: A2242, 1990. Craig PI, Mehta I, Murray M, McDonald D, Astrom A, van der Meide PH, F a r r e l l GC. Interferon down regulates the male-s p e c i f i c cytochrome P450IIIA2 i n r a t l i v e r . Mol. Pharmacol. 38: 313-318, 1990. Cross SAM. The l o c a l i z a t i o n of metiamide and cimetidine using autoradiographical techniques. In: Duncan WAM, Leonard BJ (eds.). C l i n i c a l Toxicology. Proceedings of the European Society of Toxicology. Amsterdam: Exerpta Medica, 288-290, 1977. Cusack BJ, Dawson GW, Mercer GD, Vestal RE. Cigarette smoking and theophylline metabolism: e f f e c t s of cimetidine. C l i n . Pharmacol. Ther. 37: 330-336, 1985. Dahl AR, Hodgson E. The i n t e r a c t i o n of a l i p h a t i c analogs of methylenedioxyphenyl compounds with cytochrome P-450 and P-420. Chem.-Biol. Interact. 27: 163-175, 1979. Dalton MJ, Powell JR, Messenheimer J r JA. The influence of cimetidine on single-dose carbamazepine pharmacokinetics. E p i l e p s i a 26: 127-130, 1985. Dalton MJ, Powell JR, Messenheimer J r JA, Clark J. Cimetidine and carbamazepine: a complex drug i n t e r a c t i o n . E p i l e p s i a 27: 553-558, 1986. Danhof M, Krom DP, Breimer DD. Studies on the d i f f e r e n t metabolic pathways of antipyrine i n r a t s : influence of phenobarbital and 3-methylcholanthrene treatment. Xenobiotica 9: 695-702, 1979. Dannan GA, Guengerich FP, Kaminsky LS, Aust SD. Regulation of cytochrome P-450. Immunochemical quantitation of eight isozymes i n l i v e r microsomes of rats treated with poly-brominated biphenyl congeners. J . B i o l . Chem. 258: 1282-1288, 1983. Dawson GW, Vestal RE. Cimetidine i n h i b i t s the in vitro N-demethylation of methadone. Res. Commun. Chem. Pathol. Pharmacol. 46: 301-304, 1984. 204 Dey NG, C a s t l e d e n CM, Ward J , C o r n h i l l J , McBurney A. The e f f e c t o f c i m e t i d i n e on tolbutamide k i n e t i c s . Br. J . C l i n . Pharmacol. 16: 438-440, 1983. D i s t l e r a t h LM, R e i l l y PEB, M a r t i n MV, Davies GG, W i l k i n s o n GR, Guengerich FP. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f the human l i v e r cytochromes P-450 i n v o l v e d i n d e b r i s o q u i n e h y d r o x y l a t i o n and phenacetin 0 - d e e t h y l a t i o n , two p r o t o t y p e s f o r g e n e t i c polymorphism i n o x i d a t i v e drug metabolism. J . B i o l . Chem. 260: 9057-9067, 1985. Dixon PAF, Okereke NO, Ogundahunsi OA. I n f l u e n c e o f s p e c i e s and drug pretreatment on the m e t a b o l i c o x i d a t i o n o f c i m e t i d i n e and metiamide. Biochem. Pharmacol. 34: 2028-2030, 1985. Drew R, Rowell J , G r y g i e l J J . C i m e t i d i n e : a s p e c i f i c i n h i b i t o r o f h e p a t i c a r y l hydrocarbon h y d r o x y l a s e (AHH) i n the r a t . Res. Commun. Chem. P a t h o l . Pharmacol. 33: 81-93, 1981. Dutton DR, P a r k i n s o n A. Reduction of 7 - a l k o x y r e s o r u f i n s by NADPH-cytochrome P450 reductase and i t s d i f f e r e n t i a l e f f e c t s on t h e i r O - d e a l k y l a t i o n by r a t l i v e r microsomal cytochrome P450. Arch. Biochem. Biophys. 268: 617-629, 1989. Eichelbaum M, Ekbom K, B e r t i l s s o n L, Ringberger VA, Rane A. Plasma k i n e t i c s o f carbamazepine and i t s epoxide m e t a b o l i t e i n man a f t e r s i n g l e and m u l t i p l e doses. Eur. J . C l i n . Pharmacol. 8: 337-341, 1975. Eichelbaum M, Tomson T, T y b r i n g G, B e r t i l s s o n L. Carbamazepine metabolism i n man. I n d u c t i o n and pharmacogenetic a s p e c t s . C l i n . Pharmacokinet. 10: 80-90, 1985. E l Azhary R, Renton KW, Mannering GJ. E f f e c t o f i n t e r f e r o n i n d u c i n g agents ( p o l y r o b o i n o s i n i c a c i d , p o l y r i b o c y t i d y l i c a c i d and t i l o r o n e ) on the heme t u r n o v e r of h e p a t i c cytochrome P-450. Mol. Pharmacol. 17: 395-399, 1980. Emi Y, Omura T. S y n t h e s i s of s e x - s p e c i f i c forms o f cytochrome P-450 i n r a t l i v e r i s t r a n s i e n t l y suppressed by h e p a t i c monooxygenase i n d u c e r s . J . Biochem. 104: 40-43, 1988. Favreau LV, M a l c h o f f DM, Mole JE, Schenkman JB. Responses t o i n s u l i n by two forms of r a t h e p a t i c microsomal cytochrome P-450 t h a t undergo major (RLM6) and minor 4(RLM5b) e l e v a t i o n s i n d i a b e t e s . J . B i o l . Chem. 262: 14319-14326, 1987. 205 Feely J , Pereira L, Guy E, Hockings N. Factors a f f e c t i n g the response to i n h i b i t i o n of drug metabolism by cimetidine -dose response and s e n s i t i v i t y of e l d e r l y and induced subjects. Br. J . C l i n . Pharmacol. 17: 77-81, 1984. Feldman M, Burton ME. Histamine 2-receptor antagonists. Standard therapy for acid-peptic diseases. ( F i r s t of two pa r t s ) . N. Engl. J . Med. 323: 1672-1680, 1990a. Feldman M, Burton ME. Histamine 2-receptor antagonists. Standard therapy for acid-peptic diseases. (Second of two pa r t s ) . N. Engl. J. Med. 323: 1749-1755, 1990b. Frankli n MR. I n h i b i t i o n of mixed-function oxidations by substrates forming reduced cytochrome P-450 metabolic-intermediate complexes. Pharmacol. Ther. 2: 227-245, 1977. Galbraith RA, J e l l i n c k PH. Decreased estrogen hydroxylation i n male rat l i v e r following cimetidine treatment. Biochem. Pharmacol. 