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Hepatic first-pass metabolism of metoclopramide in the rat Kapil, Ram Prakash 1982

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c. / HEPATIC FIRST-PASS METABOLISM OF METOCLOPRAMIDE IN THE RAT by RAM PRAKASH KAPIL B.Pharm; University of Delhi , 1977 M.Pharm; Banaras Hindu University, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences) Division of Pharmaceutics We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1982 @ Ram Prakash Kapi l , 1982 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f PjUaMON\^ CJAAI'CCLL S CtSjsTvC^A T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1W5 Date 13 IK fAo.il 1 ) g ) % £ , -7Q ^  i i ABSTRACT Metoclopramide (MCP), a procainamide analog, is a potent antiemetic and gastric motil ity modifier. C l i n i c a l l y , i t Is used in gastro-intestinal (GI) diagnostic examinations, treatment of various types of GI disorders and as a pre-and postoperative antiemetic-antinausea antinauseant agent. It has been found recently that, after a low i .v . dose of MCP (.$ 15 mg.kg"^) in rats, the nonrenal clearance approaches hepatic blood flow suggesting that the disposition of the drug may be perfusion limited. This further suggested that, following an oral dose, an appreciable extent of hepatic first-pass metabolism of MCP should occur. However, i t was observed by the previous workers that there was insignificant hepatic f irst-pass metabolism of MCP following oral dosing ^ 1 mg.kg~\ Hypotheses of temporary saturation of the binding sites and metabolic pathways during the f i r s t passage through the l iver as well as extrahepatic metabolism have been used to attempt to rationalize this observation. A four fold increase in the terminal ha l f - l i f e of MCP and a similar reduction in the total body clearance of MCP in rats as well as patients with renal impairment has been observed by previous investigators. These observations were unexpected since only about 20-25% of the MCP dose is excreted as intact drug in both the species suggesting that the renal elimination pathway is relat ively unimportant. i i 1 Hypotheses of reduced hepatic function secondary to renal injury or the loss of extrahepatic metabolic capabiltties due to kidney malfunction were made to explain the observed discrepancies. Prior to animal experimentation, an existing gas-liquid chromatographic-electron capture detector assay technique.for the simultaneous analysis of MCP and i t ' s mono-de-ethylated metabolite (De-MCP) was improved. The method was based on the acylation of these compounds with heptafluorobutyryl anhydride at 55°C for 60 minutes in the presence of 0.1 ml of 0.1M triethyl amine catalyst. A 1.8m x 2 mm ( i .d . ) glass column packed with 3% Silar-9CP on Chromosorfo- W ( H . P . ) ^ 100-120 mesh size was used. Complete baseline resolution between MCP and De-MCP was obtained. This thesis examines the lack of presystemic clearance (hepatic first-pass metabolism) and evidence of extra-hepatic metabolism of MCP, u t i l i z ing in-vivo and in-vi tro studies. The in-vivo experiments included two-third (2/3rd) hepatectomy and carbon tetrachloride (CCl^) pretreatment studies. An equal dose of MCP was administered intraperitoneally in test (2/3rd hepatectomised and CC1^ pretreated) as well as control (sham operated and normal saline pretreated respectively) rats, and 48 hour cumulative urinary excretion studies of intact MCP and De-MCP were made. In both studies, the total amount of intact MCP recovered from the 48 hour cumulative urine of test rats was signif icantly higher than iv that of control rats, This suggested that the rat l i ver Is involved in the metabolism of MCP. In-vitro studies involved incubation of equal amounts of J MCP (5 micromole. nil ) with various tissue homogenates ( v i z . , l i v e r , kidney and lung) and their 9000g supernatant fractions. Our studies suggest that the metabolism of MCP occurs in the rat l i ver and that De-MCP is further metabolised in the l i v e r . Also, there was no evidence suggesting the involvement of kidney or lung tissues in the metabolism of MCP. Forty-eight hour cumulative urinary excretion studies, following oral and intravenous administration of 0.1, 0.5, 1.0 and 5.0 mg.kg"^ of MCP were conducted. The bioavai labi l i ty (F) of MCP was found to be non-linear, i . e . the 'F ' value increased with increasing doses. This observation suggested that MCP undergoes dose-dependent hepatic f i r s t pass metabolism in the rat. An attempt to predict the bioavai labi l i ty of MCP, on the basis of a non-linear model was also made. The experimental values of MCP b ioavai labi l i ty , at different doses, were found to be in agreement with the predicted values, suggesting that the bioavai labi l i ty of MCP is non-linear and dose dependent. V TABLE. OF CONTENTS Chapter Page ABSTRACT i i LIST OF TABLES x LIST OF FIGURES xi LIST OF SCHEMES xiv SYMBOLS AND ABBREVIATIONS xv ACKNOWLEDGEMENT x v i i i INTRODUCTION - RATIONALE 1 1 LITERATURE SURVEY General 5 1,1 Pharmacodynamic studies on Metoclopramide (MCP) 6 1.1.1. Mechanism of Action (general 7 1.1.2. Mechanism of Antiemetic Action of ' Metoclopramide 9 1.1.3. Action on Pituitary Gland 1 0 1.1.4. Other Pharmacological Effects ^ 1.2, Cl in ica l Tr ia l s 1 2 1.2.1. Nausea and Vomiting ^ 1.2.2. Radiology 1 3 1.2.3. Gastrointestinal Intubation 13 1.2.4. Upper Gastrointestinal Endoscopy ^ 1.2.5. Gastric Stasis 1 4 1.2.6. Post Vagotomy 1 4 1.2.7. Migraine ^ 5 1,2.8 Diabetic Gastroparesis ^ 1.2.9. Gastric Ulceration ^ 1.2.10.. Reflux Esophagitis 1 6 1.2.11. Dyspepsia 1 7 1.2.12. Lactation 1 7 1.2.13. Anesthesia J 7 1.2.14. Radiation Sickness 1 8 1.2.15. Parkinson's Disease ] 8 1.2.16. Other Uses of Metoclopramide *° vi Chapter Page 1.3. Side Effects of Metocloprami.de 19 1.4. Pharmacokinetics of Metoclopramtde 21 1.4.1. Rate of Absorption 21 1.4.2. Distribution 2 2 1.4.3. Metabolism 2 4 1.4.4. Excretion 2 5 1.4.5. Systemic Bioavai labi l i ty of Metoclopramide 2 8 1.5. The Concept of Drug Bioavai labi l i ty 2 9 1.6. Pharmacokinetic Models of First-Pass ' ' Metabolism 35 1.6.1. Linear Models 3 5 1.6.1.1. Compartmental Approach 3 5 1.6.1.2. Perfusion Model Approach 3 9 1.6.2. Non-Linear Model 43 1.7. Analysis of Metoclopramide in Biological Fluids 4 4 2 EXPERIMENTAL 4 9 2.1. Supplies 4 9 2.1.1. Chemicals 4 9 2.1.2. Reagents 4 9 2.1.3. Solvents 5 0 2.1.4. Supplies for rat experiments 50 2.2. Equipment 5 0 2.3. Stationary Phases and Solid Supports 51 2.3.1. Commercially coated products 51 2.3.2. Stationary Phases 51 2.3.3. Solid Supports 52 2.4. Preparation of Hydrochloride Salt of De-MCP Base 52 2.5. Solutions 52 v i i Chapter • .'Page 2 , 5 J . Preparation of Stock and Dilute Solutions of MCP.HCl.HoO and De-MCP.HCl 52 2.5.2. Preparation of Internal Standard (Diazepam] Solution 53 2.5.3. Preparation of 0.02M, pH 7.4 phosphate Buffer Solution 53 2.6. General Procedure for Phase Coating 54 2.7. General Method of Column Cleaning and Phase Packing 55 2.8., General Method of Column Conditioning 56 2.9. Preliminary GLC-ECD Analysis 56 2.9.1. Phase Selection 57 2.9.2. Optimisation of HFBA Derivatization of De-MCP 59 2.10. Animal Studies 59 2.10.1. General Animal Handling 59 2.10.2. In-Vivo Rat Experiments gg 2.10.2.1. Techniques of Drug -Administration 60 2.10.2.2. Urine Sample Collection 62 2.10.2.3. Two-Third Hepatectomy Study 6 2 2.10.2.4. Carbon Tetrachloride Pretreatment Study 6 4 2.10.2.5. Bioavai labi l i ty Experiments 65 2.10.3. In-Vitro Metabolism Study 65 2.10.3.1. Preparation of Tissue Homogenates 65 2.10.3.2. Incubation Experiments with Whole Tissue Homogenetes and their 9000g Supernatants 66 2.10.3.3. Extraction Procedure for Incubation Mixture 67 2.11. Bioavai labi l i ty Calculations and Stat is t ical Analysis 67 vi i i Chapter Page 3 RESULTS 68 3.1. GLC-ECD Analysts 68 3.2. In-Vivo Studies 8 8 3.2.1. Two-Third Hepatectomy Study 8 8 3.2.2. Carbon Tetrachloride Pretreatment Study 95 3.3. In-Vitro Studies 9 5 3.3.1. 9000g Liver and Kidney Homogenate Study 95 3.3.2. 9000g Liver and Lung Homogenate Study 100 3.3.3. In-Vitro Experiments with Whole Fraction Tissue Homogenate 100 3.3.4. In-Vitro Hepatic Metabolism of De-MCP 105 3.4. Bioavai labi l i ty of MCP as a Funtion of Dose 105 4 DISCUSSION 121 4.1. GLC-ECD Analysis 121 4.1.1. Phase Selection 121 4.1.2. Optimisation of HFBA Derivatization of De-MCP 122 4.1.3. Appl icabi l i ty of the Assay Method 123 4.2. Animal Experiments 124 4.2.1. In-Vivo Experiments 124 4.2.1.1. Two-Thirds Hepatectomy Study 124-4.2.1.2. Carbon Tetrachloride Pretreatment Study 125 4.2.1.3. Bioavai labi l i ty Experiments 127 4.3. Prediction of Bioavai labi l i ty 129 4.3.1. Linear Models 129 4.3.2. Non-Linear Model 131 5 SUMMARY AND CONCLUSIONS 134 6 REFERENCES 136 i x Chapter Page 7 APPENDIX 156 7.1. Indteattons 156 7.2. Contraindications 156 7.3. Precautions 157 7.4. Treatment of Overdosage and Toxic Effects 160 7.5. Dosage and Administration (A.H. Robins, Canada, 1981) 160 7.6. Preparations (E.P.77; B.P.80; C.P.S. 81;P.P.G.8}) 161 7.7. Non-Linear Bioavai labi l i ty 162 X LIST OF TABLES Table Page 1 Excretion of ^C-Metoclopramide (percent] in Rats, Dogs and Humans 27 2 Lis t of physiological factors which can modify bioavai labi l i ty 31 3 List of substances subject to hepatic first-pass metabolism 34 4, General gas l iquid chromatographic conditions 73 5. Coefficient of variation of MCP and De-MCP 8 5 6 Non-linear, dose dependent bioavai labi l i ty of MCP 120 7 Theoretical Km values generated by iterative technique 133 8 Comparison of predicted and experimental values of MCP bioavai labi l i ty 133 9 Lis t of drugs where rate of gastrointestinal absorption is increased in the presence of metoclopramide 159 xi LIST OF FIGURES F igure P a g e 1 S t r u c t u r e o f metoclopramide 3 2 Hepat ic f i r s t - p a s s pharmacok inet ic models 3 7 3 Diagram i l l u s t r a t i n g the dev i ce employed i n c o l l e c t i n g u r i n e samples from r a t s w i thout f a e c a l contaminat ion 4 Chromatogram of the HFB d e r i v a t i v e o f MCP (R .T . = 3 . 0 3 m i n . ) and u n d e r i v a t i z e d diazepam ( R . T . = 5 .48 m i n . ) on a 3% 0V-17 packed g l a s s column. 5 Chromatogram o f the HFB d e r i v a t i v e s o f MCP ( R . T . = 6 .72 m i n . ) and De-MCP (R .T . = 7 .19 m i n . ) , and u n d e r i v a t i z e d diazepam ( R . T . = 16.63 m i n . ) on a 3% 0V-225 packed g l a s s column. 71 6 Chromatogram o f the HFB d e r i v a t i v e s o f De-MCP (R .T . = 20.65 min . ) and MCP (R .T . = 21.67 m i n . ) , and u n d e r i v a t i z e d diazepam ( R . T . = 27.57 m i n . ) on a SE-30 g l a s s c a p i l l a r y column. 7 5 7 B a s e l i n e r e s o l u t i o n o f the HFB d e r i v a t i v e s of MCP and De-MCP, and u n d e r i v a t i z e d diazepam on 3% S i l a r 9CP packed g l a s s column. 7 7 I R e p r e s e n t a t i v e Chromatogram of HFBA d e r i v a t i z e d d i l u t e blank r a t u r i n e e x t r a c t . II Representa t i ve chromatogram o f a s y n t h e t i c mix ture o f the HFB d e r i v a t i v e s of MCP (R .T . = 4 . 6 5 m i n . ) and De-MCP (R .T . = 6.91 min . ) - , and u n d e r i v a t i z e d diazepam ( R . T . = 9 .87 m i n . ) . 8 K i n e t i c s o f HFBA d e r i v a t i z a t i o n o f De-MCP i n the absence ( — . ) and p r e s e n c e " ^ - o f t r i ethyl amine (TEA) c a t a l y s t . 80 x i i Figure 9 Gas chromatographic peaks representing trace levels of the HFB derivatives of MCP and De-MCP and underivatized diazepam. 10 Representative chromatograms of HFB derivatives of MCP (R.T. - 6.26 min.) and De-MCP (R.T. = 9.08 min.) , and underivatized diazepam (R.T. = 17.58 min.) , i l lus trat ing quantitative reproducibil i ty of peaks and the retention times of the peaks obtained from duplicate samples, extracted and derivatized under similar conditions. 11 Calibration curve of the urine extract of MCP. 12 Calibration curve of the urine extract of De-MCP. 13 Histograms i l lus tra t ing the percent recovery of 48 hour cumulative urinary excretion of intact MCP and De-MCP (equivalent to MCP) in two-third hepatectomised and sham operated rats. An I.P. dose equivalent to 15 mg.kg-1 MCP base was adminis tered in both groups of rats. 14 Percent recovery of 48 hour cumulative urinary excretion of intact MCP and De-MCP (equivalent to MCP) in CCl/. pretreated and normal saline (N.S.) pretreated rats. An I.P. dose equivalent to 1.0 mg.kg-1 MCP base was administered in both groups of rats. 15 Representative gas chromatograms showing the different levels of MCP obtained from various incubation mixtures at the end of each incubation reaction ( I n i t i a l l y , each incubation mixture contained an equal amount of MCP). 16 Histograms representing the area ratios (derivative/diazepam) obtained from the in-vi tro metabolism of MCP in the absence and presence of 9000 g supernatants from l iver and kidney homogenates. 17 Histograms representing the area ratios (derivative/diazepam) obtained from the in--vitro metabolism of MCP in the absence and presence of 9000 g supernatants of l iver and lung homogenates. Page 83 86 89 91 93 96 98 101 103 xi i i Page Figure 18 Histograms representing the area ratios (derivative/diazepam) obtained from the in-v i tro metabolism of MCP in the absence and presence of different volumes of whole tissue homogenates of l iver (2.0 ml), kidneys (0.5, 1.0, 1.5 and 2.0 ml) and lungs (0.5, 1.0, 1.5 and 2.0 ml). 106 19 Histograms representing the area ratios (derivative/diazepam) obtained from the in-v i tro metabolism of De-MCP in the absence (control) and presence (test) of 9000 g supernatant of l i ver homogenate. 108 20 Histograms comparing the percent recovery of 48 hour cumulative urinary excretion of MCP and De-MCP (equivalent to MCP) following an oral (p.o.) or intravenous ( i . v . ) dose (MCP. HC1. H20) equivalent to 5mg.k.g-lM C P b a s e > 111 21 Histograms comparing the percent recovery of 48hour cumulative urinary excretion of MCP and De-MCP (equivalent to MCP) following an p.o. or f;v. dose (MCP. HC1. H90) equivalent to 1.0 mg. kg-1 MCP base. c 1 1 3 22 Histograms comparing the percent recovery of 48 hour cumulative urinary excretion of MCP and De-MCP (equivalent to MCP) following an p:;o; or i . v . dose (MCP. H C 1 . H70) equivalent , , c to 0.5 ,-g. ,g-l MCP base. 1 1 b 23 Histograms comparing the percent recovery of 48 hour cumulative urinary excretion of MCP and De-MCP (equivalent to MCP) following an p.o. or i . v . dose (MCP. H C L H 2 O ) equivalent to 0.1 mg.kg-1 MCP base. 1 1 8 X I V LIST OF SCHEMES Scheme Page A schematic of metabolites of metoclopramide recovered from rabbit urine. 26 Modified gas l iquid chromatographic-electron. capture detector assay technique for the simultaneous quantitation of metoclopramide and i t ' s mono-de-ethyl ated metabolite _ Scheme of Extraction and HFBA derivatization. 82 XV SYMBOLS AND ABBREVIATIONS ADP adenosine diphosphate ATP adenosine triphosphate AUC.J area under the plasma concentration Versus time curve * " after intravenous administration AUCQ area under the plasma concentration versus time curve after oral administration CCl^ carbon tetrachloride C l j B total body clearance CNS central nervous system CTZ chemoreceptor trigger zone C V . coefficient of variation D dose in milligrams De-MCP mono-de-ethylated metabolite of metoclopramide De-MCP. HCL mono-de-ethylated metoclopramide monohydrate Diaz diazepam (internal standard) ECD electron capture detector F bioavai labi l i ty (systemic) F' fraction of the administered dose actually absorbed fm fraction of the administered drug metabolised by the l iver g acceleration due to gravity (cm.sec, ) GI gastrointestinal GIT gastrointestinal tract GLC gas-liquid chromatography (or chromatographic) g gram. xvi HFB heptafluorobutyryl (or butyric) HFBA heptafluorobutyric anhydride H.P. high performance HPLC high performance l iquid chromatography (or chromatograph) hr hour HVA homovanillic acid i . d . internal diameter i.m. intramuscular i . p . intraperitoneal i . v . intravenous Ka appart f i r s t order absorption rate constant Km Michael is Menten Constant in mass units K-J2 transport rate constant from compartment 1 to compartment 2 Kpi transport rate constant from compartment 2 to compartment V. LES or LOS lower esophageal (Oesophageal) sphincter M molar (mole/litre) MCP metoclopramide ml m i l l i l i r e s MCP.HCl.H2O metoclopramide monohydrochloride monohydrate mg milligrams min minutes MS mass spectrometer (or spectrometry) xvii pNADP.Na mono sodiurn-g-nicotinamide adenine dinucleotide ng nonogram peg picograms p.o. per os (oral route) QL hepatic ( l iver) blood flow rate r correlation coefficient rpm revolutions per minute R resolution R.T. retention time SCOT support coated open tubular ±S.D. ±one standard deviation T time interval for portal blood circulation (=1 minute) th or lh biological ha l f - l i f e T time at which maximum concentration of drug in the plasma occurs following a single oral dose TCA trichloroacetic acid TEA triethyl amine TLC thin layer chromatography (or chromatographic) V- theoretical maximum rate of process describable by Michaelis-Menten kinetics , expressed in mass' terms E X U q orXUgnn, cumulative amount of unchanged drug ultimately 0 excreted in the urine after oral administration zXu. v orXU| cumulative amount of unchanged drug ultimately excreted 0 ' ' * Tn the urine after intravenous administration. 5 z cumulative McReynolds' constants (benzene, 1 1-butanoo, 2-pentanone, nitropropane and pyridine) xvi i i ACKNOWLEDGEMENT The author wishes to thank Dr, James Axel son for suggesting the problem, providing f a c i l i t i e s and for his supervision during the course of study. The author is grateful to Dr. Yun Tarn and Mrs. Barbara McErlane for their assistance during the analytical and animal experimentation. The author would l ike to acknowledge Dr. Frank Abbott, Dr. Keith McErlane, Dr. Jim Orr, Dr. Helen Burt and Dr. Basil Roufogalis for their help and encouragement. The author is deeply indebted to Wayne Riggs for his invaluable time, kindness and constant support throughout the project. This work was supported by the Medical Research Council, the Kidney Foundation and the University of Bri t i sh Columbia Research Grants. x ix Papaj i and Mummyj 1 INTRODUCTION Metoclopramide CMCP), a procainamide analog, is a. putative dopamine receptor antagonist. MCP is a potent antiemetic and gastric motil ity modifier which is used to fac i l i ta te radiological examinations, to al ter drug absorption (rate and/or extent) and to treat nausea and vomiting secondary to various pathological states. MCP has been widely used for more than a decade in Europe and has been recently introduced to Canada,, (Compendium of Pharmaceuticals and Special i t ies , 1975). Despite the substantial work done on the pharmacodynamics of MCP and i t ' s extensive use over the years, pharmacokinetic studies of this drug started appearing in the l i terature only recently. This delay was due to a lack of specific and sensitive assay methods for MCP quantitation in the biological f lu ids . Recently, various thin layer chromatographic (TLC)(Huizing 'et a l , , 1979; Schuppan et a l . , 1979), gas l iquid chromatographic-electron capture detector (GLC-ECD) (Bateman et a l . , 1978; Tarn et a l . , 1978, 1979a; Lee et a l . , 1980) and high performance l iquid chromatography (Teng et a l . , 1977; Graffner et a l . , 1979; Bateman et a l . , 1981, Block et a l . , 1981) assay methods for the quantitation of MCP in biological fluids have appeared in the l i terature. Tarn and Axel son (1979b) have also reported a GLC-ECD method for the simultaneous quantitation of MCP and i t ' s mono-deethylated metabolite (De-MCP) in rat urine. However, complete resolution was not achieved between the chromatographic peaks of these two compounds. 2 Tarn et a l . , (1981a;) have shown that the total body clearance of MCP in the rat (C1 T B = 50 ml/min/kg) approaches l iver blood flow. Based on the high clearance values from the intravenous data and assuming that MCP is mainly cleared by the l i v e r , Tarn et a l . , (1981a) have proposed that extensive hepatic f i r s t pass metabolism should occur after oral administration of the drug. However, MCP does not appear to undergo hepatic f i r s t pass metabolism in the rat (Tarn et a l . , 1981a) over the dosage range studied (Img to 35mg. kg~^). The- following hypotheses were proposed to explain the observed discrepancy: (1) There Is extra hepatic metabolism of MCP in the rat , and/or (2) The concentration of MCP in the hepatoportal vein during the absorption phase is high enough to saturate the l iver enzymes. Bateman and his coworkers (1980 and 1981) have reported a four fold increase in the biological half l i f e (T^) of MCP and a corresponding reduction in the Cl-j-g of MCP in patients with chronic renal fa i lure . A similar observation was made by Tarn et a l . , (1981b) in uremic rats. Both these observations are unexpected since only about 20-25% of the dose is excreted as intact drug in both the rat and human urine suggesting that the renal elimitation pathway is re lat ive ly unimportant. Based upon the renal dysfunction studies in the rat , Tarn et a l . , (1981b) proposed two hypotheses: 3 (1) the rat kidney tissue Is involved in excretion and metabolism of MCP, and/or (2) the metabolic capacity of the l iver is reduced in renal fa i lure . The objective of this study was to test the hypotheses proposed by previous workers u t i l i z ing the modified analytical technique for the quantitation of MCP and De-MCP in rat urine. The specific aims of the project were as follows: (1) to achieve quantitative separation of MCP and De-MCP by GLC-ECD technique, (2) to conduct -in-vivo experiments to obtain information regarding the involvement of rat l iver as the metabolic organ for MCP. This portion of the study included carbon tetrachloride pretreatment studies and two-third hepatectomy studies, (3) to investigate in -v i tro hepatic and extra-hepatic metabolism of MCP in the rat. This study involved incubation of various tissue homogenates and 9000 g supernatants ( l iver , lung and kidney), (4) to determine the bioavai labi l i ty of MCP using urinary excretion data over a wide range of low dosage levels (0.1, 0.5, 1.0 and 5.0 nig* kg"])• The 48 hour cumulative : 4 rat urine after oral administration of an equal dose was compared with the intravenous route of administration, and (5) to attempt to correlate the experimental values for bioavai labi l i ty to the predicted values calculated by using a recently published expression (Keller and Scholle, 1981) for non-linear b ioavai labi l i ty . 5 1. LITERATURE SURVEY Metoclopramide (MCP), 4-amino-5-chloro-2 methoxy-N-(2-di ethyl ami noethyDbenzamide, was introduced by Justin-Besancon and his coworkers in 1964. It's chemical structure is shown in F i g . l . Although structurally related to procainamide, MCP has negligible cardiac effects (Thorburn and Sowton, 1973) and poor local anaesthetic act iv i ty (Cheymol and Movilk, 1975). However, i t has been observed that MCP is a very potent anti-emetic agent and that i t has profound effects on the gastro-intestinal tract (Justin-Besancon and L a v i l l e , 1964; Margieson et a l . , 1966; Martin and Scobie 1967; Eisner, 1968, 1971; James and Humes 1969; Ramsbottom and Hunt, 1970; HowelIs et a l , 1971; Johnson, 1971a, 1973; Kreel, 1973; Stanciv and Bennet, 1973; Hancock et a l . , 1974; Meeroff, 1974; Paul 1 and Grant, 1974; McCallum et a l . , 1975, 1976a; Schmidt et a l . , 1978; Bauman et a l . , 1979; N-(DIETHYLAMIN0ETHYL)-4-AMIN0-5-CHL0R0-2-METHOXYBENZAMIDE (METOCLOPRAMIDE) Figure 1 6 Berkowitz and McCallum, 1980; Cotton and Smith, 1981). Excellent review art ic les on MCP have appeared in the l i terature (Robinson, 1973; Pinder et a l . , 1976; Smith and Salter, 1980; Riley, 1980; Clark, 1980; Ponte and Nappi, 1981 and Schulze-Delrieu, 1981). 1.1. Pharmacodynamic Studies The principal pharmacologic effects of MCP in humans involve the gastro-intestinal tract (GIT) and central nervous system (CNS). MCP has the following effects on the GIT, : (1) Improves resting tone of the oesophageal sphincter; i t increases the lower esophageal sphincter (LES) pressure and the force of per i s ta l t ic contractions without any effect on relaxation in man (Heitman and M i l l e r , 1970; Bremner and Bremner, 1972; Guelrud, 1974; Bauman et a l . , 1979; Cotton et a l . , 1981). (2) Improves gastric tone and peristals is of the stomach (increased gastric antral contractions) with accelerated gastric emptying (James and Humes, 1969; Johnson, 1973; Schmidt et a l . , 1978). (3) Enhances the pyloric sphincter pressure (Valenzuela, 1976) thus enhancing the pyloric act iv i ty . (4) Distends the duodenal bulb (Pinder et a l . , 1976), and (5) Enhances peristals is of the duodenum with accelerated transit through the duodenum and jejunum (Margieson et a l . , 1966; James and Humes, 1969; Johnson, 1973). 7 MCP has. l i t t l e effect on colonic motor act iv i ty in-vivo (Eisner, 1968, 1971 ; Banke et a l . , 1972; Hancock et a l . , 1974). It does not stimulate gastric acid secretion (Connell and George, 1969; McCallum et a l . , 1975; Meeroff et a l . , 1974). 1.1.1. Mechanism of action The precise mechanism of action of MCP in the GIT remains uncertain. '-Esophageal and gastric contractions induced by MCP are blocked by anticholinergic drugs such as atropine and potentiated by cholinergic drugs such as carbachol and methacholine (Jacoby and Brodie, 1967; Johnson, 1971b; Anderson and Watson, 1977). MCP has no anticholine-sterase act iv i t ies (Eisner, 1968) and i t ' s actions are unaffected by ganglionic blocking agents such as chlorisondamine. MCP has no action on the isolated human smooth muscles from the body or the antrum of the stomach, however, i t sensitizes the preparations to acetylcholine (Eisner, 1968). It is postulated that MCP acts via.the intramural cholinergic neurons responsible for modifying gastric motil i ty but not gastric secretion (Eisner, 1968). A recent study showed that MCP part ia l ly and s ignif icantly reduces the relaxation effect of adenosine-5-triphosphate (ATP), adenosine diphosphate (ADP) and adenosine, but potentiates the effect of noradrenaline on the atropine pretreated taenia c o l i , rabbit ileum and rat duodenum (Okwuasaba and Hamilton, 1975). The inhibit ing effect of ATP, ADP and adenosine on the pperistalsis of guinea-pig ileum was decreased, the 8 effect of adrenaline was potentiated and the effect of theophylline ethylene diamine was not affected by MCP. This specification of MCP was postulated to be due to i t ' s sensitive blockade, at the post synaptic s i tes , of the effect of the inhibitory purinergic transmitter (ATP and related nucleotides) released during persitalsis. (Okwuasaba and Hamilton, 1975). Such action of MCP in antagonising the action of the in tr ins ic inhibitory mechanism may well be complementary to the documented muscarinic sensitizing action reported by others (Jacoby and Brodie, 1967; Birt ley and Baines, 1973 and Fontaine and Reuse, 1974). In man, the gastric emptying time prolonged by L-dopa was reduced by MCP action on the GIT. This might be partly due to the antidopaminergic effect of MCP (Berkowitz and McCallum, 1980). According to Bauman et a l . , (1979) MCP acts on the LES by blocking the dopaminergic pressure lowering mechanism. The effects of MCP on the CNS include: (a) an antiemetic effect in animals (Pinder et a l . , 1976) and in man (Klein et a l . , 1968, Dobkin et a l . , 1968; Pinder et a l . , 1976) (b) Stimulation of prolactin secretion from the anterior pituitary gland both in animals (Carlson et a l . , 1977; J iro et a l . , 1977) and in humans (McCallum et a l . , 1976b; Delitala et a l . , 1976; :Judd et a l . , 1976; Healy and Burger, 1977; Ogihara et a l . , 1977; Sowers et a l . , 1977; Huizing et a l . , 1979a), Ijaiya, 1980; Broumers et a l . , 1980; Kitaoka et a l . , 1980; Brandes et a l . , 1981). 9 (c) release of aldosterone from the adrenal glands in rhesus monkeys (Sowers et a l . , 1981) and in humans (Norbiato et a l . , 1977; Carey et a l . , 1979,1980; Brown et a l . , 1979). However, this action was not found in dogs or rabbits (Sowers et a l . , 1981). MCP may also have antipsychotic act iv i ty in very large doses (lg.day"1)!Stanley et a l . , 1979). 1.1.2. Mechanism of antiemetic action There is evidence that the anti-emetic action of MCP may be mediated through the chemoreceptor trigger zone (CTZ) for vomiting. MCP raises i t ' s threshold of act iv i ty and prevents vomiting caused by central emetics (hydergine and apomorphine), and also, decreases the sensit iv i ty of visceral nerves which transmit Gl impulses to the central emetic center (Justin-Besancon and Lav i l l e , 1964; Klein et a l . , 1968). Cannon (1975) has reported that the stimulation of the CTZ is specific to dopamine-1ike drugs and MCP's effect in the CTZ is believed to be due to i t ' s dopamine antagonistic property. More evidence has been accumulated to support this view. It was found that after intraperitoneal injection, MCP had no effect on the dopamine concen-tration in the whole brain, but i t increased the homovanillic acid (HVA) (a dopamine metabolite) concentration both in the corpus striatum and in the mesolimbic area (Peringer et a l . , 1976). It has been postulated that MCP, as a result of dopamine receptor blockade, causes an increase in f i r ing of the dopaminergic neurones (Peringer et a l . , 1975, 1976;.Hay, 1975; Blower, 1975; Dolphin et a l . , 1975). This leads to 10 increased turnover of dopamine ( E l l i o t t et a l . , 1977). The prevention by MCP of copper sulphate emesis suggests that the drug also has a peripheral mode of action (Lav i l l e , 1964). As MCP markedly increases gastric motor act iv i ty in both man and animals and this effect probably prevents the gastric immobility which precedes the act of vomiting (Pinder et a l . , 1976). 1.1.3. Action of MCP on Pituitary gland MCP induced stimulation of prolactin secretion from the anterior pituitary gland may be a result of blocking the dopamine receptors in the hypothalamus and decreasing the prolactin inhibiting factor (Judd et a l . , 1976). This effect of MCP is abolished by CB154, a dopaminergic stimulant (Oiro et a l . , 1977). Further evidence for MCP as a dopamine receptor antagonist has been provided by Lautin et a l . (1980). The increase in plasma aldosterone concentrations in humans after MCP administration results from an increase in aldosterone secretion rather than a change in aldosterone metabolic clearance (Brown et a l . , 1979, 1981). MCP appears to affect aldosterone secretion by i t ' s known actions as a dopamine antagonist (Peringer et a l . , 1976) since dopamine administered simultaneously with MCP prevents the rise in plasma aldosterone xonceiitrations(Noth et_al_., 1980; Carey et a l . , 1980). Sowers et a l . , (1980) suggested that dopaminergic modulation of plasma aldosterone secretion occurs independently of the renin angiotensin system. Pratt et a l . (1981) have recently shown that neither the kidney nor the pituitary is involved in the mechanism for MCP stimulated 11 aldosterone production. However their observations suggest that MCP may act on the adrenals to evoke secretion of aldosterone. The antipsychotic act iv i ty of MCP in very large doses - l v (1 g.day. 1 may be due to dopamine receptor blocking act iv i ty (Stanley et a l . , 1979) . Several groups are attempting to find the exact nature of the dopamine antagonist effect of MCP. The apparent Tack of neuroleptic act iv i ty of MCP and i t ' s inabi l i ty to inhibit the ih-v i tro dopamine stimulated adenylate cyclase system suggest that the mechanism by which i t antagonises cerebral dopamine system is fundamentally different from that of classical neuroleptics ( E l l i o t et a l . , 1977). In addition to the dopaminergic antagonist ac t iv i ty , MCP has been reported to block serotonergic pathways (Fang, 1981). It is a potent antagonist of both s tr iata l (Jenner et a l . , 1975) and peripheral (Goldberg et a l . , 1978; Brodde and Schemutt, 1978) dopamine receptors. It is an effective antagonist of cardiovascular (Day, 1975) and renal (Hahn and Wardell, 1980) dopamine receptors. "MCP .has also-been.reported to.be acselective blocking agent of pre-and post-synaptic dopamine receptors (Alander et a l . , 1980). 1.1.4. Other pharmacological effects MCP increases urinary excretion of potassium in normal volunteers (Bevilacqua et a l . , 1980). The investigators believe that the dopaminergic system has a role in the control of serum potassium. 12 MCP does not influence the secretion of pituitary glycoprotein hormones (Spitz et a l . , 1979). It has been shown that MCP is a pre-synaptic and adrenoceptor antagonist in the rat vas deferens (Spedding, 1980). 1.2. Cl in ical Tr ia l s MCP has been in c l in i ca l use in Europe since 1964. There are more than 1500 art ic les published on MCP, the majority of which describe results of c l in ica l observation and evaluation. 1.2.1. Nausea and Vomiting Extensive studies over the past two decades indicate that MCP is very effective in the symptomatic management of nausea and vomiting of varied orig in . In 82 patients, in a dosage of 30 to 50 Mg daily of MCP by mouth or by intramuscular injection, reduced the intensity and severity of post operative nausea and vomiting (Trafford, 1967). Similar results were reported by other investigators (Handley, 1967; Lind and Brevik, 1970; Cooke et a l . , 1979; Boghaert et a l . , 1980; Diamond and Szanto, 1980). According to Dundee and Clark (1973), pre-operative nausea and vomiting are not s ignif icantly relieved by MCP. But, Assaf et a l . , (1974) have reported that MCP given with pethidine or morphine pre-operatively reduced the incidence of post operative nausea and vomiting. 