38: 313-319, 1989. Galbraith RA, Michnovicz J J . The e f f e c t s of cimetidine on the oxidative metabolism of e s t r a d i o l . N. Engl. J. Med. 321: 269-274, 1989. Gibson GG, Orton TC, Tamburini PP. Cytochrome P-450 induction by c l o f i b r a t e . P u r i f i c a t i o n and properties of a hepatic cytochrome P-450 r e l a t i v e l y s p e c i f i c f o r the 12-and 11-hydroxylation of dodecanoic acid ( l a u r i c a c i d ) . Biochem. J . 203: 161-168, 1982. Gibson GG, Skett P. Introduction to Drug Metabolism. London: Chapman and H a l l Ltd., 1986. Goldstein JA, Linko P, Luster MI, Sundheimer DW. P u r i f i c a t i o n and characterization of a second form of hepatic cytochrome P-448 from rats treated with a pure polychlorinated biphenyl isomer. J . B i o l . Chem. 257: 2702-2707, 1982. Gontovnick LS, Bellward GD. Sex and age dependence of the "s e l e c t i v e " induction of rat hepatic microsomal epoxide hydratase following trans-stilbene oxide, 1-alpha-acetyl-methadol or phenobarbital treatment. Biochem. Pharmacol. 29: 3245-3251, 1980. Gonzalez FJ, Skoda RC, Kimura S, Umeno M, Zanger UM, Nebert DW, Gelboin HV, Hardwick JP, Meyer UA. Characterization of the common genetic defect i n humans d e f i c i e n t i n debrisoquine metabolism. Nature 331: 442-446, 1988. 206 G r i f f i t h s R, Lee RM, Taylor DC. Kinetics of cimetidine i n man and experimental animals. In: Burland WL, Simkins MA (eds.). Proceedings of the Second International Symposium on Histamine H 2-receptor antagonists. Amsterdam: Excerpta Medica, 38-51, 1977. Grogan DE, Lane M, Smith FE, Bresnick E, Stone K. Interaction of f l a v i n s and chloramphenicol with microsomal enzyme systems. Biochem. Pharmacol. 21: 3131-3144, 1972. Grygiel J J , Miners JO, Drew R, B i r k e t t DJ. D i f f e r e n t i a l e f f e c t s of cimetidine on theophylline metabolic pathways. Eur. J . C l i n . Pharmacol. 26: 335-340, 1984. Guengerich FP. Cytochrome P-450 enzymes and drug metabolism. In: Bridges JW, Chasseaud LF, Gibson GG (eds.). Progress i n Drug Metabolism. Vol. 10. London: Francis and Taylor, 1-54, 1987. Guengerich FP. Characterization of human microsomal cytochrome P-450 enzymes. Ann. Rev. Pharmacol. Toxicol. 29: 241-264, 1989. Guengerich FP, Dannan GA, Wright ST, Martin MV, Kaminsky LS. P u r i f i c a t i o n and characterization of l i v e r microsomal cytochrome P-450: electrophoretic, s p e c t r a l , c a t a l y t i c , and immunochemical properties and i n d u c i b i l i t y of eight isozymes i s o l a t e d from rats treated with phenobarbital or 6-naphthoflavone. Biochemistry 21: 6019-6030, 1982a. Guengerich FP, Kim DH, Iwasak M. Role of human cytochrome P-450 2E1 i n the oxidation of many low molecular weight cancer suspects. FASEB J . 5: A1515, 1991. Guengerich FP, Wang P, Davidson NK. Estimation of isozymes of microsomal cytochrome P-450 i n r a t s , rabbits, and humans using immunochemical s t r a i n i n g coupled with sodium dodecyl sul f a t e - polyacrylamide gel electrophoresis. Biochemistry 21: 1698-1706, 1982b. Gut J, Gasser R, Dayer P, Kronbach T, Catin T. Debrisoquine polymorphism of drug oxidation: p u r i f i c a t i o n from human l i v e r of a cytochrome P-450 isozyme with high a c t i v i t y for b u f u r a l o l hydroxylation. FEBS Lett 173: 287-290, 1984. Halpert J. Covalent modification by l y s i n e during the suicide i n a c t i v a t i o n of rat l i v e r cytochrome P-450 by chloramphenicol. Biochem. Pharmacol. 30: 875-881, 1981. 207 Halpert J, Balfour C, Miller NE, Morgan ET, Dunbar D, Kaminsky LS. Isozyme selectivity of the inhibition of the rat l i v e r cytochrome P-450 by chloramphenicol in vivo. Mol. Pharmacol. 28: 290-296, 1985a. Halpert J. Jaw JY, Balfour C. Specific inactivation by 176-substituted steroids of rabbit and rat li v e r cytochomes P-450 responsible for progestoerone 21-hydroxylation. Mol. Pharmacol. 34: 139-147, 1989a. Halpert J, Jaw JY, Balfour C, Kaminsky LS. Selective inactivation by chlorofluoroacetamides of the major phenobarbital-inducible form(s) of rat li v e r cytochrome P-450. Drug Metab. Dispos. 18: 168-174, 1990. Halpert J, Jaw JY, Cornfield LJ, Balfour C, Mash EA. Selective inactivation of rat li v e r cytochromes by 21-chlorinated steroids. Drug Metab. Dispos. 17: 26-31, 1989b. Halpert J, Miller NE, Gorsky L. On the mechanism of the inactivation of the major phenobarbital-inducible isozyme of rat li v e r cytochrome P-450 by chloramphenicol. J. Bi o l . Chem. 260: 8397-8403, 1985b. Halpert J, Naslund B, Betner I. Suicide inactivation of rat li v e r cytochrome P-450 by chloramphenicol in vivo and in vitro. Mol. Pharmacol. 23: 445-452, 1983. Halvorson M, Greenway D, Eberhart D, Fitzgerald K, Parkinson A. Reconstitution of testosterone oxidation by purified rat cytochrome P-450p (IIIAl). Arch. Biochem. Biophys. 277: 166-180, 1990. Hansen JM, Chistensen LK. Drug interactions with oral sulphonylurea hypoglycaemic drugs. Drugs 13: 24-34, 1977. Heuman DM, Gallagher EJ, Barwick JL, Elshourbagy NA, Guzelian PS. Immunochemical evidence for induction of a common form of hepatic cytochrome P-450 in rats treated with pregnenolone-16a-carbonitrile or other steroidal or non-steroidal agents. Mol. Pharmacol. 21: 753-760, 1982. Hietanen E, Ahotupa M, Bereziat JC, Park SS, Gelboin HV, Bartsch H. Monoclonal antibody characterization of hepatic and extraphepatic cytochrome P-450 activity in rats treated with phenobarbital or methylcholanthrene and fed various cholesterol diets. Biochem. Pharmacol. 36: 3973-3980, 1987. 208 H i l l SJ. Distribution, properties, and functional characteristics of three classes of histamine receptor. Pharmacol. Rev. 42: 45-83, 1990. Hoensch HP, Hutzel H, Kirch W, Ohnhaus EE. Isolation of human hepatic microsomes and their inhibition by cimetidine and ranitidine. Eur. J. Clin. Pharmacol. 29: 199-206, 1985. Hostetler KA, Wrighton SA, Molowa DT, Thomas PE, Levin W, Guzelian PS. Coinduction of multiple hepatic cytochrome P-450 proteins and their mRNAs in rats treated with imidazole antimycotic agents. Mol. Pharmacol. 35: 27 9-285, 1989. Imai Y, Inada M, Tamura S, Kawata S, Minami Y, Tarui S. Comparative effects of famotidine and cimetidine on 7-ethoxycoumarin O-deethylase activity in human liv e r s . Br. J. Clin. Pharmacol. 22: 495-496, 1986. Ioannides C, Rodrigues AD, Aryton AD, Barnett CR, Chown J, Parke DV. Induction of the rat hepatic microsomal mixed-function oxidases by cimetidine. Toxicol. Lett. 49: 61-68, 1989. Ioannoni B, Mason SR, Reilly PEB, Winzor DJ. Evidence for induction of hepatic microsomal cytochrome P-450 by cimetidine: binding and kinetic studies. Arch. Biochem. Biophys. 247: 372-383, 1986. Ishizaki H, Yoo JSH, Guengerich FP, Yang CS. The metabolism of N-nitrodimethylamine and N-nitrosodiethylamine by human and rat l i v e r microsomal cytochromes P450. FASEB J. 5: A1516, 1991. Jansson I, Mole J, Schenkman JB. Purification and characterization of a new form (RLM2) of li v e r microsomal cytochrome P-450 from untreated rat. J. Biol. Chem. 260: 7084-7093, 1985. Jarewenko MV, Van Buren CT, Kramer WG, Lorber MI, Flechner SM, Kahan BD. Ranitidine, cimetidine and the cyclosporine-treated recipient. Transplantation 42: 311-312, 1986. Jensen JC, Gugler R. Cimetidine interaction with li v e r microsomes in vitro and in vivo. Involvement of an activated complex with cytochrome P-450. Biochem. Pharmacol. 34: 2141-2146, 1985. 20 9 Johansson I, Ekstrom G, Scholte B, Puzycki D, Jornvall H, Ingelman-Sundberg M. Ethanol-, fasting- and acetone-inducible cytochrome P-450 in rat l i v e r : regulation and characteristics of enzymes belonging to the IIB and HE gene subfamilies. Biochemistry 27: 1925-1934, 1988. Jonen HG, Huthwohl B, Kahl R, Kahl GF. Influence of pyridine and some pyridine derivatives on spectral properties of reduced microsomes and on microsomal drug metabolizing activity. Biochem. Pharmacol. 23: 1319-1329, 1974. Jonsson KA, Eriksson SE, Kagevi I, Norlander B, Bodemar G, Walan A. Cimetidine, but not oxmetidine, penetrates into the cerebrospinal f l u i d after a single intravenous dose. Br. J. Clin. Pharmacol. 14: 815-819, 1982. Kamataki T, Maeda K, Shimada M, Kitani K, Nagai T, Kato R. Age-related alteration in the acti v i t i e s of drug-metabolizing enzymes and contents of sex-specific forms of cytochrome P-450 in li v e r microsomes from male and female rats. J. Pharmacol. Exp. Ther. 233: 222-228, 1985. Kaminsky LS, Fasco MJ, Guengerich FP. Comparison of different forms of liv e r , kidney and lung microsomal cytochrome P-450 by immunological inhibition of regio-and stereoselective metabolism of warfarin. J. Biol. Chem. 254: 9657-9662, 1979. Kao LR, Wilkinson CF. Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole. Xenobiotica 17: 793-805, 1987. Kelley M, Hantelle P, Safe S, Levin W, Thomas PE. Co-induction of cytochrome P-450 isozymes in rat l i v e r by 2 , 4 , 5 , 2',4'5'-hexachlorobiphenyl or 3-methoxy-4-aminoazobenzene. Mol. Pharmacol. 32: 206-211, 1987. Klein A, Sami M, Selinger K. Mexiletine kinetics in healthy subjects taking cimetidine. Clin. Pharmacol. Ther. 37: 669-673, 1985. Knodell RG, Holtzman JL, Crankshaw DL, Steele NM, Stanley LN. Drug metabolism by rat and human hepatic microsomes in responses to interaction with H2-receptor antagonists. Gastroenterology 82: 84-88, 1982. Koop DR, Tierney DJ. Multiple mechanisms in the regulation of ethanol-inducible cytochrome P-450IIE1. BioEssays 12: 429-435, 1990. 210 Kupfer D. Endogenous substrates of monooxygenases: fatty acids and prostaglandins. Pharmacol. Ther. 11: 469-496, 1980. Lai C. UBC SPSS-X. Stat i s t i c a l Package for Social Sciences - Extended Version Release 2.0 (Under MTS). Vancouver: University of British Columbia Computing Centre, 1986. Larrey D, Distlerath LM, Dannan GA, Wilkinson GR, Guengerich FP. Purification and characterization of the rat l i v e r microsomal cytochrome P-450 involved in the 4-hydroxylation of debrisoquine, a prototype for the genetic variation in oxidative drug metabolism. Biochemistry 23: 2787-2795, 1984. Larrey D, Tinel M, Letteron P, Geneve J, Descatoire V, Pessayre D. Formation of an inactive cytochrome P-450Fe(II)-metabolite complex after administration of amiodarone to rats, mice and hamsters. Biochem. Pharmacol. 