13 MCP, with fewer side effects, has been found to be equal or superior to the established antiemetics such as trimethobenzamide (Dobkin et a l . , 1968) and phenothiazines (McGarry, 1971; Robinson, 1973). Intravenous administration of high doses (1-3 mg.kg"^ body weight) of MCP have been found to beneficial in the prevention of nausea and vomiting associated with chemotherapy. (Gralla et a l . . , 1981; Green and Brown, 1981). 1.2.2. Radiology Radiological barium meal examination is usually a time consuming and expensive procedure, particularly for those patients who have pyloric obstruction which makes visualization of the duodenum more d i f f i c u l t . MCP, accelerates gastric emptying by increasing per is ta l t ic act iv i t ies and relaxes the pyloric canal thus greatly fac i l i ta t ing the examination, reducing the time required and minimizing radiation exposure (Howarth et a l . , 1969; James and Melrose, 1969; Kreel, 1970). A further benefit is that nausea and regurgitation of barium suspension are prevented. MCP is also useful in shortening the duration of small bowel transit (James and Hume, 1969). 1.2.3. Gastro-intestinal Intubation A 10 Mg dose of MCP given . v.m. or i . v . fac i l i tated the passage of a biopsy capsule or aspirating catheter through the pylorus into the proximal jejumum(Pirola, 1967; Bol in , 1969) and duodenum (Delcourt and Wettendorff, 1968). This is due to the relaxant 14 effect of MCP on the pyloric sphincter. 1.2.4. Upper gastro-intestinal endoscopy. Intravenous administration of MCP is useful during emergency endoscopy for upper gastro-intestinal hemorrhage, part icularly when blood and other gastric contents have restricted the visual f i e ld (Bader, 1973). MCP acts by increasing the strength of ^peristalsis of the gastro-intestinal tract. 1.2.5. Gastric stasis Gastric stasis is a condition associated with sensations of abdominal fullness and dis tent ion sometime accompanied by nausea and vomiting (Clarke, 1980). It may be present in association with a number of underlying conditions. 1.2.6 Post Vagotomy When the vagus nerve is cut to reduce acid secretion in the stomach, unless the procedure is highly selective, those para-sympathetic fibres concerned with gastric motil ity are also cut. MCP is useful in al leviating post-vagotomy symptoms such as stasis of gastric contents, post-prandial vomiting, belching, epigastric distress and diarrhoea (Stadaas and Aune, 1972. Davidson et a l . , 1977, Perkel et al. ,1979, 1980; Wright and Macgregor, 1979). 15 1.2.7. Migraine MCP has been reported to have beneficial effect in the treatment of migraine (Matts, 1974). The delayed gastric emptying due to impaired gastro-intestinal motil ity during migraine attacks is controlled by intramuscular administration of MCP (Matts, 1979; Volans, 1975). Similar observations have been reported by Hughes (1977) with slow intravenous injection of 10 Mg MCP. 1.2.8. Diabetic -gastropar.es-fs • • Diabetic gastroparesis is a feature of autonomic neuropathy, which affects some diabetics. It manifestes as impaired gastric emptying and hypotonicity of the stomach. MCP may be useful in relieving the discomfort associated with this condition (Brown!ee and Kroffpf, 1974; Longstretg et a l . , 1977; Brady and Richardson, 1977; Hartong et a l . , 1977; Braverman and Bogoch, 1978; Berkowitz et a l . , 1976). 1.2.9. Gastric Ulceration Gastric ulceration may be associated with impaired gastric emptying; the prolonged contact between acidic gastric contents and the gastric epithelium contributing to gastric epithelial damage (Pinder et a l . , 1976). Results of controlled studies are inconclusive so far but MCP may have a role in the management of gastric ulceration (Haskins, 1973). It is found to be highly effective in preventing 16 relapse of duodenal ulceration, but no beneficial effects are shown with patients having acute exacerbation of duodenal ulcer (Moshal, 1973). MCP is postulated to exert i t ' s effect by fac i l i ta t ing gastric emptying thereby reducing the acid content next to the site of hemorrhage (Moshal, 1973). 1.2.10. Reflux Esophagitis Several studies (Glanville and Walls, 1972; Venables et a l . , 1973; Stanciv and Bennett, 1973; Paul 1 and Grant, 1974) indicate that MCP may not improve the condition of patients with severe symptomatic reflux esophagitis. However a recent study conducted by McCallum et a l . , 1975 suggests that MCP is effective in the management of this condition. Several of the drug's actions may be important in this context: the most significant effect is probably the increase in lower esophageal sphincter pressure. However, the increased tone of esophageal peristals is improves clearing of the refluxed contents from the esophagus, and accelerated gastric emptying may contribute by indirect ly preventing reflux. Hey et a l . , 1981 have indicated that MCP is a useful adjunct in the prevention of reflux in preparation for, and after, surgery in patients who have been given pethidine for pain re l i e f . 17 1.2.11. Dyspepsia Usefulness of MCP in the symptomatic r e l i e f in patients with flatulent dyspepsia has been validated by the results of two double blind t r i a l s (Uzeta Mejia, 1971; Johnson, 1971). 1.2.12. Lactation Improved lactation after MCP administration (10 mg thrice daily) has been reported in 5 lactating mothers who had suffered severe decrease in milk volume (Sousa, 1975). Similar results are reported by other investigators (Lewis et a l . , 1980; Kauppila et a l . , 1981). 1.2.13. Anesthesia MCP is useful in anesthesia for emergencies (Davis and Howells, 1973; Dundee et a l . , 1974) and for labor and delivery (McGarry, 1971; Howard, 1973; Howard and Sharp, 1973; Schulze-Delrieu, 1981) in which i t ' s gastrokinetic effects reduce the risk of vomiting and aspiration of gastric contents. Thus, the incidence of mortality caused by pulmonary aspiration of stomach contents (Mendelson's syndrome) has been reduced (Schulze-Delrieu, 1981). The usefulness of MCP may be diminished in anesthesia because of the drug's short duration of action and the poss ibi l i ty of prior administration of atropine (Assaf et a l . , 1974; Pinder, et a l . , 1976)_ 18 1.2.14 Radiation Sickness. In controlled t r i a l s , MCP alleviated nausea and vomiting associated with radiation sickness in 86% of patients, (Boisson and Albot, 1966). The effectiveness of the drug (10 mg thrice daily) was further confirmed in a double blind cross over study where radiation sickness was relieved in 20 or 38 patients with established sickness (Ward, T973). 1.2.15. Parkinson's disease The use of MCP in Parkinson's disease is not completely established. In one double bl ind, randomized crossover study, MCP made the tremors worse in two patients, although this effect appears to be dose related (Bateman et a l . , 1978a). On the contrary, several studies support the use of MCP in Parkinson's disease (Tarsy et a l . , 1975; Berkowitz et a l . , 1980). 1.2.16. Other uses of MCP As a result of wide c l in i ca l t r i a l s , MCP has been found to be useful in the treatment of various conditions such as : lump in the throat (Lane, 1980); orthostatic hypotension (Kuchel et a l . , 1980) ; children's behaviour problems (Robertson, 1981) and Tardive dyskinesia (Karp et a l . , 1981). It is also found to increase the sperm output in normal healthy volunteers for a short term' (Jecht et a l . , 1981) . MCP is also been used in the management of gastrointestinal intolerance in rheumatoid ar thr i t i c patients. (Awerbuch et a l . , 1981). 19 1.3. Side Effects MCP is a relat ively safe drug. The side effects of this drug are usually mild, transient and easily reversed with discontinuation of the drug (Pinder et a l . , 1976; Kataria et a l . , 1978). MCP may cause drowsiness, lassitude, insomnia, gastro-intestinal disturbances such as constipation or diarrhea and extrapyramidal reactions (Robinson, 1973). Uncommon side effects include galactorrhea (secondary to prolactin release), dizziness, faintness, agitation, anxiety, aggresion, oedema of the tongue, periorbital oedema and skin rashes. Although uncommon at normal dosage, various extra-pyramidal reactions, usually of the dystonic type occur in about 1% of patients, (Robinson, 1973). These reactions include spasm of the facial muscle, trismus, rhythmic protrusion of the tongue, t o r t i c o l l i s , a bulbar type of speech, spasm of the extra-ocular muscles including oculogyric c r i s i s , unnatural positioning of the head and shoulders, opisthotonos and akathasia (Pinder et a l . , 1975; Daneshmend and Manning, 1979; Wandless et a l . , 1980). Akathasia which is a feeling of restlessness is the most common (Ponte and Nappi, 1981). Dystomia occurs more frequently in females (Pinder, et a l . , 1975) and in young patients (Daele et a l . , 1970, Pinder et a l , 1976; Low and Goel, 1980). Also, facial dyskinesia (Melmed and Bank, 1975) and tetanus l ike motor disorder resembling phenothiazine induced "pseudo-tetanus" have been documented.(Cochlin, 1974; Venkatswaren and Otto, 1972). The majority of reactions occur within 36 hours of starting treatment and the effects usually disappear within 24 hours of 20 withdrawal of the drug (Pinder et a l . , 1976, P . P . G . , 1981; A.H. Robins, 1981). An increase in the frequency and severity of seizures has been reported in conjunction with the administration of MCP to epileptic patients (A.H. Robins,, 1981). Hypotension following the intravenous injection of MCP have been reported in four patients undergoing surgery for repair of a ruptured intracranial aneurysm (Park 1978) and in healthy volunteers (Park, 1980). MCP is a stimulant of prolactin release in both sexes under normal health conditions (Pinder et a l . . , 1976; Judd et a l . , 1976; McCallum and Sowers, 1976; Healy and Burger, 1977). Recent investigations have provided further evidence that MCP is a good and reproducible stimulator of prolactin secretion after acute treatment (Ijaiya, 1980; Brouwers et a l . , 1980; Kitaoka et a l . , 1980; Brandes et a l . , 1981) as well as after chronic treatment (Tamagna et a l . , 1979); Brouwers et a l . , 1980). The hyperprolactinemic response due to MCP administration is exaggerated in conditions such as renal fai lure (Leroith et a l . , 1979), primary test icular fai lure (Spitz et al.,1981) and in 1,2-dibromo-3-chloropropane induced azoospermia (Leroith et al.,1981). Hyperprolactinemia, may be, in part the cause of both galactorrhea and menstrual abnormalities since these symptoms can be reversed by stopping the treatment, provided that patients have not taken the drug for longer than a year (Aono et a l . , 1978; Larsen et a l . , 1979). Hyperprolactinemia has been reported to be associated with hypogonadism (Thorner et a l . , 1974) and amenorrhea (Anderson e t a ] . , 1981). 21 MCP was reported to cause a reduction in seminal volume, total sperm count and l ibido and a loss of spontaneous erections after subchronic administration (Falashi et a l . , 1978). This was also related to MCP induced hyperprolactinemia (Falaschi et a l . , 1978). 1.4. Pharmacokinetics of Metoclopramide 1.4.1. Rate of absorption The rapid appearance of peak concentrations of MCP in plasma after oral administration ( t . , „ in the range of 45-150 mins.) in max rabbit, dog (Bakke and Segura, 1976), mouse (Ingrand and Boulu, 1970) and rat (Tarn et a l . , 1981ia) suggests that the drug is rapidly absorbed from the gastrointestinal tract . It has also been shown that MCP is well absorbed from the rectal mucosa (Hucker et a l . , 1966). Human pharmacokinetic studies indicate that MCP is rapidly absorbed from the gut and peak plasma concentrations are achieved within 40 to 120 minutes (Teng et a l . , 1977; Graffner et a l . , 1979; Schuppan et a l . , 1979; Batemah et a l . , 1979, 1980; Block et a l . , 1981) The ,:onset of action of MCP in humans varies from one to three minutes following intravenous doses and from three to five minutes following intramuscular injections (Ponte and Nappi, 1981). The onset of action after oral administration is approximately 15-20 minutes. Absorption has been found to be more rapid from drops than from dragees or tablets (Block et a l . , 1981). This indicates that therdissolution 22 rate of MCP from the dosage form is the rate l imiting factor in i t ' s gastrointestinal absorption. 1.4.2. Distribution 3 Animal experiments with radioactive ( H-MCP) MCP have shown that after intramuscular doses, i t is concentrated in the intestinal mucosa, l i v e r , b i l iary tract and the salivary glands (Pinder et a l . , 1976; Neeb : et a l . , 1979). Smaller amounts were found in the heart, thymus, suprarenal glands, fat and bone marrow. It has also been shown to cross the blood brain barrier (Ingrand and Boulu, 1970). MCP is believed to be located in the chemo-receptor trigger zone within the central nervous system. Although no data are available, the relat ively small molecule size of MCP and i t ' s l i p o p h i l l i c properties suggest i t would also cross the placental barrier (Smith and Salter, 1980; Ponte and Nappi, 1981). Equilibrium dialysis studies indicate that MCP is weakly bound (13-22%) to human serum proteins (mainly albumin) (Hucker et a l . , 1966; Pagnini and Dicarlo, 1972). Until recently, the accurate quantitation of therapeutic dose levels of MCP in plasma was not possible because of lack of sensitive analytical methods. (Tunon et a l . , 1974; Bakke and Segura, 1976). However, recent advances in the analytical techniques for quantitating plasma samples have considerably enhanced our present day 23 knowledge of MCP pharmacokinetics. The i n i t i a l distribution after intravenous administration of MCP has been shown to be very rapid in the rat (Tarn et a l . , 1981), dog (Bateman et a l . , 1980a) and man (Teng et a l . , 1977; Graffner et a l . , 1979; Schuppan et a l . , 1979; Bateman et a l . , 1979, 1980; Lee et a l . , 1980; Block et a l . , 1981). These investigators have adequately shown that upon intravenous administration, MCP follows biexponential distribution-elimination kinetics represented by a two compartment open model. The apparent volume of distribution (VQ) of MCP is quite large (2-4l .kg!) in a l l the species studied as would be expected for a bas ic , l ip id soluble compound such as MCP. The plasma terminal half l i f e of MCP after 10mg intravenous dose in normal human volunteers has been found to-be in the range of 3-6 hours (Teng et a l . , 1977; Graffner et a l . , 1979; Schuppan et a l . , 1979; Bateman et a l . , 1979, 1980; Lee et a l . , 1980, 1981; Block et a l . , 1981; Tarn et a l . , 1,981a).. The kinetics of MCP in man have been reported to be dependent on the dose and the route of administration (Graffner et a l . , 1979; Bateman et a l . , 1980a). These observations are based on highly variable and limited experimental data. The high var iabi l i ty observed in the study of the effect of route of administration may in part, be due to the tablet dosage form employed rather than a solution (Bateman et a l . , 1980a). A thorough study of the effect of route of administration and dose on the kinetics of MCP in normal human volunteers is presently underway in our laboratory. 24 In rats , at a high dose of MCP (35 mg,kg"')"the drug transiently reduces perfusion to the eliminating organs (viz. l iver kidney etc.) thereby reducing i t ' s total body clearance (Tarn et a l . , 1981a). The pharmacokinetics of MCP have also been studied after intravenous and oral dosing (10 Mg) in patients with chronic renal fai lure (Bateman et a l . , 1980, 1981). The mean ha l f - l i f e was 13.9 h after oral administration. In comparison to previous studies on normal subjects (Bateman et a l . , 1978, 1979, 1980),these results indicate that the total body clearance of MCP in renal fai lure is approximately 30% of normal (Bateman et a l . , 1981). This difference is not accounted for by the change in renal clearance and suggests either impaired metabolism or an alteration in enterohepatic circulation of metoclopramide in renal dysfunction (Bateman et a l . , 1981). Similar observations of decreased total body clearance of MCP have been found in rats with experimental renal fai lure (Tarn et a l . , 1981b). Although further evidence is required, i t is proposed that extra-hepatic metabolism of MCP might be present in rats (Tarn et a l . , 1981b). 