35: 2213-2220, 1986. LeBlanc GA, Waxman DJ. Feminization of rat hepatic P-450 expression by cisplatin. Evidence for pertubations in the hormonal regulation of steroid-metabolizing enzymes. J. Bi o l . Chem. 263: 15732-15739, 1988. LeBlanc GA, Waxman DJ. Interaction of anticancer drugs with hepatic monooxygenase enzymes. Drug Metab. Rev. 20: 395-439, 1989. LeBlanc GA, Waxman DJ. Mechanisms of cyclophosphamide action on hepatic P-450 expression. Cancer Res. 50: 5720-5726, 1990. Leclercq V, Desager JP, Horsmans Y, Van Nieuwenhuyze Y, Harvengt C. Influence of rifampicin, phenobarbital and cimetidine on mixed function monooxygenase in extensive and poor metabolizers of debrisoquine. Int. J. Clin. Pharmacol. Ther. Toxicol. 27: 593-598, 1989. Lennard MS, Crewe HK, Tucker GT, Woods HF. Metoprolol oxidation by rat l i v e r microsomes. Inhibition by debrisoquine and other drugs. Biochem. Pharmacol. 35: 2757-2761, 1986. Lertratanangkoon K, Horning MG. Metabolism of carbamazepine. Drug Metab. Dispos. 10: 1-10, 1982. Lesca P, Rafidinarivo E, Lecoite P, Mansuy D. A class of strong inhibitors of microsomal monooxygenases: the e l l i p t i c i n e s . Chem.-Biol. Interact. 24: 189-198, 1979. 211 Levin W, Lu AYH, Jacobson M, Kuntzman R. L i p i d peroxidation and the degradation of cytochrome P-450 heme. Arch. Biochem. Biophys. 158: 842-852, 1973. Levin W, Ryan D, Kuntzman R, Conney AH. Neonatal imprinting and the turnover of microsomal cytochrome P-450 i n rat l i v e r . Mol. Pharmacol. 11: 190-200, 1975. Levin W, Thomas PE, Ryan DE, Wood AW. Isozyme s p e c i f i c i t y of testosterone 7a-hydroxylase i n rat hepatic microsomes: i s cytochrome P-450a the sole catalyst? Arch. Biochem. Biophys. 258: 630-635, 1987. Levine M, Jones MW, Sheppard I. D i f f e r e n t i a l e f f e c t s of cimetidine on serum concentrations of carbamazepine and phenytoin. Neurology 35: 562-565, 1985. L i n JH. Pharmacokinetic and pharmacodynamic properties of histamine H 2-receptor antagonists. Relationship between i n t r i n s i c potency and e f f e c t i v e plasma concentrations. C l i n . Pharmacokinet. 20: 218-236, 1991. Lu AYH, Levin W. P a r t i a l p u r i f i c a t i o n of cytochrome P-450 and P-488 from rat l i v e r microsomes. Biochem. Biophys. Res. Commun. 46: 1334-1339, 1972. Lubet RA, Mayer RT, Cameron JW, Nims RW, Burke MD, Wolff T, Guengerich FP. Dealkylation of pentoxyresorufin: a rapid s e n s i t i v e assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics i n the r a t . Arch. Biochem. Biophys. 238: 43-48, 1985. Ma Q, Dannan GA, Guengerich FP, Yang CS. S i m i l a r i t i e s and differences i n the regulation of hepatic cytochrome P-450 enzymes by diabetes and f a s t i n g i n male r a t s . Biochem. Pharmacol. 38: 3179-3184, 1989. Mahgoub A, Dring LG, Idle JR, Lancaster R, Smith RL. Polymorphic hydroxylation of debrisoquine i n man. Lancet 2: 584-586, 1977. Mansuy D. Formation of reactive intermediates and metabolites: , e f f e c t s of macrolide a n t i b i o t i c s on cytochrome P-450. Pharmacol. Ther. 33: 41-45, 1987. Mathews JM, Bend JR. N-Alkylaminobenzotriazoles as isozyme-s e l e c t i v e suicide i n h i b i t o r s of rabbit pulmonary microsomal cytochrome P-450. Mol. Pharmacol. 30: 25-32, 1986. M i t c h e l l SC, Idle JR, Smith RL. The metabolism of 1 4C-cimetidine i n man. Xenobiotica 12: 283-292, 1982. 212 Miura T, Iwasaki M, Komori M, Ohi H, Kitada M, M i t s u i H, Kamataki T . Decrease i n a c o n s t i t u t i v e form of cytochrome P-4 50 by macrolide a n t i b i o t i c s . J. Antimicrob. Chemother. 24: 551-559, 1989. Morgan ET, Norman CA. P r e t r a n s l a t i o n a l suppression of cytochrome P-450h (IIC11) gene expression i n rat l i v e r a f t e r administration of interferon inducers. Drug Metab. Dispos. 18: 649-653, 1990. Mosca P, Freddara U, Lorenzini I, Venturini C, Jezequel AM, Orlandi F. The ef f e c t s of H 2-receptor antagonists on hepatic microsomal drug metabolism and f i n e morphology of r a t l i v e r . Pharmacol. Res. Commun. 17: 513-524, 1985. Murray M. Complexation of cytochrome P-450 isozymes i n hepatic microsomes from SKF 525-A induced r a t s . Arch. Biochem. Biophys. 262: 381-388, 1988. Murray M, Reidy GF. S e l e c t i v i t y i n the i n h i b i t i o n of mammalian cytochrome P-450 by chemical agents. Pharmacol. Rev. 42: 85-101, 1990. Murray M, Ryan AJ. The i n t e r a c t i o n of arylalkylbenz-imidazoles and related compounds with microsomal oxidation. Xenobiotica 13: 707-714, 1983. Murray M, Zaluzny L, F a r r e l l GC. Selective r e a c t i v a t i o n of s t e r o i d hydroxylases following d i s s o c i a t i o n of the i s o s a f r o l e metabolite complex with r a t hepatic cytochrome P-450. Arch. Biochem. Biophys. 251: 471-478, 1986. Nakajima T, Elovaara E, Park SS, Gelboin HV, Hietanen E, Vainio H. Monoclonal antibody-directed characterization of benzene, ethoxyresorufin and pentoxyresorufin metabolism i n r a t l i v e r microsomes. Biochem. Pharmacol. 40: 1255-1261, 1990. Nash T. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J . 55: 416-421, 1953. Nebert DW, Nelson DR, Adesnik M, Coon MJ, Estabrook RW, Gonzalez FJ, Guengerich FP, Gunsalus IC, Johnson EF, Kemper B, Levin W, P h i l l i p s IR, Sato R, Waterman MR. The P450 superfamily: updated l i s t i n g of a l l genes and recommended nomenclature for the chromosomal l o c i . DNA 8: 1-13, 1989. Okey AB. Enzyme induction i n the cytochrome P-450 system. Pharmacol. Ther. 45: 241-298, 1990. 