1.4.3. Metabolism MCP is extensively metabolised in rabbit (Arita et a l . , 1970b; Bakke and Segura, 1976; Cowan et a l . , 1976), dog, rat (Teng et a l . , 1977) and man (Teng et a l . , 1977; Bateman et a l . , 1978, 1980a and Tarn et a l . , 1979). Sulfate and glucuronic acid conjugation are 25 the major metabolic pathways in man and rabbit. However, the sulfate and glucuronide conjugates of MCP have not been found in the dog and the rat where, de-ethylation of MCP is the dominant metabolic pathway (Teng et. a l . , 1977). An ' in-v i tro study in rabbits (Beckett and Huizing, 1975) showed that MCP had eight metabolites (Scheme V). Later, Cowan et a l . (1976) were able to identify four additional metabolites which exist in small quantities (< 1% of a dose). 1.4.4. Excretion The early studies on human urinary excretion (Hucker et a l . , 1966) indicated that 24% of the dose was excreted unchanged in the f i r s t 24 hours following intramuscular administration of 40 Mg MCP. After oral doses of 10 Mg, however, about 50% of the dose was excreted unchanged in urine over 8 hours (Kumada 1965). The urinary and fecal 14 excretion of radio-activity after an oral dose of C-MCP in the three species is presented in Table 1 (Teng et a l . , 1977). These investigators found that about one half of the total amount excreted in. human urine was unchanged MCP (i.e. about 40% of the dose administered). The lack of a sensitive and specific analytical technique might explain, in part, the var iab i l i ty of these ear l ier investigations. Recently, u t i l i z ing a sensitive Gas Chromatograph - Mass Spectrometric analytical technique, Bateman et a l . (1978) have reported that 20% of the dose is excreted unchanged in human urine over 36 hours. In the rat , about 20 to 30% of the dose is excreted unchanged in the oxidised Cl D CM^CMj OCM. Cl CM,CMj Cl 5 , 0 OCM, " V _ / C ~ N - C H 1 C M 1 . - N / I \ CM. CM^CMj OCM, tCM, IX VII VIII Cl \ v O CM.CM. M tN y- C - N - C M^M,. - N V\ 1? M M1 . M A N ) - C - H - C M 1 C M l - M x V==y CM^CM, OM V OCM, CH tCM^ M ^ H - ^ V c - N - C M ^ M t - N ' — * M . N - ^ V c - N -(Cowan et a l . , 1976) ro OCM, C M l T C M l P M OCM, I V Cl M M , N \ / C - N - C M x C M t - H ^ \ = { CM^CMj OM \J1 Cl M . N ^ ^ C - K - C M ^ M j - V OCM, 111 Scheme 1. Metabolism of metoclopramide i n - v i t r o Incubations using f o r t i f i e d 9000g supernatant of rabb'it l i v e r TABLE 1 Excretion of 1 4C-Metoclopramide (Percent) in Rats, Dogs, and Humans Rat a Dog Human Hours Urine Feces Urine Feces Urine Feces 0 r 2 4 7 1 . 9 5 . 7 6 5 . 3 - 7 7 . 8 2 . 0 2 4 - 4 8 8 . 5 4 . 4 6 . 9 1 8 . 3 5 . 7 1 . 0 4 8 - 7 2 1 . 0 1 . 7 1 . 0 1 . 0 0 . 8 1 . 6 7 2 - 9 6 NCC NC NC NC - 0 . 7 Subtotal 8 1 . 4 1 1 . 8 7 3 . 2 1 9 . 3 8 4 . 3 5 . 3 Total Excretion 9 3 . 2 9 2 . 5 8 9 . 6 a Average of 3 rats b Average of 2 human subjects CNC = not collected (Teng, L . ; Bruce, R .B . ; Dunning, L .K . 1 9 7 7 ) 28 urine (Tam et a l . , 1981a).. The extent of unchanged MCP excreted in rat urine is independent of dose and route of administration within the dosage range studied. 1.4.5. Systemic Bioavai labi l i ty of Metoclopramide The total body (plasma) clearance of MCP has been shown to approach l iver plasma flow rate both in rat (50 ml/min/kg; Tam et a l . , 1981) and in man (10.9 ml/min/kg; Bateman et a l . , 1978). Based on the high clearance values from the intravenous data and assuming that MCP is mainly cleared by the l i v e r , investigators have speculated extensive hepatic first-pass metabolism after oral administration of the drug (Bateman et a l . , 1978; Tam et a l . , 1981). If the preceding assumptions are true, then only a fraction of the oral ly administered drug wi l l reach the systemic circulation in spite of complete absorption. This wil l result in low systemic bioavai labi l i ty . Recent studies have reported that MCP undergoes substantial hepatic f i r s t pass metabolism in normal human volunteers (Schuppan et a l . , 1979; Graffner et a l . , 1979; Block et a l . , 1981). These investigators have reported the oral b ioavai labi l i ty of various dosage forms of MCP (drops, dragees, tablets and capsules) to be between 50-80%. The bioavai labi l i ty of MCP in patients with chronic renal fai lure has been found to be 71.8% (Bateman et a l . , 1981). The b ioavai labi l i ty of MCP tablets as reported by Bateman et a l . , (1980) is highly variable (32 - 97%). This may be due to the 29 inter-individual differences in gastrointestinal absorption from the dosage form (Bateman et a l . , 1980). These authors speculated that the wide differences in the bioavai labi l i ty of MCP might contribute to the unpredictable occurence of side effects. In rats , MCP does not undergo hepatic first-pass metabolism over the dose range studied (Tam et a l . , 1981a). Possible explanations such as transient saturation of the binding sites and metabolic path-ways during the f i r s t passage through the l iver and extra hepatic metabolism have been put forward (Tam et a l . , 1981a). However, further studies are required to test these hypotheses. 1.5. The Concept of Drug Bioavai labi l i ty The plasma drug concentration versus time profi le i . e . the bioavai labi l i ty of an oral ly administered drug depends on the rate and extent of drug absorption (Wagner, 1971; Chodos and Disanto, 1972) which in turn govern the time of onset, intensity and duration of pharmacologic act iv i ty of a drug (Barr, 1968). According to the Academy of Pharmaceutical Sciences (A.Ph.A. , Washington, D.C. 1972), bioavai labi l i ty is defined as a measure of both the relative amount of an administered dose that reaches the general c irculat ion ( i . e . the extent of absorption of a given dose) and the rate at which this occurs. The assessment of the rate of ava i lab i l i ty is one of the most d i f f i c u l t problems faced in developing a pharmacokinetic profi le of a 30 drug due to model dependency (Gibaldi and Perrier, 1975). However, the time when peak plasma.concentration occurs, the peak time, corrected for lag period should reflect the rates of absorption of test dosage forms relative to a standard dosage form (Gibaldi and Perrier, 1975). The extent to which an oral ly administered dose of a drug reaches the systemic circulation intact , can be defined as i t ' s systemic or physiologic ava i lab i l i ty (Perrier et a l . , 1973). For an oral ly administered drug to be total ly available systemically, i t must be fu l ly released from the dosage form, be completely absorbed from the gastrointestinal tract and enter the hepato-portal circulation (via mesenteric veins) without being altered (Gibaldi and Perrier, 1975). Table 2 l i s t s some of the dosage form and physiological factors which can modify bioavai labi l i ty (Riegelman and Rowland, 1973). The most rel iable quantitative assessment of systemic ava i lab i l i ty is obtained by comparing the total area under the blood or plasma con-centration versus time curve after oral administration to the corresponding area obtained following intravenous administration of an equivalent dose. Similar comparisons can be made using total amounts of unchanged drug in the urine following equivalent doses via the two routes (Rowland, 1972, Gibaldi and Perrier, 1975, Balant and McAinsh, 1980): 31 TABLE 2 Physiological factors Dosage form factors Properties of luminal fluids hydrogen ion concentration mucous interaction completing components surface act iv i ty bi le interaction Factors affecting gastrointestinal transit gastric emptying food effects bed rest motil ity enterohepatic cycling Factors at s ite of absorption surface area permeability of barrier specialized transport local blood flow intestinal metabolism Metabolic aspects hepatic metabolism enzyme levels hepatic portal blood flow drug binding proteins extrahepatic metabolism saturation phenomena gut wall metabolism kidney metabolism Distribution effects plasma protein levels obesity Disease states achlorhydria thyrotoxicosis b i l i ary atresia congestive heart fai lure Pharmacological effects of drugs modification of blood flow parasympatholytic act iv i ty Physical properties of drug water so lubi l i ty l i p i d so lubi l i ty partit ion coefficient pK. Properties of dosage form disintegration time dissolution rate surface area of particles crystal size polymorphic form solvates salt form excipients Manufacturing variables granulating process lubricant concentration compression pressure • tablet coatings (Riegelman and Rowland, 1973). 32 (AUC) (X u ) F = ORAL _ ORAL ^ ( A U C ) K V > " ( X U " ) I > V > Where F = Apparent Systemic Bioavai labi l i ty ( A U C ) Q ^ ^ and (AUC)j y are the area :under the plasma concentration versus time curve for oral and intravenous routes respectively, . OO. 00 (X u ) and (X u )j y are the total amounts of intact drug eliminated in the urine following oral and intravenous drug administration, (at time = 7 half l ives ) . Frequently, the extent of drug absorption has been determined by comparing the relative area under the plasma concentration versus time curves (AUC) after oral and intravenous administration of an equivalent dose. This approach is based on the assumption that the AUC is proportional to the dose of the drug administered. The propor-t ional i ty betweehidose and AUC has been referred to as "The law of corresponding areas" (Dost, 1958 and Gladtke 1964, a,b) and is independent of route of administration i f the absorption, distribution and elimination (metabolism and excretion) are f i r s t order kinetic processes. The l i terature however contains many reports on drugs being poorly available ( i . e . the AUC's are not equivalent following administration of equal doses of a drug via oral route compared to the intravenous route) in spite of being well absorbed (Gibaldi and Perrier, 1975). These findings reveal that the disposition of many drugs is markedly dependent on the route of administration. Drugs 33 administered by peripheral routes (eg..subcutaneous, intravenous, intramuscular or sublingual) enter direct ly into the systemic c irculat ion. Distribution into various tissues and organs is ini t iated and only about 30% of the dose passes through the l iver during the f i r s t circulatory pass. However, for drugs given by a hepatic route (oral , intraperitoneal, splenic or portal vein), absorption occurs across that portion of the gastrointestinal epithelium which is drained by veins forming part of the hepato-portal system (Blaschke, 1979). Before reaching the systemic circulat ion these drugs must pass through the l iver and are therefore exposed to hepatic metabolic enzymes. There-fore, the assumption that drug administered both intravenously and oral ly i n i t i a l l y enters the same vascular pool is not valid (Gibaldi et a l . , 1971). For certain drugs which are susceptible to hepatic degradation, a substantial portion of the oral dose may be metabolised before even reaching the systemic circulat ion and the site(s) of pharmacological action. This process is referred to as hepatic f i r s t pass metabolism or f i r s t pass effect (Harris and Riegelman, 1969). Any drug which undergoes hepatic metabolism theoretically is subject to f i r s t pass metabolism. However, this term is usually only applied to those drugs which undergo substantial (eg. 50%) removal during their f i r s t pass through the l i v e r . For those drugs eliminated by hepatic metabolism, the pharmacokinetic models based upon the assumption that the site of elimination is an integral part of the same compartment as the sampled plasma may not be val id under a l l circumstances. Table 3 l i s t s numerous substances which are subject to hepatic f i r s t pass metabolism (Riegelman and Rowland, 1973; Wilkinson and Shand, 1975): 34 TABLE 3 Lis t of Substances Subject to Hepatic F irs t Pass Metabolism propranolol methyl phenidate alprenolol acetaminophen pindolol pheniprazine metoprolol morphine 1idocaine methyl testosterone desmethyl imipramine propoxyphene nortryptyline salicylamide oxyphenbutazone aspirin tryptophane hexobarbital dopami ne phenytoin serotonin chlorpromazi ne pentazocine pethidine isoprenaline and others Many drugs cannot be administered intravenously and ava i lab i l i ty could be determined relative to some standard oral dosage form such as an aqueous solution (Wagner et a l . , 1972). Although the test dosage form may have a relative bioavai labi l i ty of 100%, i t ' s systemic ava i lab i l i ty may be substantially less i f the drug .under investigation is subject to appreciable metabolism on i t ' s f i r s t passage through the l i ver (Perrier et a l . , 1973). 35 The use of metabolite levels in plasma or urine as an index of systemic ava i lab i l i ty of a drug has been suggested where a suitable and sensitive assay method is not available for the parent drug (Gibaldi and Perrier, 1975). However, during such studies, the possible existence of hepatic f i r s t pass must be considered. The modern concept of b ioavai labi l i ty (United States Food and Drug Administration, 1974) also considersthe pharmacological act iv i ty of the metabolite(s) along with that of the parent drug. If the metabolite shows the same pharmacological act iv i ty as the parent drug, b ioavai labi l i ty measurement based on metabolite levels wi l l be appropriate (Balant and McAinsh, 1980). On the other hand, i f the.metabolite is inactive, this method wil l lead to an overestimate of the true bioavai labi l i ty of the therapeutic moeity. The determination of b ioavai labi l i ty based on metabolite levels is most appropriate in the case of prodrugs where the metabolite being measured is the molecule of therapeutic interest. 1.6. Pharmacokinetic Models of F irs t Pass Metabolism 1.6.1. Linear Models: 1.6.1.1. Compartmental Approach: Gibaldi et a l . (1969, 1971 and 1975) proposed a three compartment open system to explain the influence of route of administration ( i . e . intravenous versus oral) on the AUC. The essential feature of the model is that elimination is assumed to occur, at least in part, from a compartment dist inct from that 36 c o n t a i n i n g the v a s c u l a r sampl ing s i t e and t h a t t h i s compartment (which i s analogous to the hepatopor ta l system) r e c e i v e s the drug d i r e c t l y upon a d m i n i s t r a t i o n v i a . a hepat i c r o u t e . The model i s based on the assumption t h a t the system i s l i n e a r w i th respec t to drug d i s t r i b u t i o n and drug e l i m i n a t i o n . The model i s shown i n f i g u r e ( 2 ) . The f o l l o w i n g r e l a t i o n s h i p was deduced: _ ( A U C ) p R A L _ K 2 1 0 m ~ W C J L V . " K 2 1 + K 2 0 where, F rep resents the p r o p o r t i o n o f the drug a d m i n i s t e r e d i n t o compartment 2 t h a t a c t u a l l y reaches the plasma or c e n t r a l compartment ( i . e . the sys temic a v a i l a b i l i t y ) , i s the apparent f i r s t order f o r drug movement from hepatopor ta l compartment to c e n t r a l compartment, l<2Q i s the apparent f i r s t o rder r a t e constant f o r metabol ism from the hepatopor ta l compartment. Equat ion ( 2 ) shows c l e a r l y t h a t i n t h e o r y , the AUC a f t e r o r a l a d m i n i s t r a t i o n w i l l i n v a r i a b l y be l e s s than t h a t observed a f t e r in t ravenous a d m i n i s t r a t i o n . The importance of f i r s t - p a s s metabol ism w i l l depend upon the r e l a t i v e magnitude of l^-j and KgQ f o r a g iven drug or a g iven p a t i e n t . I f IC^ » K ^ Q , F i s e s s e n t i a l l y u n i t y and ! l i t t l e or no f i r s t pass metabol ism i s observed . On the other hand, i f l<2Q >> K 2 1 , then v i r t u a l l y a l l o f the o r a l l y a d m i n i s t e r e d dose w i l l be metabo l i sed by the l i v e r before reach ing the sys temic 37 Figure 2. Hepatic first-pass pharmacokinetic models i.v.,i.m., I orol, i.p., etc. I etc. k i 2 plosmo K21 Compartment 2 Comportment 1 (Hepoto-Portol) (Centrol) k 10 l k 2 0 Model I I i.v., i.m. orol, i.p. etc. I etc. k i 3 k,2 plosmo Compartment 3 k31 k21 Comportment 2 (Peripheral) Compartment 1 (Hepoto-Portol) (Centrol) | k10 l k 2 0 Model II Note: The rate constants K^Q and K-jQ characterize hepatic metabolism and urinary excretion, respectively. The central compartment of Model I includes a l l accessible body compartments except the "liver". (Gibaldi and Perrier, 1975) 38 c irculat ion , and the systemic ava i lab i l i ty wil l be negligible. If l^ -j and I^Q are of the same order of magnitude, then one wil l encounter a f i r s t pass effect ranging from modest to substantial (Gibaldi and Perrier, 1975). While Gibaldi and Perrier (1975) believe Equation (2) to be of considerable theoretical interest, i t is of l i t t l e practical usefulness i f only data following oral administration are available. The c r i t i c a l pharmacokinetic parameters (K-^ ^ and l ^ ) cannot be determined from plasma level data since i t is l ike ly that for v ir tual ly a l l drugs there is an exceedingly rapid equilibrium between the plasma or blood and the hepatoportal system. Accordingly, the l iver cannot be distinguished as a separate compartment of a multi-compartment model. A solution to this problem however, may be made possible by introducing certain physiological considerations into the interpretation of the data. Thus, assuming that the clearance from one compartment to another is equal in both directions and that the transfer between compartments (1) and (2) is blood flow rate l imited, the bioavai labi l i ty can be described by Equation (3) (This is true only for high clearance drugs (Nies et a l . , 1976)): QL F = n , l D 0 S E >0RAL ( 3 ) where, * = l iver blood flow rate Equation (3) assumes complete gastro intestinal absorption and i f absorption is not complete, then, *Ideally, plasma flow rate should be used because AUC is the area under the plasma concentration versus time curve. 