213 Okey AB, Bondy GP, Mason ME, Kahl GF, Eisen HJ, Guenthner TM, Nebert DW. Regulatory gene product of the Ah locus. Characterization of the c y t o s o l i c inducer-receptor complex and evidence for i t s nuclear t r a n s l o c a t i o n . J . B i o l . Chem. 254: 11636-11648, 1979. Omura T, Sato R. The carbon monoxide binding pigment of l i v e r microsomes. I I . S o l u b i l i z a t i o n , p u r i f i c a t i o n , and properties. J . B i o l . Chem. 239: 2379-2385, 1964. O r t i z de Montellano PR. Oxygen a c t i v a t i o n and t r a n s f e r . In: O r t i z de Montellano PR (ed.). Cytochrome P-450. Structure, Mechanism, and Biochemistry. New York: Plenum Press, 217-271, 1986. O r t i z de Montellano PR. Suicide substrates f o r drug metabolizing enzymes: mechanisms and b i o l o g i c a l consequences. In: Gibson GG (ed.). Progress i n Drug Metabolism, Vol. 11. London: Taylor & Francis Ltd., 99-148, 1988. O r t i z de Montellano PR, Correia MA. S u i c i d a l destruction of cytochrome P-450 during oxidative drug metabolism. Ann. Rev. Pharmacol. T o x i c o l . 23: 481-503, 1983. O r t i z de Montellano PR, Reich NO. I n h i b i t i o n of cytochrome P-450 enzymes. In: Or t i z de Montellano PR (ed.). Cytochrome P-450. Structure, Mechanism and Biochemistry. New York: Plenum Press, 273-314, 1986. Park SS, Fujino T, West D, Guengerich FP, Gelboin HV. Monoclonal antibodies that i n h i b i t enzyme a c t i v i t y of 3-methylcholanthrene-induced cytochrome P-450. Cancer Res. 42: 1798-1808, 1982. Pasanen M, Taskinen T, Sotaniemi EA, Kairaluoma M, Pelkonen O. I n h i b i t o r panel studies of human hepatic and placental cytochrome P-450 associated monooxygenase a c t i v i t i e s . Pharmacol. T o x i c o l . 62: 311-317, 1988. Pederson TC, Aust SD. Aminopyrine demethylase. K i n e t i c evidence f o r multiple microsomal a c t i v i t i e s . Biochem. Pharmacol. 19: 2221-2222, 1970. Pelkonen O, Puurunen J . The e f f e c t of cimetidine on i n vitro and in vivo microsomal drug metabolism i n the r a t . Biochem. Pharmacol. 29: 3075-3080, 1980. Pershing LK, Frankl i n MR. Cytochrome P-450 metabolic-intermediate complex formation and induction by macrolide a n t i b i o t i c s , a new class of agents. Xenobiotica 12: 687-699, 1982. 214 Pessayre D, Descatoire V, Konstantinova-Mitcheva M, Wandscheer JC, Cobert B, Level E, Banhamou JP, Jaouen M, Mansuy D. Sel f - i n d u c t i o n by triacetyloleandomycin of i t s own transformation into a metabolite forming a stable 456-nm absorbing complex with cytochrome P-450. Biochem. Pharmacol. 30: 553-558, 1981. P h i l l i p s o n CE, Ioannides C, Barrett DCA, Parke DV. The homogeneity of rat l i v e r microsomal cytochrome P-448 a c t i v i t y and i t s r o l e i n the a c t i v a t i o n of benzo[a]-pyrene to mutagens. Int. J . Biochem. 17: 37-42, 1985. Poland A, Glover E, Kende AS. Stereospecific high a f f i n i t y binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosols. Evidence that the binding species i s a receptor for induction of a r y l hydrocarbon hydroxylase. J . B i o l . Chem. 251: 4936-4946, 1976. Pounder RE. Histamine H 2-receptor antagonists and g a s t r i c acid secretion. Pharmacol. Ther. 26: 221-234, 1984. Puurunen J, Sotaniemi E, Pelkonen 0. E f f e c t of cimetidine on microsomal drug metabolism i n man. Eur. J . C l i n . Pharmacol. 18: 185-187, 1980. Rando RR. Mechanism-based enzyme i n a c t i v a t o r s . Pharmacol. Rev. 111-143, 1984. Reichen J, H o i l i e n C, Kirshenbaum GR. Cimetidine induces hepatic heme oxygenase a c t i v i t y without a l t e r i n g hepatic heme catabolism. Experientia 42: 942-945, 1986. Reidy GF, Mehta I, Murray M. I n h i b i t i o n of oxidative drug metabolism by orphenadrine: i n v i t r o and i n vivo evidence f o r isozyme-specific complexation of cytochrome P-450 and i n h i b i t i o n k i n e t i c s . Mol. Pharmacol. 35: 736-743, 1989. Reik LM, Levin W, Ryan DE, Maines SL, Thomas PE. Monoclonal antibodies d i s t i n g u i s h among isozymes of the cytochrome P-450b subfamily. Arch. Biochem. Biophys. 242: 365-382, 1985. R e i l l y PEB, Carrington LE, Winzor DJ. The i n t e r a c t i o n of cimetidine with r a t l i v e r microsomes. Biochem. Pharmacol. 32: 831-835, 1983. R e i l l y PEB, Ivey DE. I n h i b i t i o n by chloramphenicol of the microsomal monooxygenase complex of rat l i v e r . FEBS Le t t . 97: 141-146, 1979. 215 R e i l l y PEB, Mason SR, G i l l i a m EMJ. D i f f e r e n t i a l i n h i b i t i o n of human l i v e r phenacetin O-deethylation by histamine and four histamine H 2-receptor antagonists. Xenobiotica 18: 381-387, 1988. R e i l l y PEB, Winzor DJ. Adverse drug interactions with cimetidine: competitive i n h i b i t i o n of monooxygenase-dependent N-demethylation of morphine. Biochem. Pharmacol. 33: 1151-1153, 1984. Rekka E, Sterk GJ, Timmerman H, Bast A. I d e n t i f i c a t i o n of s t r u c t u r a l c h a r a c t e r i s t i c s of some po t e n t i a l H 2-receptor antagonists that determine the i n t e r a c t i o n with rat hepatic P-450. Chem.