39 F'Q, F = L _ _ ( 4 ) Q L + F' (DOSE) ORAL (AUC>ORAL where F 1 = fraction of the administered oral dose actually absorbed. Under certain conditions, F' may be determined independently using urinary excretion data based on total metabolites or an isotopic label . The value of systemic ava i lab i l i t y , 1 F ' has been successfully predicted from Equation (3) for propranolol, propoxyphene and alprenol (Gibaldi et a l . , 1969 and 1971). F can be calculated from the AUC data obtained from i . v . administration of a drug. This is accomplished by rearranging Equation (3) and substituting F from Equation (1), Hence, (DOSE)j v F = 1 " Q. (AUC)j ' v < ( 5 ) 1.6.1.2. Perfusion model approach: Rowland (1972) independently accomplished the derivation of Equation (5) by means of a "perfusion model" approach rather than the compartmental approach used by Gibaldi et al.(1969, 1971 and 1975). Rowland has introduced the importance of clearance concepts in b ioavai labi l i ty studies. He has noted that for drugs where elimination occurs by hepatic metabolism and renal excretion, and i f absorption is complete, then fm. (DOSE)j v  F = 1 " Q. ( A U O j ^ ' ( 6 ) where, fm is the fraction of dose metabolised-; by l iver 40 Therefore, systemic bioavai labi l i ty can be predicted from Equation (6) u t i l i s ing intravenous data alone. If the foregoing assumptions and approximations are reasonable ( i . e . dose independent bioavai labi l i ty and elimination), the systemic bioavai labi l i ty predicted by Equation (6) should agree with that found experimentally ( i . e . Equation 1). If the actual area ratio is smaller than predicted, probably, either absorption after oral administration is incomplete or metabolism occurs in the gastrointestinal contents or in the gut wal l . Alternatively, if: the.experimentally observed area ratio is larger than predicted, the poss ibi l i ty exists that the concentration of drug in the hepatic portal vein following oral administration may be suff ic iently high to saturate.the hepatic! : enzymes and require that the rate of metabolism to be described by nonlinear (Michaelis Menten) kinetics rather than Linear ( f i r s t order) kinetics . In this case, the ava i lab i l i ty of drug to the systemic circulation becomes a function of dose and rate of absorption (Rowland, 1972). After oral administration, i t is usual to have high drug concentrations in the hepato-portal system during the f i r s t passage through the l i v e r , part icularly i f the absorption rate is re lat ively rapid. Therefore, i t is possible to observe saturable kinetics with apparent zero order elimination^during the absorption phase,'.followed by f i r s t order elimination in the post absorptive phase .when the drug concentration in the l i ver is relat ively low (Balant and McA.insh, 1980). 41 In such a s i tuation, the systemic bioavai labi l i ty wil l depend on the extent of saturation of l iver enzymes during the absorptive phase and hence wil l be dependent on dose as well as the absorption rate. The evidence of dose-dependent bioavai labi l i ty exists in the l i terature. Suzuki et a l . , (1972) have presented a portal vein carinulation method (in rats) to investigate the influence of the f i r s t pass effect on the bioavai labi l i ty of propranolol following portal vein infusion. The areas for the portal vein infusion curves were found to be 7.8% (for 2.5 .mg.kg - 1) and 90.0% (for 12.5mg.kg~ 1) of those obtained for the corresponding intravenous (femoral vein) infusion curves. This i l lustrates that the AUC was 12 times larger following intravenous administration of a 2.5 mg.kg"1 dose-than after portal vein ahfusion of an equal dose. The reduction in AUC following portal vein infusion is attributed to the fact that significant f i r s t pass metabolism occured during the f i r s t passage of propranolol through the l iver at the 2.5 mg.kg - 1 dose leve l . The difference at the 12.5.mg.kg"1 dose however, was not s ignif icant. The evidence for the non-linear dose-dependent hepatic metabolism of propranolol was further provided by a subsequent study by Suzuki et a l . , (1974). The hepatic elimination of propranolol, during the f i r s t pass through the l iver after portal vein infusion (in rat) was highly dose dependent at relat ively low doses and rate dependent at higher doses. The unusual disposition of propranolol ( i . e . dose dependent avai labi l i ty ) depending on the route of admini-stration has been presumed to result from i t ' s concentration dependent 42 hepatic clearance as evidenced by in vitro perfusion of the rat l i ver (Evans et a l . , 1973; Shand et a l . , 1973). Suzuki et a l . , (1980) have presented an in vivo study in rats to measure the hepatic extraction ratio of propranolol. The hepatic extraction ratio was found to be dependent on the route and rate of administration. The non-linear hepatic extraction was further confirmed by determing the markedly decreased hepatic extraction ratio of labelled ( 1 4 C ) -propranolol, given intravenously after pretreatment with or during portal venous administration of unlabelled propranolol. Dose-dependent disposition of oral propranolol in normal subjects have also been studied recently (Machichan et a l . , 1980) In the dosage range studied, the amount of propranolol reaching the systemic circulat ion increased with dose, while the biologic half l ives remained, unchanged. The apparent intr ins ic clearance values were shown to decrease with increase in dose. These data suggest the saturation of a low capacity enzyme system in the l i ver and are consistent with theoretical characteristics of a drug that is exten-sively metabolised in the l i v e r . A new approach to measure the hepatic ftrs.t-pas.s effect of model drugs by whole body autoradiography has been proposed by Ri ki hi sa et a l . , (1981). It was shown that the rat ioact iv i ty of propranolol that reached the systemic circulat ion was highly dependent on both the route of administration and dose. The pharmacokinetic studies of alprenolol also indicate that the hepatic extraction ratio of this drug after oral administration is 43 non-linear and decreases with increasing dose in the dosage range studied (Ablad et a l . , 1972 and 1974). For drugs, where bioavai labi l i ty is dose dependent, i t is possible to determine an apparent threshold dose below which they wi l l be completely extracted by the l iver prior to reaching the systemic circulation (Balant and McAinsh, 1980). In contrast, for higher doses a linear relationship wi l l be observed between the amount of drug administered oral ly and the amount of unchanged drug reaching the systemic c irculat ion . The pharmacological act iv i ty of the "first pass metabolites" wil l be of practical importance. If the metabolites are inactive and i f there is a threshold concentration in the hepatoportal system under which no unchanged drug reaches the systemic c irculat ion , i t is possible that sustained released formulations of the drug wi l l lead to inefficacy because the drug concentration in the hepato-portal system during the absorption phase wi l l always be lower than the threshold value. 1.6.2. Non-Linear Hepatic Firs t Pass Metabolism Keller and Scholle (1981) have recently derived an integrated form of the Michael is Menten equation to describe the non-linear hepatic f i r s t pass metabolism of an oral ly administered drug. Accordingly, the f i r s t pass effect (F) or the systemic bioavai labi l i ty of a metabolised drug is described by the following relationship: 44 Vm F = 1 - Ka.D D.Ka.T. + Km 10LN(2) + Ln D.Ka.T. + K m . e . 1 0 L n ^ 17) where, Vm and Km are the Michael is Menten constants described in mass terms (lmg:...,hr~1 and mg.respectively). T = Time interval for portal blood circulat ion (1 minute) Ka = Absorption rate constant ( h r - 1 ) and, D = Oral dose of the drug administered. For the calculation of non linear f i r s t pass effect, the constant parameters (Vm, Km and Ka) have to be determined in-vivo. The true gastrointestinal absorption rate constant (Ka) of a drug is d i f f i c u l t to determine because of i t ' s model dependency and inter-intra-subject variation (Gibaldi and Perrier, 1975). However, an estimate of the absorption rate constant (Ka) is calculated from the combination of oral and intravenous data (of equal doses) f i t ted to a two compartment open model. The estimation of Km and Vm is possible by iterative techniques. 1.7. Analysis of MCP in Biological Fluids Until recently, very l i t t l e information of the pharmacokinetics of MCP was available. The inabi l i ty to measure 45 very low concentrations of this substance has been the major reason for the lack of comprehensive studies of this drug. However, improved analytical techniques for the quantitation of MCP in biological samples have brought about a better understanding of the pharmacokinetics of MCP. In the past, colorimetric (Arita et a l . , 1970a; Tunon et a l . , 1974) and thin layer chromatography photodensitometric (Bakke and Segura, 1976) assay methods were used to measure MCP levels in bio-logical f lu ids . These techniques suffer from lack of sensit ivity in the nanogram range and therefore require large volumes ($ 1 ml) of plasma samples for suitable quantitation. This characteristic rendered continuous sampling in small animals (eg. rats) impossible. Further more, interference from structurally related metabolites l imits the scope of the colorimetric method.' A gas l iquid chromatographic (GLC) assay was employed in forensic chemistry (Kaempe, 1974) to isolate and identify MCP qual i tat ively . The sensit iv i ty of this method was not reported. Baeyens and Moerloose (1978) presented a fluorimetric assay to quantitate MCP in various pharmaceutical dosage forms. The detection l imit was reported to be 30 ng. ml.~^. The appl icabi l i ty of this method has yet to be established in biological samples containing MCP. A rapid TLC-densitometric method u t i l i z ing an in-s i tu diazo coupling technique has been shown to be satisfactory for the 46 -1 selective measurement of low levels of MCP (20 ng.mV )in biological fluids (Huizing et a l . , 1979b). Schuppan et a l . , (1979) have reported preliminary pharmacokinetics of MCP in normal human volunteers u t i l i z i n g a TLC assay method. The non specific nature of the assay may,in part, account for the large variation in their pharmacokinetic data for MCP. In the past few years, various high performance l iquid chromatographic methods have been reported for the quantitation of MCP in human biological samples (Teng et a l . , 1977; Graffner et a l . , 1979; Bateman et a l . , 1981; Block et a l . , 1981). The lowest detect-able concentration range of these HPLC techniques is between 5-10 ng of MCP per ml of plasma, provided a 5 ml. sample was being analysed (detection l imit - 5 rig)- Bateman et a l . , (1981) claim their HPLC method is more sensitive compared to others. This, however, is achieved only by u t i l i s ing a smaller volume of solvent for f inal reconstitution and injecting the whole sample, thereby sacrif ic ing the advantage of repeated sampling. The aforementioned HPLC techniques are rapid and f a i r l y sensitive and are capable of detecting MCP levels in human plasma upto eight hours after 20 mg oral dosing. The majority of the pharmacokinetic work reported in the l i terature (discussed elsewhere) is based on eight hour blood sampling protocols, 47 According to Gibaldi and Weintraub (197.1), premature termination of pharmacokinetic studies may y ie ld erroneous under-estimates of true biological half l i f e of certain drugs. Since the plasma levels of MCP after administration of a single therapeutic dose (10 nig) may be so; low upon attainment of the terminal exponen-t i a l phase^ that they are not detected by presently available HPLC methods, the biological half l i f e may be indeterminable from such single dose studies. Various workers have independently developed highly sensitive and specific GLC electron capture detector (ECD) assays for the detection of trace amounts of MCP in rat (Tam and Axel son, 1978) and in man (Tam and Axelson, 1979b, Lee et a l . , 1980). The lowest quantifiable MCP concentration has been reported to be in the range of 2 . 5 - 5 ng.ml provided 0.5 ml of human plasma was used (detection l imit - 20 peg). The method reported by Tam and Axelson (1978) permitted serial blood sampling (0.1 - 0.2 ml) for pharmacokinetic studies in rat . A highly sensitive and specific GLC mass spectro-meter (MS) method has also been reported by Bateman et a l . , (1978b) for the measurement of MCP in dog and in man. Preliminary investigation of 24 hour human plasma data u t i l i z ing the highly sensitive and specific GLC-ECD method (Tam and Axelson, 1979b) have clearly shown that the use of 8 hour plasma data results in a significant underestimation of MCP half l i f e tn man. An extensive study of various pharmacokinetic parameters including 48 the determination of true biological half l i f e of MCP in normal human volunteers is presently underway in our laboratory. Tarn and Axelson (1979a) have also reported a sensitive GLC-ECD assay for the simultaneous quantitation of MCP and i t ' s mono de-ethylated metabolite in rat urine. Although baseline resolution between the two peaks was not attained, the investigators have reported that when a 3% SP2250-D8-:phase. was used, the electronic integration method provided consistent results . 49 2, EXPERIMENTAL 2 J . Supplies 2.1.1. Chemicals 4-Ami no-5-chloro-2-methoxy-N-(2-ethylami noethyl)benzami de (De-MCP) (Lot No.3137) 1, 4-amino-5-chloro-2-methoxy-N-(2-diethyl aminoethyl) benzamide monohydrochloride monohydrate(MCP.HC1.h^O) (Lot No.9207) , monosodium-g-nicotinamide adenine dinucleotide . , 3 4 phosphate (g-NADP.Na) , D-Glucose-6-phosphate monosodium sa l t . Diazepam , carbontetrachloride (CCl^) . 2.1.2 Reagents Heptafluorobutyric anhydride(HFBA) 7, triethylamine [ (C 2 H 5 ) 3 N, T E A ] 8 , ammonium hydroxide (NH 40H) 9, hydrochloric acid (HC1) 1 0 , dibasic sodium phosphate ( N a ^ P O ^ H ^ ) 1 1 , monobasic phosphate (NaH 2 P0 4 .H 2 0) 1 2 , sodium hydroxide (NaOH) 1 3, sodium chloride ( N a C l ) 1 4 , potassium chloride ( K C I ) 1 5 , magnesium chloride (MgCl 2 . 6H 2 0) 1 6 , Trichloracetic acid ( C C 1 3 C00H, T C A ) 1 7 . 1 , 2 A.H. Robins Co. , Richmond, V a . , U.S.A. 3 ' 4 Sigma(R) Chemical Co. , St. Louis, MO., U.S.A. Hoffmann-La Roche, Montreal, Canada 6 North American Scient i f ic Chemical L t d . , Vane , B.C. Canada 7 ' 8 P ierced) Chemical Co. , Rockford, 111., U.S.A. 9 ' 1 0 Reagent A . C . S . , A l l i ed Chemical, Canada L t d . , Point Cla ire , Quebec, Canada. Fisher^ K ' Science C o . , New Jersey, U.S.A. 13-17 The Bri t i sh Drug House L t d . , Toronto, Canada 50 2.1.3. Solvents Benzene1^, ether 1^. 2.1.4. Supplies for rat experiments 20 21 22 Ether solvent , heparin , polyethylene tubing (PE50) , (R) 23 (R) 24 s i l a s t i c v ; medical grade tubing , Dermaseptv ' skin cleaner . 2.2. Equipment Hewlett-Packard model 5830A reporting gas chromatograph CO Or / D ^  equipped with Ni-electron-capture detector , Vortex-genie^ mixer , Teflon pestle glass tube homogenizer with Dynamix^ ' , ID) po (r>\ pq Accumetv ; pH meter model 600 , Isotempv ; oven model 350 , (R) 30 (R) Burrell wrist action v ; shaker , Refrigerated Damonv ' 32 (R) 32 Centrifuge model B-26 , Polytron v ' homogemizer , Water bath with 34 shaking device model 02156 . 18 Dis t i l l ed in glass, Caledon Laboratories L t d . , Georgetown, Ontario, Canada 19 Analytical reagent, Mallinkrodt Canada L t d . , Montreal,- Canada 20 North Amer. Sc i . Chem. L t d . , Vane , B . C . , Canada 2 1 Sigma(R) chemical Co. , St. Louis, M0. U.S.A. IntramedicW Beckton, Dickinson and Co. , Parsippany, New York,U.S.A. 23 Dow Corning Corporation, Medical Products, Midland, Michigan, U.S.A. 24 Germiphene Co. L t d . , Brantfort, Ont. Canada. 