-Biol. Interact. 67: 117-127, 1988. R e l l i n g MV, Aoyama T, Gonzalez FJ, Meyer UA. Tolbutamide and mephenytoin hydroxylation by human cytochrome P-450s i n the CYP2C subfamily. J . Pharmacol. Exp. Ther. 252: 442-447, 1990. Rendic S, Kajfez F, Ruf HH. Characterization of cimetidine, r a n i t i d i n e , and related structures' i n t e r a c t i o n with cytochrome P-450. Drug Metab. Dispos. 11: 137-142, 1983. Rendic S, Ruf HH, Weber P, Kajfez F. Cimetidine and r a n i t i d i n e : t h e i r i n t e r a c t i o n with human and pig l i v e r microsomes and with p u r i f i e d cytochrome P-450. Eur. J . Drug Metab. Pharmacokinet. 9: 195-200, 1984. Rendic S, Sunjic V, Toso R, Kajfez F. Interaction of cimetidine with l i v e r microsomes. Xenobiotica 9: 555-564, 1979. Renton KW. Factors a f f e c t i n g drug biotransformation. C l i n . Biochem. 19: 72-75, 1986. Renton KW, Armstrong S, Hawa R. P r e t r a n s l a t i o n a l depression of cytochrome P4 50IA1 during the a c t i v a t i o n of host defence mechanisms. Proc. Can. Fed. B i o l . Soc. 34: 83, 1991. Riddick DS, McGilvray I, Marks GS. Inactivation of r a t l i v e r microsomal s t e r o i d hydroxylations by 4-alky1 analogues of 3,5-diethoxycarbonyl-l,4-dihyro-2,4,6-trimethylpyridine: evidence for s e l e c t i v i t y among steriod-inducible cytochrome P-450IIIA forms. Can. J . Physiol. Pharmacol. 68: 1533-1541, 1990. Rodrigues AD, Lewis DFV, Ioannides C, Parke DV. Spectral and k i n e t i c studies of the i n t e r a c t i o n of imidazole a n t i -fungal agents with microsomal cytochrome P-450. Xenobiotica 17: 1315-1327, 1987. 216 Ryan DE, Iida S, Wood AW, Thomas PE, Lieber CS, Levin W. Characterization of three highly p u r i f i e d cytochrome P-450 from hepatic microsomes of adult male r a t s . J . B i o l . Chem. 259: 1239-1250, 1984. Ryan DE, Levin W. P u r i f i c a t i o n and characterization of hepatic microsomal cytochrome P-4 50. Pharmacol. Ther. 45: 153-239, 1990. Ryan DE, Ramanathan L, Iida S, Thomas PE, Haniu M, Shively JE, Lieber CS, Levin W. Characterization of a major form of r a t hepatic microsomal cytochrome P-450 induced by i s o n i a z i d . J . B i o l . Chem. 260: 6385-6393, 1985. Ryan DE, Thomas PE, Korzeniowski D, Levin W. Separation and characterization of highly p u r i f i e d forms of l i v e r microsomal cytochrome P-450 from rats treated with poly-chlorinated biphenyls, phenobarbital and 3-methylcholan-threne. J . B i o l . Chem. 254: 1365-1374, 1979. Ryan DE, Thomas PE, Levin W. Hepatic microsomal cytochrome P-4 50 from rats treated with i s o s a f r o l e . P u r i f i c a t i o n and charac t e r i z a t i o n of four isozymic forms. J . B i o l . Chem. 255: 7941-7955, 1980. Ryan DE, Thomas PE, Levin W. P u r i f i c a t i o n and charac t e r i z a t i o n of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls. Arch. Biochem. Biophys. 216: 272-288, 1982a. Ryan DE, Thomas PE, Reik M, Levin W. P u r i f i c a t i o n , c h aracterization and regulation of f i v e r a t hepatic microsomal cytochrome P-450 isozymes. Xenobiotica 12: 727-744, 1982b. Schenkman JB, Wilson BJ, C i n t i DL. Diethylaminoethyl 2,2-diphenylvalerate HC1 (SKF 525-A) - i n vivo and in vitro e f f e c t s of metabolism by rat l i v e r microsomes -formation of an oxygenated complex. Biochem. Pharmacol. 21: 2373-2383, 1972. Schentag J J , Cerra FB, C a l l e r i GM, L e i s i n g ME, French MA, Bernhard H. Age, disease, and cimetidine d i s p o s i t i o n i n healthy subjects and c h r o n i c a l l y i l l patients. C l i n . Pharmacol. Ther. 29: 737-743, 1981. Schuetz EG, Guzelian PS. Induction of cytochrome P-450 by glucocorticoids i n r a t l i v e r . I I . Evidence that gluco-c o r t i c o i d s regulate induction of cytochrome P-450 by a nonclassical receptor mechanism. J . B i o l . Chem. 259: 2007-2012, 1984. 217 S e r l i n MJ, Sibeon RG, Mossman S, Breckenridge AM, Williams JRB, Atwood JL, Willoughby JMT. Cimetidine: i n t e r a c t i o n with o r a l anticoagulants i n man. Lancet 2: 317-319, 1979. Sesardic D, Boobis AR, Edwards RJ, Davies DS. A form of cytochrome P-450 i n man, orthologous to form d i n the r a t , catalyzes the 0-deethylation of phenacetin and i s inducible by cigarette smoking. Br. J . C l i n . Pharmacol. 26: 363-372, 1988. Sesardic D, Cole KJ, Edwards RJ, Davies DS, Thomas PE, Levin W, Boobis AR. The i n d u c i b i l i t y and c a t a l y t i c a c t i v i t y of cytochrome P-450c (P450IA1) and P450d (P450IA2) i n rat t i s s u e s . Biochem. Pharmacol. 39: 499-506, 1990a. Sesardic D, Edwards RJ, Davies DS, Thomas PE, Levin W, Boobis AR. High a f f i n i t y phenacetin O-deethylase i s catalyzed s p e c i f i c a l l y by cytochrome P450d (P450IA2) i n the l i v e r of the r a t . Biochem. Pharmacol. 39: 489-498, 1990b. Shimada M, Murayama N, Yamauchi K, Yamazoe Y, Kato R. Suppression i n the expression of a male-specific cytochrome P-450, P450-male: differences i n the e f f e c t of chemical inducers on P450-male mRNA and protein i n r a t l i v e r s . Arch. Biochem. Biophys. 270: 578-587, 1989. Slusher LB, Park SS, Gelboin HV, V e s e l l ES. Studies on the metabolism of aminopyrine, antipyrine and theophylline using monoclonal antibodies to cytochrome P-450 isozymes p u r i f i e d from r at l i v e r . Biochem. Pharmacol. 36: 2359-2367, 1987. Smith SR, Kendall MJ. Ranitidine versus cimetidine. A comparison of t h e i r p o t e n t i a l to cause c l i n i c a l l y important drug i n t e r a c t i o n s . C l i n . Pharmacokinet. 15: 44-56, 1988. Somogyi A, Gugler R. C l i n i c a l pharmacokinetics of cimetidine. C l i n . Pharmacokinet. 8: 463-495, 1983. Somogyi A, Muirhead M. Pharmacokinetic interactions of cimetidine 1987. C l i n . Pharmacokinet. 12: 321-366, 1987. Somogyi A, Rohner HG, Gugler R. Pharmacokinetics and b i o a v a i l a b i l i t y of cimetidine i n g a s t r i c and duodenal u l c e r p a r t i e n t s . C l i n . Pharmacokinet. 5: 84-94, 1980. Sonne J . Luhdorf K, Larsen NE, Andreasen PB. Lack of i n t e r a c t i o n between cimetidine and carbamazepine. Acta Neural. Scand. 68: 253-256, 1983. 218 Speeg J r KV, Patwardhan RV, Avant GR, M i t c h e l l MC, Schenker S. I n h i b i t i o n of microsomal drug metabolism by histamine H 2-receptor antagonists studied in vivo and in vitro i n rodents. Gastroenterology 82: 89-96, 1982. Steiner E, Spina E. Differences i n the i n h i b i t o r y e f f e c t of cimetidine on desipramine metabolism between rapid and slow debrisoquine hydroxylators. C l i n . Pharmacol. Ther. 42: 278-282, 1987. Stevens JC, Halpert J . Selective i n a c t i v a t i o n of four rat l i v e r microsomal androstenedione hydroxylases by chloramphenicol analogs. Mol. Pharmacol. 33: 103-110, 1988. Stockley C, Keal J, Rolan P, Bochner F, Somogyi A. Lack of i n h i b i t i o n of tolbutamide hydroxylation by cimetidine i n man. Eur. J . C l i n . Pharmacol. 31: 235-237, 1986. Tanaka E, Misawa S, Kuroiwa Y. E f f e c t s of cimetidine and diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A) on trimethadione metabolism i n the r a t . J . Pharmacobio-Dyn. 8: 767-772, 1985. Taylor DC, Cresswell PR, B a r t l e t t DC. The metabolism and elimination of cimetidine, a histamine H 2-receptor antagonist, i n the r a t , dog, and i n man. Drug Metab. Dispos. 6: 21-30, 1978. Testa B, Jenner P. Inhibitors of cytochrome P-450s and t h e i r mechanism of action. Drug Metab. Rev. 12: 1-117, 1981. Thomas PE, Bandiera S, Maine SL, Ryan DE, Levin W . Regulation of cytochrome P-450j, a h i g h - a f f i n i t y N-nitrosodimethylamine demethylase, i n r a t hepatic microsomes. Biochemistry 26: 2280-2289, 1987. Thomas PE, Reik LM, Ryan DE, Levin W. Regulation of three forms of cytochrome P-4 50 and epoxide hydrolase i n rat l i v e r microsomes. E f f e c t s of age, sex and induction. J. B i o l . Chem. 256: 1044-1052, 1981. Thomas PE, Reik LM, Ryan DE, Levin W. Induction of two immunochemically related rat l i v e r cytochrome P-450 isozymes, cytochromes P-450c and P-450d, by s t r u c t u r a l l y diverse xenobiotics. J . B i o l . Chem. 258: 4590-4598, 1983. Thomas RC, Ikeda GJ. The metabolic fate of tolbutamide i n man and r a t . J . Med. Chem. 9: 507-510, 1966. 219 Tukey RH, Johnson EF. Molecular aspects of regulation and structure of the drug-metabolizing enzymes. In: Pratt WB, Taylor P (eds.). P r i n c i p l e s of Drug Action. The Basis of Pharmacology. 3rd Ed. New York: C h u r c h i l l Livingstone, 423-467, 1990. V e s e l l ES. The antipyrine t e s t i n c l i n i c a l pharmacology: conceptions and misconceptions. C l i n . Pharmacol. Ther. 26: 275-286, 1979. Vestal RE, Cusack BJ, Mercer GD, Dawson GW, Park BK. Aging and drug i n t e r a c t i o n s . I. E f f e c t of cimetidine and smoking on the oxidation of theophylline and C o r t i s o l i n healthy men. J . Pharmacol. Exp. Ther. 241: 488-500, 1987. Vyas KP, Kari PH, Wang RW, Lu AYH. Biotransformation of l o v a s t a t i n . I I I . E f f e c t of cimetidine and famotidine on i n vitro metabolism of l o v a s t a t i n by rat and human l i v e r microsomes. Biochem. Pharmacol. 39: 67-73, 1990. Walsh C. Suicide sustrates: mechanism-based enzyme i n a c t i v a t o r s . Tetrahedron L e t t . 38: 871-909, 1982. Wang RW, Miwa GT, Argenbright LS, Lu AYH. In vitro studies on the i n t e r a c t i o n of famotidine with l i v e r microsomal cytochrome P-450. Biochem. Pharmacol. 37: 3049-3053, 1988. Waterman MR, John ME, Simpson ER. Regulation of synthesis and a c t i v i t y of cytochrome P-450 enzymes i n phy s i o l o g i c a l pathways In: O r t i z de Montellano PR (ed.). Cytochrome P-450. Structure, Mechanism and Biochemistry. New York: Plenum Press, 345-386, 1986. Watkins PB, Wrighton SA, Schuetz EG, Maurel P, Guzelian PS. Macrolide a n t i b i o t i c s i n h i b i t the degradation of the glucocorticoid-responsive cytochrome P-450p i n rat hepatocytes in vivo and i n primary monolayer culture. J . B i o l . Chem. 261: 6264-6271, 1986. Waxman DJ. Rat hepatic cytochrome P-450 ieoenzyme 2c. I d e n t i f i c a t i o n as a male-specific, developmentally induced s t e r o i d 16a-hydroxylase and comparison to a female-specific cytochrome P-450 isoenzyme. J . B i o l . Chem. 259: 15481-15490, 1984. Waxman DJ. Rat hepatic cytochrome P-450. Comparative study of multiple isozymic forms. In: Ortiz de Montellano PR (ed.). Cytochrome P-450. Structure, Mechanism and Biochemistry. New York: Plenum Press, 525-539, 1986. 