25 Avondale, Pennsylvania, 19311, U.S.A. 26-29p i . s | i e r <-c_j ^ Springfield, Mass., U.S.A. 3 0 Burrell Corp. Pittsburgh, PA., U.S.A. 3 1 '"^International Equip. Co. ( I . E . C . ) , Needham, Hts. , Mass., U.S.A. 33 Brinkmann Instruments, Westbury, N .Y. , 11500, U.S.A. 34 American Optical Corp. , Sc i . Instrument. Div . , U.S.A. 51 2.3. Stationary Phases and Solid Supports 2.3.1. Commercially coated products 3% OV-225 (25% cyanopropyl, 25% phenyl, methyl silicone) on Gas Chrom-Q,^ 230/270 mesh size (Ultra Pak), 3% OV-225 3 6 on Chromosorb.-. W (H.P.) 100/120 jnesh size, 37 3% Silar-9CP (cyanopropyl methyl phenyl sil icone) on Chromosorb-W^ (H.P. 100/120 mesh size. 2.3.2. Stationary phases (in the order of increasing polari 2% 0V-101 3 8 (methyl sil icone) 3% 0V-17 3 9 (50% methyl phenyl. sil icone) 3% OV-225 4 0 3% S i lar -5CP 4 1 3% S i l a r - 9 C P 9 2 3% S i l a r - l O C 4 3 5% Carbowax 20 M 4 4 (Polyethylene, glycol) 35, 37, 41-43, 46. Chromatographic : S p e c i a l i t i e s , Br.ockville, Ont., Canada 36, 38-40, 45 Applied Sc i , PA. U.S.A. 44 Altech Associates, Arlington, 111., .U.S.A. 52 2.3.3. Solid supports Chromosorb-W ( R )(H.P.) 4 5 , 100/120 mesh size, Gas .ChromQ ( R), ( 4 6 ) 8O.-100 mesh,size. Note: (a) The coated phases were packed in a 1.8 meter x 2 mm ( i .d . ) glass column. (b) In addition to packed glass columns, a 24 meter x 0.5 mm ( i .d . ) SE30 glass capi l lary (SCOT) column 4 7 was also used. 2.4. Preparation of Hydrochloride Salt of De-MCP .Base A saturated solution of hydrogen chloride (HC1) gas in ether was prepared by bubbling HC1 gas through 25 ml of ether in a 100 ml conical flask for 5 minutes. The saturated ethereal solution of HC1 gas was then added dropwise to a 50 ml ethereal solution of 100 mg De-MCP base in a 100 ml conical flask. The product (De-MCP.HC1) was collected by f i l t r a t i o n , and washed twice with 10 ml aliquots of ether. 2.5. Solutions 2.5.1.- Preparation of Stock and Dilute,Solutions of MCP.HC1.H?0 : . and De-MCP.HC1 ' " MCP.HCl.H2O (approximately 10.0 mg) was weighed accurately, transfered into a 100 ml volumetric f lask, and dissolved in deionised d i s t i l l e d water to y ie ld a 100 ml solution. One ml of this solution 47 Scient i f ic Glass Engineering L t d . , Austin, Texas, U.S.A. 53 was diluted to 50 ml with, deionised d i s t i l l e d water in a 50 ml volumetric flask. Different volumes (0.1 to 1.0 ml) of this solution were used for extraction' (0.2 yg.ml.~V to 2-.yg.-m.l~1 of MCP.HC1 .H 2 0) . A similar method was used to make 50 ml of dilute De-MCP solution (2 y g . m l - 1 ) . Five ml. of this solution was further diluted to 50 ml,with deionised d i s t i l l e d water in a separate 50 ml volumetric flask. Different volumes (0.1 to 1.0 ml) of this solution were used for extraction (0.02 yg.ml" 1 to 0.2 yg .ml - 1 of De-MCP.HCl). 2.5.2. Preparation of internal standard (diazepam) solution  Diazepam (approximately 10.0 irig) was weighed,accurately, transferred into a 50 ml volumetric f lask, and dissolved in benzene to y ie ld a 50 ml solution. Ten ml of this solution was diluted to 1000 nil with benzene in a 1000 ml volumetric flask. One ml of this solution (2 yg.ml" 1) was used.for one yl injection samples containing MCP (sj 500 peg) and De-MCP (s 5J3 peg). The internal standard solution (2 ug-ml"1) was diluted five times with benzene to y ie ld a concentration of 0.4 yg of diazepam per ml for the five yl injection samples. 2.5.3. Preparation of 0.02M, pH 7.4 phosphate buffer (Parott and Saski, 1977)  Monobasic sodium phosphate (Nah^PO^.h^O) (0.552g) and dibasic sodium phosphate (Na9HP0A.7H?0) (1.07g) were weighed accurately 54 and dissolved separately in sufficient quantity of deionised d i s t i l l e d water to y ie ld 200 ml solutions (0.02m) of each substance. Twenty ml of 0.02M monobasic sodium phosphate was combined with 80 ml of 0.02M dibasic sodium phosphate to furnish a 0.02M phosphate buffer of pH 7.4, which was verif ied by u t i l i s ing a pH meter. 2.6. General Procedure for Phase Coating Ten grams of sol id support (Chromosorb W (H.P.) V ' 100-120 mesh size) was weighed accurately and spread evenly in a Petri-dish. A 20 ml chloroform solution containing 0.3 gms of stationary phase ( v i z . , Silar-5CP, Silar-9CP, Carbowax-20M) was added dropwise in the Petri-dish containing the sol id support. The chloroform solution was added in a manner such that the f i r s t drop was allowed to soak into the sol id support before adding a second drop on the same spot of support surface. This process was repeated until the surface was thoroughly wetted. The coated sol id support was then transferred to a H i - E F F ^ 4 8 cyl indrical f luidised bed dryer. The base of the dryer was heated with a hot plate to a temperature of 60°C. A low nitrogen flow was applied sufficient to suspend the sol id support particles (viz. f luidization) and was maintained until a l l the solvent had evaporated [- 10 mins). The dryer was then removed from the hot plate and nitrogen flow maintained until the powder bed had reached room temperature. The coated support was transferred to and stored in a t ightly capped, l ight resistant container until required for column packing. Applied Science Co. L t d . , State College, PA.16801, U.S.A. 55 2.7. General Method of Column Cleaning and Phase Packing Deteriorated stationary phase was blown out of the glass column using N2 gas under pressure and gentle hand tapping. The emptied (R) 49 column was subsequently f i l l e d with 0.025% Alcojet v detergent solution in deionised d i s t i l l e d water and soaked overnight. The next day, the column was rinsed thoroughly with tap water followed by deionised d i s t i l l e d water (five minutes each) by applying suction. The column was then f i l l e d with and allowed to soak in deionised d i s t i l l e d water overnight. The following day, deionised d i s t i l l e d water was drained from the column and the column was dried in a hot air oven (100°C). Upon cooling, the column was washed with 250 ml of methanol by applying light suction and then dried by heating in the oven at 100°C and cooling to room temperature with a low flow of N2. A similar washing and drying cycle was carried out using 250 ml of toluene. The column 50 was then f i l l e d with a 5% solution of dimethyl dichlorosilane in toluene. The solution was l e f t in the column for 30 min at room temperature, then drained off and the column rinsed with 250 ml of toluene. The column was dried by heating with a low flow of N 2 > After cleaning and silanization, one end of the column was plugged with silanized glass wool and light suction was applied. Fresh solid support (coated with phase as previously described) was added in small amounts through a funnel to the open end of the column and i t ' s passage aided by gentle tapping (by hand) until the column was completely 49 ^ Alconox Inc., New York, N.Y., 100003 U.S.A. 50 Pierce Chemical Co. 56 and firmly packed. Tight packing was avoided. 2.8. General method of column conditioning The newly packed column was transfered to the oven of the gas chromatograph and one end was connected to the injection port. The detector end of the gas chromatograph was plugged to avoid contamination from i n i t i a l column bleed. The oven temperature was raised from 50°C to 175°C at a rate of l°C.m\in. while maintaining a carrier gas [argon::methane(19:l)] flow of 40 ml. m i n - 1 through the column. The column was held at 175°C overnight. Five y l of S i l y l - 8 ^ ' ^ was slowly injected five times into the column at intervals of one minute. The column temperature was then raised from 175°C to 250°C at a rate of l°C l;rrrin7'Iand held at this temperature for at least 2 days for 0V phases and 7 days" for S i lar phases. 2.9. Preliminary GLC-ECD Analysis Tarn and Axel son (1979b) reported a GLC-ECD assay for the.simul taneous quantitation of MCP and De-MCP in rat urine. However, using this method, only partial resolution was achieved between the chromatographic peaks of HFB derivatives of these two compounds. The i n i t i a l objective then was to attempt further improvement upon the resolution of the two peaks. The existing assay was evaluated in terms of the following aspects: (a) phase selection, and (b) optimisation of De-MCP derivatization with HFBA. Pierce Chemical Co. Rockford, 111., U.S.A. 57 2.9.1. Phase selection Various phases ranging from relat ively nonpolar (OV-101, and OV-17) to polar (OV-225; Si lar-5 CP, S i lar 9CP, Si lar- lOC and Carbowax-20M) phases were examined for peak shape and resolution of HFB derivatives of MCP and De-MCP. The testing of columns containing the aforementioned phases was- carried out by injecting test samples of HFB derivatives of MCP and De- MCP. The extraction and derivatization of these compounds were based on the method developed by Tam and Axelson (1979b): In a 50 ml centrifuge glass tube containing 0.05 ml blank rat urine, 0.5 nil of aqueous solutions of MCP;HC1SH20 (2.0'ug.ml" 1) and De.MCP.HCl (1.0 yg.ml - 1) were added. The volume was made upto 2 ml with deionised d i s t i l l e d water. One ml of IN NaOH and 6 ml of benzene was added in the same centrifuge tube and the mixture was shaken on a horizontal wrist action shaker for 20 minutes to extract MCP and De-MCP. After centrifugation, 5 ml of the organic layer was transferred to a 15 ml centrifuge tube. Evaporation of the organic layer was carried out under a gentle stream of N 2 . Three quarters of the centrifuge tube was immersed in a water bath held at 40°C to fac i l i ta te evaporation. The residue was reconstituted with 1 ml of internal standard solution (2 ug.nrT1 v of diazepam in benzene) and 20 ul of HFBA was added. After thorough vortex mixing for 10 sees.., the reaction mixture was incubated at 55°C for 20 minutes and then allowed to cool to room temperature. Excess derivatizing agent was removed by hydrolysis with 0.5 Ml of deionised d i s t i l l e d water, vortex for 10 sees., and 58 subsequent neutralisation of HFB acid formed with 0.5 Ml of 4% NH^ OH solution, vortex for 20 sees. The mixture was then centrifuged for 2 mins at 1000 rpm to separate the organic and aqueous layers. One ul of the organic layer was injected onto a particular column for the examination of shape and resolution of HFB derivatives of MCP and De-MCP. The general GLC conditions used were as follows: Instrument: Hewlett - Packard 5830 A ft 3 reporting gas chromatograph equipped with Ni-electron capture detector. Oven temperature: Isothermal in the range of 230°C to 255°C. Injection port temperature: 250°C Detector temperature : 350°C Chart speed: 0.3 cm.min-'' Carrier gas (Argon: Methane:: 19:1 ) : 30-50 mil:.min-1 2.9..2 Optimisation of HFBA Derivatization of DE-MCP In addition to the primary amino group on the aromatic r ing, the mono de-ethylated metabolite of metoclopramide (De-MCP) also has a secondary amino function in the alkyl side chain. Both the primary as well as secondary amino groups are derivatized by HFBA as evidenced by GC-MS analysis (Tam and Axelson, 1979b). The primary amino group of MCP is derivatized instantly (Tam and Axelson, 1978). However, the kinetics of simultaneous HFBA derivatization of the primary and secondary amino groups of De-MCP has not been reported. 59 The effect of different incubation times on the y ie ld of HFB derivative of De-MCP was monitored at a constant temperature of 55°C. De-MCP was extracted as mentioned in the preceding section. Then 20 y l of HFBA was added to the reaction mixture along with 1 Ml of internal standard solution (0.2 ygiml" 1 ) . Samples, after thorough vortexing for 10 sees., were incubated for different periods of time (0, 15, 30, 45, 60, 75, 90, 120, 180, 240, 300 and 360 min.). After hydrolysing the excess HFBA with 0.5 ml deionised d i s t i l l e d water and neutralising the heptafluorobutyric acid formed with 0.5 ml of 4% NH^OH, 1 ml of the organic layer was injected onto the column (3% Si lar 9CP on Chromosorb W (H.P.) 100-120 mesh size) and the response (area counts) monitored. A similar method of derivatization and incubation was carried out in the presence of a catalyst (0.1 .ml of 0.1 M triethylamine solution in benzene). 2.10. Animal Studies 2.10.1. General animal handling 51 Adult male Wistar rats ranging in weight from 225-275 grams (mean of 250G) were used in a l l of the experiments. Al l newly received animals were maintained in metal cages (41 x 34 x 18 Cm) (6 to 8 per cage) in a controlled environment (temperature: 22°C, relative humidity : 30%) for at least three days. This allowed them to acclimatize to the new surroundings of the animal care f a c i l i t i e s before being handled for an UBC Animal Care Unit, Vancouver, B.C. Canada. 60 experiment. The animal room illumination was controlled by providing darkness from 20.00 hrs .to 06.00 hrs and l ight from 06.00 hrs to 52 20.00 hrsi. .. The rats were fed with Lab Chow and tap water was allowed ad-1ibitum. 2.10.2. In-vivo rat experiments 2.10.2.1. Techniques of drug administration Aqueous solutions of MCP'HCl-h^O in normal saline were used for administration via intravenous ( i . v . ) , intraperitoneal ( i .p . ) and oral (gavage) routes. A total volume of 0.5 Ml of MCP solution per 250 G rat was administered by a l l routes (Therefore, the amount of MCP-HCl-h^O was weighed such that the total Dose.Kg - 1 required to be administered by any route would be contained in 0.5 Ml of normal saline s o l u t i o n „ p e r 250 g rat) The i . v . and oral routes of adminis-tration were used for the bioavai labi l i ty experiments, whereas the i . p . route was ut i l i sed for the two-thirds hepatectiomised and CCl^ pretreated rats. (a) Intravenous route I n i t i a l l y , a le f t jugular vein cannula (Weeks and Davies, 1964, Venkataramanan, 1978) was implanted for two purposes: (1) to administer the drug solution, and (11) to col lect blood samples. However, from the GLC analysis, i t was found that the blood levels of MCP after low doses (<5Mg.Kg -1) were not detectable. Therefore blood sampling was discontinued and jugular vein cannulation 52 Purina's Laboratory Chow, Rolston Purina Co. , St. Louis, Missouri, 63188, U.S.A. 61 was no longer necessary. The i . v . administration for the urinary excretion studies was carried out via the caudal vein (Bergstrom, 1971). The rat was put under l ight ether anaesthesia. A forceps (designed for 53 wound cl ips) was clamped on the base of the t a i l . The ta i l was rotated 90 degrees (lateral positioning) for injection convenience. 54 The injection needle (24 gauge) was inserted in the v is ible dark caudal vein. After being sure that the tip of the needle had freely entered the venous blood stream (by t i l t i n g the needle up and down), the forceps were removed and the injection was accomplished. After the injection and withdrawal of needle, the ta i l was held at the point of needle insertion with a small piece of gauze for one minute to prevent bleeding. (b) Oral route The oral administration of drug solution by gastric intubation was carried out using a blunted,curved oral intubation needle and syringe. Care was taken to ensure that the gavage needle entered the esophagus avoiding passage into the lungs. (c) Intraperitoneal i route Two-thirds hepatectomised as well as CC14 pretreated rats were very susceptible to ether overdose and the incidence of death was high among these populations. Therefore, these animals were not anaesthesized before administration of the drug solution. A protective "53 Becton, Dickinson and Co. , (supplied by B.C. Stevens, Vane. B.C.,Canada). 54 Yale needle (Luer lok hub) Beckton, Dickinson and Co.,Canada Ltd. Miss . , Ont., Canada 62 mitt was used to hold the rat p r i o r to injecting the drug solution 55 into the peritoneum with a syringe equipped with 23 gauge needle, 2.10.2.2. Urine sample collection The experimental design for the urinary excretion studies required a cumulative 48 hour urine collection following I . V . , i . p . and oral administration of different doses (equivalent to 0.1 -0.5, 1.0, 5.0 and 15.1 mg-k'gV MCP base) of MCP-HC1-h^O. Dosed rats were housed in individual stainless steel metabolic cages (24.5x17.5x18cm) (Fig. 3 ), equipped with a fine mesh screen floor to prevent faecal contamination of the urine. Urine was collected in an attached l ight resistant amber colored bottle. Forty-eight hours after dosing, the floor,and sides of the cage were rinsed with deionised d i s t i l l e d water to maximize urine recovery. The diluted urine samples thus collected were frozen (-20°C) until analysed. 2.10.2.3. Two-thirds hepatectomy study The technique of Higgins and Anderson (1931) was followed for the surgical removal of two-thirds of the rat l i ver . The animals were fasted for a period of 8-12 hours prior to the surgery. Following ether anaesthesia, median line incision reaching 3-4 cm posteriorly from the xiphoid process of the sternum was made. Large portions of the median lobe together with the left lobe of the l iver were securely 55 Glasspak 1 c c . tuberculin syringe/B-D Yale tuberculin 1 c c . glass syringe. Beckton, Dickinson and Co. , Rutherford, N . J . , U.S.A. 63 METABOLIC CAGE FOR SEPARATE COLLECTION OF URINE AND FECES Fig. 3 Diagram to show the device employed In collecting urine samples from rats without fecal contamination. 64 ligated with '000' silk"1" and then carefully excised. In this way, portions of the parenchyma ranging from 65 to 75% of the total l iver were removed, leaving within the peritoneum. the right lateral and the small caudate lobe. The peritoneum and the abdominal muscles were 56 sutured with chromo '000' gut and the skin was closed with '000' s i lk . There was no special post operative care. Lab Chow and water were allowed ad libitum. For control rats, sham operations were carried out by making a similar median l ine incis ion. The peritoneal organs 57 were gently disturbed with a Q-tip and the wound closed as previously described. One day following surgery, i . p . administration of an isotonic solution (0.5 Ml/250 G rat) of MCP-HC1-H20 (containing . .-1 equivalent to 15 Mg.Kg of MCP base) was carried out in two-thirds hepatectomised as well as sham operated rats. Urine was collected for 48 hours after dosing and frozen (-20°X) until analyzed. 2.10.2.4, Carbon tetrachloride pretreatment study The animals were randomly separated into two groups the control and experimental group received an oral dose of 0.5 Ml normal saline or 1.0 M l . K g - 1 CCl^ (Hirano et a l . , 1975) respectively. After saline or CCl^ administration, the rats were fasted overnight. On the following day, an i . p . dose of MCP-HCl-h^O equivalent to 15 Mg.Kg"1 MCP base was administered to control and test animals. Food was limited to 2-3 Lab Chow pellets a day and water was allowed 56" 7 ^  Ethicon(R), Ethicon sutures L.td.Peterborough, Ont. Canada 57 Chesebrough-Pond's Ltd. ' , -Ont. , Canada 65 ad libitum. Cumulative urine samples, were collected up to 48 hours dosing and stored ( -20°C) until analyzed. 2.10.2.5. Bioavai1abi1ity experiments Urinary excretion studies were carried out following i . v . and oral administration of a range of MCP-HCl-^O doses (equivalent to 0.1, 0.5, 1.0 and 5.0 mg.kg"1 MCP base). Urine samples were collected as described in the preceding section (2-11-2-2), 2.10.3 In-vitro Metabolism study A method of similar to Cowan et a l . (1976) was ut i l i sed to study the in-vi tro metabolism of MCP in the rat. 2.10.3.1. Preparation of tissue homogenates Rats (n=4) were k i l l ed by stunning followed by decapitation, their organs ( l ivers , kidneys and lungs) immediately removed and placed in a fresh, ice cold, isotonic (1.15% w / v ) aqueous solution of potassium chloride (KC1). Al l subsequent operations were carried out at 0 -4°C. The excised tissues were blotted dry, weighed and minced with scissors. The l iver tissue was homogenised in four volumes of fresh ice cold isotonic KC1, using 4-5 passes with a motorised teflon pestle glass tube homogeniser. The lung and kidney tissues were homogenised seperately using 4-5 passes with a ' P o l y t r o n , v ' equipped 66 with toothed metallic homogen.ts.er, A similar l iver tissue to KC1 solution ratio (1:4) was used for lung and kidney tissues. The homogenates were transferred to plastic tubes and centrifuged at 9000g for 20 minutes to remove nuclei , mitochondria and cel l debris. Aliquots (0.5, 1.0, 1.5 and 2.0 ml) of supernatants as well as whole tissue homogenates were used for the incubation reactions described below. 2.10.3.2. Incubation experiments with whole tissue homogenates and their 9000g supernatants MCP-HCl'r^O (5 riiicromole./ml \"n deionised d i s t i l l e d water was incubated i.n uncapped glass tubes at 37°C for 30 minutes with whole tissue homogenates (Liver, kidney and lung) as well with 9000g tissue supernatant preparations. Each incubation tube contained the following ingredients: 1 ml of substrate solution, one of the four volumes (0.5, 1.0, 1.5 and 2.0 ml) of whole homogenate or 9000g supernatant, one of the corresponding volumes (3.5, 3.0, 2.5 and 2.0 ml) of 0.02M jJH 7.4 phosphate buffer, and 1 ml of cofactor solution which contained: 6 Mg of glucose-6-phosphate, 3.4 Mg of NADP-Na, 0.2 Mg of 0.1 M MgCl 2-6H 20 and 0.8 Ml of deionised d i s t i l l e d water. 67 The one Ml of substrate solution was replaced with. 1M1 of .1.15% KC1 in the control experiments which were carried 0 L , t concurrently, The incubation reactions were terminated by the addition of 0.5 Ml of 3% trichloroacetic acid (TCA) in a l l tubes. The tubes were then vortexed for 10 sees, and centrifuged (.5 mins. at 2000 rpm) at room temperature. A similar experiment was also carried out with De-MCP r l (2.5 micromole.ml of deionised d i s t i l l e d water) using the li v e r tissue homogenate only. 2.10.3.3. Extraction procedure for incubation  mixture 0.5 Ml of centrifuged incubation mixture was transferred into a clean 50 Ml centrifuge glass tube and the volume adjusted to 1 Ml by adding 0.5 Ml of deionised d i s t i l l e d water. The extraction procedure from this point on was the same as the one described previously. 2.12. Bioavailability calculations and Statistical Analysis The bioavailability of MCP at different dose levels was calculated by comparing the percent of MCP recovered in 48 hour cumulated rat urine after oral dosing to the percentage of MCP recovered after an equal I.V. dose. Predictions of MCP bioavailability based on linear and nonlinear models were made. The unpaired, two tailed Student's 't' test was employed to s t a t i s t i c a l l y test the difference between groups of data. The level of significance was chosen to be p = 0.05 for a l l analyses. 68 3, RESULTS 3.1. A stepwise approach to obtain a suitable l iquid phase for the simultaneous determination of HFB derivatives of MCP and De-MCP was made. From the preliminary experimentation, i t was found that 3% OV-17 with cumulative McReynolds constants (E ) equal to 884, was 1 not a suitable choice for the desired purposes. An unacceptable broad, f lat chromatographic peak of the HFB derivative of MCP [Retention Time (R.T.) = 3.03 min.] (Fig. 4) was obtained. It was therefore decided to try phases with increasing se lect iv i ty . Figure 5 represents a gas chromatogram obtained from a synthetic mixture of dilute rat urine, MCP, De-MCP and the internal standard diazepam. Identification of the peaks was made by individually injecting the standard compounds of interest onto the gas chromatograph. The chromatographic peaks with R .T . ' s at 6.72 and 7.19 min. were Identified as the HFB derivatives of MCP and De-MCP respectively. The peak at 16.63 min. represented that of underivatized diazepam. The peaks with R.T. ' s 5.57 and 9.69 min. were of unidentified compounds. 37o OV-225 U = 1813) was used on an Ultra Pak Gas Chrom-Q^ 1 (230/270 mesh size) sol id support. The peaks of HFB derivatives of MCP and De-MCP were symmetrical but unresolved. The results contained in this chromatogram (Fig. 5) were similar to those reported by previous workers (Tam and Axelson, 1979a). A relat ively better degree of resolution (R=0.4) was expected from this particular combination of J . Chromatogr. S c i . , 8:685 (1970). 69 Figure 4 : Chromatogram of the HFB derivative of MCP (R.T. = 3.03 min.) and underivatized diazepam (R.T. = 5.48 min.) on a 3% OV-17 packed glass column. Chromatographic conditions: 1.8 m x 2 mm ( i .d . ) , glass column; Stationary phase, 3% OV-17; Solid support, Chromosorb - W (H.P.)( R ) 100-120 mesh; Injection temperature, 250°C; Column temperature, 255°C; Detector (ECD) temperature, 350°C; Carrier gas (Argon: Methane : : 19:1) flow, 40 ml/min; Chart speed, 0.3 cm/min. 71 Figure 5 : Chromatogram of the HFB derivatives of MCP (R.T. = 6.72 min.) and De-MCP (R.T. = 7.19 min.) , and underivatized diazepam (R.T. = 16.63 min.). on a 3% OV-225 packed glass column. Chromatographic conditions: .1.8 m x 2 mm ( i .d . ) glass column; Stationary phase, 3% OV-225; Solid support, Gas chrom-Q (ultra pak)(R) 230-270 mesh. 72 3%OV-225 on ultra pak gas chrom Q (230/270) ro 73 Table 4, General ga,s Itqufd chromatographic conditions Oven/column temperature Isothermal in the range of 230PC to 255QC Injection port temperature 250°C Detector (electron capture) temperature 350°C Chart speed 0.1 to 0.5 Cm.Min"1 Carrier gas (Argon:Methane : : 19:1) flow 30 to 50,ml.min"1 Volume of sample injection 1 and 5 yl Slope sensit ivi ty 0.5 to 1.5 Attenuation 7 to 10 Instrument: Hewlett-Packard 5830 A reporting gas chromatograph equipped with 63Ni- electron capture detector. 74 l iquid phase and sol id support because of the manufacturer's claim regarding the high efficiency [3270.-4250. plates per meter) of the ultra fine (230/270 mesh size) sol id support. This suggested that the l iquid phase OV-225, might s t i l l not be selective enough for the separation of the two compounds. Glass capi l lary columns with high efficiency were compared to conventional packed columns to attempt to overcome the resolution problem. Figure 6 represents a chromatogram obtained on a 24 meter SE30 (support coated open tubular, SCOT) coated glass capi l lary column. The stationary phase SE30, though a relat ively non polar phase 5 ( E = 217), successfully resolved De-MCP, (R.T. = 20.65 min.) , MCP, 1 (R.T. = 21.67 min.) and diazepam, (R.T. = 27.57 min.). However, the MCP peak exhibited characteristic and significant t a i l i n g . This observation implied that a more selective phase was required. Surprisingly, one could not obtain peaks for MCP and De-MCP on a 50 meter OV-225 coated glass capil lary column. Deteriorated phase (OV-225) was suspected as the reason for this observation. In spite of encouraging preliminary results , further experiments with glass capi l lary columns were truncated due to the unavailabil ity of an adequate capi l lary column l ibrary . Based on previous observations, i t was decided to examine the relat ively more polar S i lar phases. The results obtained with represents a chromatogram of a synthetic mixture of HFB derivatives 75 Figure 6 : Chromatogram of the HFB derivatives of De-MCP (R.T. = 20.65 min.) and MCP (R.T. = 21.67 min.) , and underivatized diazepam (R.T. =27.57 min.) on a SE-30 glass capi l lary column. 76 24 METER SE 30 GLASS CAPILLARY COLUMN 77 Figure 7 : Baseline resolution of the HFB derivatives of MCP and De-MCP, and underivatized diazepam on 3% S i lar 9CP packed glass column. I Representative Chromatogram of HFBA derivatized dilute blank rat urine extract. II Representative chromatogram of a synthetic mixture of the HFB derivatives of MCP (R.T. = 4.65 min.) and De-MCP (R.T. = 6.91 min.) , and underivatized diazepam (R.T. = 9.87 min.) . Chromatographic conditions: Column 1.2 m x 2 mm ( i .d . ) glass column; Stationary phase. 3% Si lar 9CP; Solid support, Chromosorb - W ( H . P . ) w l 100-120 mesh. 78 3% Silar 9CP on Chromosorb W (100/120) 79 of MCP CR.T. = 4.65 min.) and De-MCP (R.T. = 6.91 min.) and underivatized diazepam (R.T. = 9,87 min.) on a 3% Silar-9CP l iquid phase coated on r ! R ) Chromosorb W (H.P.) , (-100/120 mesh size). / The peaks were sharp and symmetrical. There was complete resolution among the three peaks. No endogenous interference was observed in the chromatogram from blank dilute urine sample (Fig.7-1). Figure 8 shows the kinetics of HFBA derivatization of De-MCP at 55°C in the absence ( ) of and presence ( — ) of 0.1 Ml of 0.1M triethyl amine (TEA) catalyst. The derivatization of De-MCP in the absence of catalyst was a slow process as evident from the constant increase in the area ratio of HFB derivative of De-MCP and diazepam over a period of six hours. The rate of derivative formation was faster in the presence of TEA catalyst. The reaction reached.repeatable equilibrium within sixty minutes. A modified method for the simultaneous analysis of MCP and De-MCP is shown in Scheme 2. The reproducibility of the assay method was good. The coefficient of variation ( C V . ) for the detection of small quantities of MCP (846 peg.) and De-MCP (88 peg.) was found to be 6.79% (n=6) and 6.39% (n=6) respectively (Fig. 9, Table 5). Fig. .10 represents the reproducibil ity of chromatographic peaks of MCP and DeMCP obtained from duplicate samples, extracted and derivatized under similar conditions. Linearity was observed in the ranges 0.2 yg to 80 Figure 8'.: Kinetics of HFBA derivatization of De-MCP in the absence (—) and presence (;?--) of triethylamine (TEA) catalyst. The Y axis represents the area rat io , di-hepta-fluoro butyryl derivative of De-MCP/diazepam. The X axis represents time, in minutes, after addition of reagent (S). Each sample contained extracted De-MCP base (= 0.67 yg of De-MCP. HC1); 1.0 ml of diazepam (0.2 yg/ml) in benzene; 20 y l of HFBA; 0.1 ml of TEA (0.1M) in benzene (This was replaced with 0.1 ml of benzene in the samples without TEA). 5 y l samples were injected onto the gas chromatograph. 81 KINETICS OF HFBA DERIVATIZATION OF DE-MCP 82 SCHEME 2 ECD - GLC Analysis of MCP and De-MCP Urine 0.1-2ml centrifuge tube IN NaoH 1 ml Deionised Dis t i l l ed H?0 q.s.2ml Benzene 6ml Shaken for 20 mins. Centrifuged to separate layers (Discard aqueous layer) 5,ml benzene layer Evaporate Dissolve residue in 1 ml Benzene solution of internal standard containing diazepam concentration of 0.4 ug.ml-1 < 0.1 MTEA 0.1 ml 4% NH4oH 0.5 ml * I Vortex (20 sees at speed 10) Centrifuged —> • Vortex Reaction allowed to proceed for 60 min at 55°C Vortex H20 ^  Cool io room 10 sees at speed 10 0.5 ml Temperature 5 y l of organic layer injected in GLC 83 Figure 9: Gas chromatographic peaks representing trace levels of the HFB derivatives of MCP and De-MCP and underivatized diazepam. Each y l of sample injected onto the gas chromatograph represent: MCP (R.T. = 6.75 min.) 846 peg, De-MCP (R.T. = 9.41 min.) 88 peg, Diazepam (R.T. = 21.07 min.) 2 ng. Column, 3% Silar-9CP on Chromosorb-W(H.P.)(R) 100/120 mesh 84 TABLE 5 Coefficient of Variation ( C V . ) of Peak Heights HFBA Derivatives of MCP and De-MCP 5 yl Injection of Each Sample Contained: MCP = 846 peg, De - MCP = 88 peg and Diazepam = 2000 peg Sample No. Peak heights (mm) MCP De-MCP Diaz. MCP/DIAZ. De-MCP/DIAZ 1 b* 14.5 14.5 1.5 1.5 10.5 10.3 1.381 1.4078 0.1429 0.1456 2 b 14.0 14.0 1.5 1.3 10.3 10.0 1.3592 1.400 0.1456 0.1300 13.8 13.8 1.5 1.5 10.3 10.5 1.3398 1.3143 0.1456 0.1429 15.0 15.0 1.5 1.5 9.7 9.5 1.5464 1.5789 0.1546 0.1579 5 9 b 14.3 14.0 1.5 1.5 10.3 10.3 1.3883 1.3592 0.1456 0.1456 6 b 14.3 14.3 1.5 1.5 9.5 9.0 1.5053 1.5889 0.1579 0.1667 ** x ± S.D. 1 .4308±0.0971 0 . 1 4 8 4 ± 0 . 0 0 9 5 * Duplicate injections from the same sample ** Mean peak height ratio ± one standard deviation <~ n Coefficient of variation. : MCP/DIAZ = f-^- x 100 = 6.79% x De-MCP/DIAZ.= 6.4% 86 Figure 10 Representative chromatograms of HFB derivatives of MCP (R.T. - 6.26 min.) and De-MCP (R.T. = 9.08 min.) , and underivatized diazepam (R.T. = 17.58 min.) , i l lus tra t ing quantitative reproducibil ity of peaks and the retention times of the peaks obtained from duplicate samples, extracted and derivatized under similar conditions. Column, 3% Silar-9CP on Chromosorb-W(H.P.) 100/120 mesh S T"*5 .a* f T r 9 9 : ' 9 . B 8 B O T T L E S T ( 3 4 ) ="T7.58 5 0 3 f W » CO 1^ 4. 29 £.23 5 . B 8 13.37 =—17.57 BOTTLE (jS> AREA v. 88 1.0. yg.ml MCP-HC1-H20 and 0.025 to.0,5 yg-ml De-MCP-HCl in dilute rat urine. Regression analysis of standard curves of MCP (Fig, 11) and De-MCP (Fig. 12) showed that the l ines of best f i t through the data points were described by: Y = 2.9018 X - 0.0309 for MCP, and Y = 2.5473 X + 0.0009 for De-MCP with correlation coefficient, r = 0.999 in both cases. The HFB derivatives of MCP and De-MCP were found to be stable for at least 30 hours at room temperature.as verified by the unchanged area ratios (HFB derivative/diazepam). 3.2. In vivo Studies 3.2.1. Two-thirds hepatectomy study The total percentage of MCP and one of i t ' s metabolites, De-MCP, recovered in 48 hour.cumulative, urine from two-third (2/3rd) hepatectomised and sham operated rats after i . p . administration of MCP-HC1-H20 (equivalent to 15 mg.kg^CP base) is shown in Figure 13. The levels of intact MCP recovered in the 48 hour cumulative urine from 2/3rd hepatectomised rats were s ignif icantly higher than those of sham operated rats. There was no significant difference in the percentage of De-MCP recovered in the urine of 2/3rd hepatectomised rats compared to that of sham operated rats. 89 Figure 11: Calibration curve of the urine extract of MCP. Each point on the curve is the average of two samples, both of which were chromatographed in duplicate. 90 CALIBRATION CURVE OF MCP AMOUNT OF MCP(Mcg) 91 Figure 12: Calibration curve of the urine extract of De-MCP. Each point on the curve is the average of two samples, both of which were chromatographed in duplicate. 92 CALIBRATION CURVE OF DEMCP AMOUNT OF DE-MCP (Meg) 93 Figure 13 Histograms i l l u s t r a t i n g the percent recovery of 48 hour cumulat ive u r i n a r y e x c r e t i o n of i n t a c t MCP and De-MCP ( e q u i v a l e n t to MCP) i n t w o - t h i r d hepatectomised and sham ODerated r a t s . An i . p . dose e q u i v a l e n t to 15 mg .kg - 1 MCP base was a d m i n i s -te red i n both groups of r a t s . 