220 Waxman DJ. Interactions of hepatic cytochromes P-450 with s t e r o i d hormones. Re g i o s e l e c t i v i t y and s t e r e o s p e c i f i c i t y of s t e r o i d metabolism and hormonal regulation. Biochem. Pharmacol. 37: 71-84, 1988. Waxman DJ, Attisano C, Guengerich FP, Lapenson DP. Human l i v e r microsomal steroi d metabolism: i d e n t i f i c a t i o n of the major microsomal steroi d hormone 6p~hydroxylase cytochrome P-450 enzyme. Arch. Biochem. Biophys. 263: 424-436, 1988a. Waxman DJ, Dannan GA, Guengerich FP. Regulation of rat hepatic cytochrome P-450: age-dependent expression, hormonal imprinting, and xenobiotic i n d u c i b i l i t y of sex-s p e c i f i c isoenzymes. Biochemistry 24: 4409-4417, 1985. Waxman DJ, Ko A, Walsh C. R e g i o s e l e c t i v i t y and stereo-s e l e c t i v i t y of androgen hydroxylations catalyzed by cytochrome P-450 isozymes p u r i f i e d from phenobarbital-induced rat l i v e r . J . B i o l . Chem. 258: 11937-11947, 1983. Waxman DJ, Lapenson DP, Park SS, Attisano C, Gelboin HV. Monoclonal antibodies i n h i b i t o r y to r a t hepatic cytochromes P-450: P-450 form s p e c i f i c i t i e s and use as probes from cytochrome P-450-dependent s t e r o i d hydroxylations. Mol. Pharmacol. 32: 615-624, 1987. Waxman DJ, LeBlanc"" GA, Morrissey J J , Staunton J, Lapenson DP. Adult male-specific and neonatally programmed r a t hepatic P-450 forms RLM2 and 2 a are not dependent on p u l s a t i l e plasma growth hormone for expression. J . B i o l . Chem. 263: 11396-11406, 1988b. Waxman DJ, Morrissey J J , LeBlanc GA. Female-predominant rat hepatic P-450 forms j (IIEl) and 3 (IIAl) are under hormonal regulatory controls d i s t i n c t from those of the sex - s p e c i f i c P-450 forms. Endocrinology 124: 2954-2966, 1989. Waxman DJ, Walsh C. Phenobarbital-induced r a t l i v e r cytochrome P-450. P u r i f i c a t i o n and characterization of two c l o s e l y r e l a t e d isozymic forms. J. B i o l . Chem. 257: 10446-10457, 1982. Waxman DJ, Walsh C. Cytochrome P-450 isozyme 1 from phenobarbital-induced r a t l i v e r : p u r i f i c a t i o n , c h a r a c t e r i z a t i o n , and interactions with metyrapone and cytochrome b 5. Biochemistry 22: 4846-4855, 1983. 221 Webster LK, Mihaly GW, Jones DB, Smallwood RA, P h i l l i p s JA, Vajda FJ. E f f e c t of cimetidine and r a n i t i d i n e on carbamazepine and sodium valproate pharmacokinetics. Eur. J. C l i n . Pharmacol. 27: 341-343, 1984. Weiner IM, Roth L. Renal excretion of cimetidine. J . Pharmacol. Exp. Ther. 216: 516-520, 1981. Wolfe MM, S o i l AH. The physiology of g a s t r i c acid secretion. N. Engl. J. Med. 319: 1707-1715, 1988. Wood AW, Ryan DE, Thomas PE, Levin W. Regio- and stereo-s e l e c t i v e metabolism of two C 1 9 steroids by f i v e highly p u r i f i e d and reconstituted r at hepatic cytochrome P-4 50 isozymes. J. B i o l . Chem. 258: 8839-8847, 1983. Wright AWE, Winzor DJ, R e i l l y PEB. Cimetidine: an i n h i b i t o r and an inducer of rat l i v e r microsomal cytochrome P-450. Xenobiotica 21: 193-203, 1991. Wrighton SA, Maurel P, Schuetz EG, Watkins PB, Young B, Guzelian PS. I d e n t i f i c a t i o n of the cytochrome P-450 induced by macrolide a n t i b i o t i c s i n rat l i v e r as the gl u c o c o r t i c o i d responsive cytochrome P-450p. Biochemistry 24: 2171-2178, 1985a. Wrighton SA, Schuetz EG, Watkins PB, Maurel P, Barwick J, Bailey BS, Hartle HT, Young B, Guzelian P. Demonstration i n multiple species of inducible hepatic cytochrome P-450 and t h e i r mRNAs rela t e d to the glucocorticoid-inducible cytochrome P-450 of the r a t . Mol. Pharmacol. 28: 312-321, 1985b. Yamazoe Y, Shimada M, Murayama N, Kato R. Suppression of l e v e l s of phenobarbital-induced rat l i v e r cytochrome P-450 by p i t u i t a r y hormone. J. B i o l . Chem. 262: 7423-7428, 1987. Yee NS, Shargel L. E f f e c t of cimetidine or r a n i t i d i n e pretreatment on hepatic mixed function oxidase a c t i v i t y i n the r a t . Drug Metab. Dispos. 14: 580-584, 1986. Yeowell HN, Waxman DJ, LeBlanc GA, Linko P, Goldstein JA. Suppression of male-specific cytochrome P-450 2c and i t s mRNA by 3,4,5,3',4',5'-hexachlorobiphenyl i n rat l i v e r i s not causally related to changes i n serum testosterone. Arch. Biochem. Biophys. 271: 508-514, 1989. 222 Yeowell HN, Waxman DJ, Wadhera A, Goldstein JA. Suppression of the c o n s t i t u t i v e , male-specific r a t hepatic cytochrome P-450 2c and i t s mRNA by 3,4,5,3',4',5'-hexachloro-biphenyl and 3-methylcholanthrene. Mol. Pharmacol. 32: 340-347, 1987. Zanger UM, V i l b o i s F, Hardwick JP, Meyer UA. Absence of hepatic cytochrome P-450bufI causes g e n e t i c a l l y d e f i c i e n t debrisoquine oxidation i n man. Biochemistry 27: 5447-5454, 1988. Zbaida S, Silman-Greenspan J, Yosselson-Superstine S, Merin E. In vitro studies on the metabolism of cimetidine by r a t l i v e r microsomes - i d e n t i f i c a t i o n of a new N-desmethylcimetidine metabolite. Biopharm. Drug Dispos. 5: 415-419, 1984. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0100471/manifest

Comment

Related Items