94 TWO-THIRDS HEPATECTOMY STUDY •slTRAPERITONEALOPj DOSE OF 16.0 MG/KG MCP TWO-THIRDS HEPATECTOMISEDversus SHAM OPERATED RATS (n* 6 ) • S at p<0.05 NS at p<0.05 MCP TWO-THIRDS MCP SHAM OPERATED DE-MCP TWO-THIRDS DE-MCP SHAM OPERATED 95 3.2,2, Carbon tetrachloride (CCI4) pretreatment study The results of CC1^ pretreatment study with respect to the percentage of intact MCP recovery in the 48 hour cumulative urine was similar to that of the two-thirds hepatectomy study (Fig. 1:4).,'.The total amount of De-MCP recovered in the urine of C C 1 4 pretreated rat was also s ignif icantly higher when compared to that of normal saline pretreated rats. 3.3. In-vitro Studies 3.3.1. 9000g l iver and kidney homogenate study. Figure 15 represents a series of gas chromatograms obtained from the extraction of various incubation mixtures, followed by HFBA derivatization in the presence of the internal standard diazepam (R.T. = 3 2 . 1 5 ± 0 . 2 mins). A l l incubation mixtures contained an equal amount of MCP prior to in i t ia t ing the incubation reaction (Section 2.11.3.2.). The f i r s t chromatogram (a) represents the extent of MCP (R.T. = 7.85 min) present in the 9000g supernatant after the incubation reaction, in the absence of any tissue homogenate. The second chromatogram (b) represents the level of MCP present in the 9000g supernatant of l iver homogenate after the incubation reaction. There was: an additional peak in this second chromatogram (Fig. 15b) (R.T. = 10.44 min) which was subsequently identified to be that of the mono-de-ethylated metabolite, De-MCP, as shown in the third 96 Figure 14 Percent recovery of 48 hour cumulative urinary excretion of intact MCP and De-MCP (equivalent to MCP) in CCl^ pretreated and normal saline (N.S.) pretreated rats. An i . p . dose equivalent to 1.0 mg.kg-1 -•• MCP base was administered in both groups of rats. 97 CARBON TETRACHLORIDE PRETREATMENT STUDY WTRAPERITONEALOPJ DOSE OF 1.0 MG/KG MCP PRETREAT ED vertut CONTROL RATS (nc5 ) MCP CCL MCP control DE-MCP DE-MCP CCL contro l . S at p<0.05 S at p<0.05 98 Figure 15 Representative gas chromatograms showing the different levels of MCP obtained from various incubation mixtures at the end of each incubation reaction ( I n i t i a l l y , each incubation mixture contained an equal amount of MCP). Peaks with R.T. ' s of 7.85, 10.44 and 32.21 mins. in the i l lustrated chromatograms represent HFB derivatives of MCP, De-MCP, and underivatized internal standard diazepam respectively. a : in the absence of any tissue homogenate preparation, b : in the presence of 9000 supernatant of l iver homogenate (note the presence of De-MCP peak), c : representative chromatogram of De-MCP, d : in the presence of 9000 g supernatant of kidney homogenate. 66 '6 100 chromatogram (c). The relative amount of MCP present in the incubation mixture after the incubation reaction, in the presence of 9Q00g supernatant of kidney tissue is shown in the fourth chromatogram (d) in Fig. 15. Figure 16 i l lustrates the relative amount of MCP remaining after various treatment procedures. Area ratios (HFB derivative of MCP/diazepam] were used for this purpose. The relative amounts of MCP in the presence of l iver tissue preparations were signif icantly lower than those of controls. In addition, appreciable amounts of De-MCP were formed in the incubation mixture containing l iver tissue. The relative amounts of MCP in the presence of the kidney tissue preparation were not s ignif icantly different from those of controls and the metabolite, De-MCP,was absent. 3.3.2. 9000g lung homogenate study The results of 9000g supernatant of lung tissue homogenate are shown in Figure 17. Experiments involving l iver tissue preparations were also conducted at the same time to check the v iab i l i t y of the enzymes involved. The results of this study suggest that lung tissue is not measurably involved in the metabolism of MCP in the rat. 3.3.3. In Vitro experiments with whole fraction tissue homogenates A study u t i l i z ing whole fraction of tissue homogenates was conducted to examine involvement of the lungs and/or kidneys in 101 Figure 16 Histograms representing the area ratios (derivative/diazepam) obtained from the in-v i tro metabolism of MCP in the absence and presence of 9000 g supernatants from l iver and kidney homogenates. 102 EXTRA-HEPATIC METABOLISM OF MCP 8 0 0 0 - g IN-VITRO STUDY Cn « 8 ) l.5^ J -OH 0-B control control MCP DE-MCP " S at p<0.05 • NS at p<0.05 Iver MCP Iver DE-MCP kidney MCP kidney DE-MCP 103 Figure 17 Histograms representing the area ratios (derivative/diazepam) obtained from the in-v i tro metabolism of MCP in the absence and presence of 9000 g supernatants of l iver and lung homogenates. 104 EXTRA-HEPATIC METABOLISM OF MCP 9000-Q IN-VITRO STUDY <n» 6 ) 20 -1 o cc < o z < < 2 CC LU LU > r-< > CC LU o US A i-oH O 0.5-1 < CC < LU CC < J±L control control MCP DE-MCP " S at p<0.05 • NS at p<0.05 Iver MCP iver DE-MCP lung MCP king DE-MCP 105 the metabolism of MCP, Whole fraction tissue homogenates were used because the exact location of MCP metabolising enzymes in lung and/or kidney was not identif ied. Also, as the optimum tissue-protein concentration for the metabolic reaction was not known, a wide range of whole tissue homogenate volumes (.0.5, 1.0, 1.5 and 2.0 Ml) was used. The results of the aforementioned experiments are shown in Fig. 18, There was no significant difference in.the area ratios (MCP-HFBA/Diazepam) of lung and kidney tissue preparations, compared to that of controls. The area ratios of l iver homogenate preparations were s ignif icantly Tower compared to the controls (Fig. 18). 3.3.4. In-rvltro metabolism of De-MCP The results of in vitro metabolism of De-MCP are shown in Fig. 19. The levels of De-MCP in the test incubation mixture containing 9000g supernatant of l iver tissue homogenate after the incubation reactions are s ignif icantly ( p « 0 . 0 5 ) lower compared to those of controls (without l i ver t issue). 3.4. Bioavai labi l i ty of MCP as a Function Of Dose The pharmacokinetic behaviour of MCP in the rat was studied as a function of dose and route of administration. Forty-eight'hour cumulative urinary excretion data of Intact MCP and De-MCP was ut i l i sed to study the effect of route of administration at different dose levels. The bioavai labi l i ty of MCP after an oral dose was calculated using 106 Figure 18 Histograms representing the area ratios (derivative/diazepam) obtained from the in-v i tro metabolism of MCP in the absence and presence of different volumes of whole tissue homogenates of l iver (2.0 ml), kidneys (0.5, 1.0, 1.5 and 2.0 ml) and lungs (0.5, 1.0, 1.5 and 2.0 ml). 107 EXTRA-HEPATIC METABOLISM OF MCP TISSUE HOMOGENATE STUDY (n = A ) Q cc < z < cc L U < CC < L U CC < 2.2H • S at p<0.05 NS at p<0.05 I 1.8-1 L U > < > CC U J i.6H 1.4H rii control Iver Iver 0.5 1.0 1 .5 2 .0 0.5 1.0 1 5 2.0 MCP MCP DEMCP kidney MCP lung MCP 108 Figure 19 Histograms representing the area ratios (derivative/diazepam) obtained from the in-Vitro metabolism of De-MCP in the absence (control) and presence (test) of 9000 g supernatant of l iver homogenate. 109 9000-g IN-VITRO STUDY HEPATIC METABOLISM OF DE-MCP (n .6 ) O < CO < CC LU LU > < > CC LU o < cc < LU CC < 20n 1.5H 1-0^ o,sH 0*0 CONTROL LIVER . S at p<0.05 110 the following equation. CEXU) F = Bioavai labi l i ty = (gxu).j where, (EXU)QJ^  and (EXU)-J ^ are the percentages of intact MCP recovered in the 48 hour cumulative rat urine samples after administration of an equal dose of MCP.HCl.^O via . oral and i . v . routes respectively. Figure 20 shows the percent recovery of intact MCP and De-MCP in 48 hour cumulative urine after administration of an equal dose -1 of MCP-HC1-H20 (equivalent to 5 mg.kg MCP base) via oral and i . v . routes in two randomly selected groups of rats. The mean percent recovery of intact MCP-(n=9) after the oral route was lower than that of the intravenous route suggesting a sl ight hepatic first-pass effect, provided that complete absorption had occurred. The levels of De-MCP recovered were less than 5% for both routes. S ta t i s t i ca l l y , the levels of both, intact MCP as well as De-MCP, recovered after oral administration were not s ignif icantly different from those of the i . v . route. Similar results were obtained after a lffig.kg"1 dose (Fig. 21). The percent of dose recovered as intact MCP and De-MCP after the oral route was not s ignif icantly different from that seen after an i .v . route of administration. These results are consistent with the work done by previous investigators (Tam et a l . , 1981a). I l l Figure 20 Histograms comparing the percent recovery of 48 hour cumulative urinary excretion of MCP and De-MCP (equivalent to MCP) following an oral (p.o.) or intravenous ( i . v . ) dose (MCP. HC1. H2O) equivalent to 5mg.kg-l^p base: 112 48 HOUR CUMULATIVE RAT URINE STUDY 5.0mg/kg MCP ORAL(P.O.) versus INTRAVEN0US(I.VJ (n = 8 ) UJ 35n cr 3 O UJ QC UJ > O o UJ OC o UJ Q OC o Q. O UJ CO o Q U. o r -Z UJ o oc UJ a. 33 H 25 A 2 20H 15H 18H MCP-P.O. MCP-LV. DE-MCP-P.O. DE-MCP-LV. NS at p<0.05 NS at p<0.05 113 Figure 21 Histograms comparing the percent recovery of 48hburt;tumulati ve urinary excretion of MCP and De-MCP (equivalent to MCP) following an p.o. or i . v . dose (MCP. HC1. H?0) equivalent to 1.0 ITO.kg-1 MCP base. 114 48 HOUR CUMULATIVE RAT URINE STUDY 1.0 mg/kg MCP ORALXP.O.) versus INTRAVENOUSd.VO (n = 8 ) 35n 33 A 2SA 2 0 H 15H UJ MCP-P.O. MCP-LV. DE-MCP-P.O. DE-MCP-LV. NS at p<0.05 NS at p<0.05 115 To test the hypothesis of temporary saturation of l iver enzymes during the absorptive stage after relat ively high doses QslMg. K g - 1 ) , the 48 hour cumulative urinary excretion pattern at lower doses IMg.Kg - 1 ) were studied. The results of the 0.5 and 0.1 -1 Mg.Kg studies are shown in figure 22 and 23 respectively. In both cases, the total amount of intact MCP recovered after oral dosing were signif icantly lower than those following I.V. administration. The total amount of De-MCP recovered was less than 5% by either route and there was no significant difference between them. Table 6 represents the compilation of experimentally determined bioavai labi l i ty data at different dose levels. It is observed that the bioavai labi l i ty of MCP increases with increasing dose. In other words, the bioavai labi l i ty of MCP has been found to be nonlinear, and is dose dependent. 116 Figure 22 Histograms comparing the percent recovery of 48 hour cumulative urinary excretion of MCP and De-MCP (equivalent to MCP) following an p,o. o r , i . v . dose (MCP. HC1. H20) equivalent to 0.5 mg.kg-1 MCP base. 117 48 HOUR CUMULATIVE RAT URINE STUDY 0.5 mg/kg MCP ORAL(P.O.) versus INTRAVENOUSO.VJ (n = 5 ) 35-i S 30H S at p<0.05 NS at p<0.05 25 H 20 H 15H 10H sH MCP-P.O. MCP-IV. DE-MCP-P.O. DE-MCP-W. 118 Figure 23 Histograms comparing the percent recovery of 48 hour cumulative urinary excretion of MCP and De-MCP (equivalent to MCP) following an p.o. or i .v . dose (MCP. HCI.H2O) equivalent to 0.1 ing.kg-1 MCP base. 119 48 HOUR CUMULATIVE RAT URINE STUDY 0.1 mg/kg MCP ORAL(P.O.) versus INTRAVENOUS(I.V0 (n = 6 ) L U 3 5 -Z cr Z O 3 3 -L U tr U J > CO 2 5 -L U rr *—s C L MC 2 0 -L U a cc o Q . 15-O L U OS 10-Q L U O H Z 5-L U O cr L U C L I MCP-P.O. MCP-LV. DE-MCP-P.O. DE-MCP-IV. . S at p<0.05 - NS at p 0.05 120 TABLE 6 NON LINEAR DOSE DEPENDENT BIOAVAILABILITY(F) OF MCP DOSE, fog,'kg" BIOAVAILABILITY (F) a 15 .0 0.91 5 . 0 0 .83 1.0 0.777 0 . 5 0.750 0.1 0 .488 B i o a v a i l a b i l i t y of MCP c a l c u l a t e d from 48 hour cumulat ive u r i n a r y e x c r e t i o n data f o l l o w i n g o r a l and i . v . a d m i n i s t r a t i o n of d i f f e r e n t doses . a . C a l c u l a t e d by comparing area under plasma c o n c e n t r a t i o n versus t ime curve a f t e r o r a l route to t h a t of i . v . route of a d m i n i s t r a t i o n . (Data obta ined by Tam et a l . , 1981) . 121 4, DISCUSSION 4.1. GLC-ECD Analysis 4.1.1. Phase selection After considerable investigation to find a suitable stationary phase to resolve the HFB derivatives of MCP and De-MCP, 3% Silar-9CP was selected as the best choice. Complete baseline resolution between the derivatives of the two compounds was obtained using a 1.8 m x 2 mm ( i .d . ) glass column packed with 3% Silar-9CP on Chromosorb-W(H.P. ) ^ 100-120 mesh. This column permitted the simultaneous quantitation of MCP and De-MCP in the same sample. There are however, some limitations associated with the use of Silar-9CP, namely: (a) It takes a long time to condition. At least seven days at 250°C with a constant carrier gas flow of 40 Ml.-Min.^ are required for conditioning before any quantitation can be made. (b) It has a short l i f e span. In the absence of oxygen pre-fiIters (unavailable during the course of study), the deterioration of the phase was quite rapid. This was indicated by a rapid decline in retention times of the peaks of interest which usually started appearing after one month of routine use. This might explain the differences in the relative retention times ( d i a z e p a m V a t 1 V e ) f o r M C P ( 0 ' 3 2 ' 0 , 4 7 a n d ° ' 2 4 ) a n d D e _ M C P ( 0 ' 4 4 , 0.70, and 0.32) in figures 7 (p.78), 9(p.87) and 15(p.99) respectively. 122 4.1.2. Optimisation of HFBA derivatization of De-MCP The present work on the kinetics of HFBA derivatization of De-MCP have demonstrated that the rate of derivative formation is faster in the presence of triethylamine (TEA) catalyst. Aryl amines are weaker bases than primary alphatic amines, due to the tendency of the lone electron pair on the nitrogen atom to conjugate with the pi(n) electrons of the nucleus (Lyons et a l . , 1965). Tarn and Axelson (1979b),using mass spectrometric analysis, have deduced that both, the primary amino group on the aromatic ring as well as the secondary amino group on the side chain of the De-MCP molecule are derivatized with HFBA. It is speculated that the rate of derivatization of the secondary amine group on the side chain of De-MCP is considerably slower than that of the primary amine group on the aromatic ring of De-MCP. This is based on steric hindrance. In the present studies, 0.1 Ml of 0.1 M TEA in benzene was used as a catalyst to increase the overall rate of De-MCP derivative formation. Triethylamine (TEA), a proton acceptor, enhances the reaction in the forward direction (Walle and Ehrsson, 1970 and 1971). 123 4.1.3. Applicabi1ity of the assay method The present assay has been found to be sensitive and specif ic . It has been shown to be applicable in the simultaneous quantitation of MCP and De-MCP in rat urine samples. The coefficients of variation ( C V . ) at the lower levels of MCP (846 peg) and De-MCP (88 peg) were found to be 6.79% and 6.39% respectively (Table 5_), reflecting the fact that the assay is reproducible. Reproducibility of the assay was also validated from the very low var iab i l i ty between duplicate samples. Excellent correlation (r > 0.999) was obtained between the area under peak count and the.amount of drug (^  80 peg) injected onto the gas chromatograph. 124 4.2. Animal Experiments 4.2.1. In-vivo experiments 4.2.1.1. Two thirds (2/3rd) hepatectomy study The 2/3rd hepatectomy study was performed to further assess the involvement of the l i ver as a major metabolic organ for MCP. The study was based on the assumption that surgical removal of 2/3rds of the rat's l i v e r , would decrease hepatic function. If the l i ver is involved in MCP metabolism, then one would expect increased amounts of intact drug in the cumulative urine compared to the non-hepatectomised rats. Surgical removal of 2/3rd of rat l i ver is a major operation, and ideally one should allow the animal to recuperate from surgically associated stress and trauma for at least three days prior to the begining of any experiment. However, Higgins and Anderson (1931) found that the regeneration of l iver tissue in rats is very rapid. They reported that i t took only three days from the day of surgery to restore l i ver mass from 29% to about 70% of control. Therefore, in the present study, the animals were allowed to recuperate for only one day following surgery. Sham operated rats were used for controls. The intraperitoneal (I .P.) route of drug administration without ether anaesthesia, as stated ear l i e r , was preferred over other 125 routes ( v i z . , i . v . or oral) which, required the use of ether anaesthesia. This was done to minimise mortality due to increased ether toxic i ty in 2/3rd hepatectomised rats. The total amount of intact MCP in urine at 48 hour was signif icantly higher in 2/3rd hepatectomised rats compared to that found in the urine from sham operated rats (Fig. 13). This strongly suggests that the l iver is involved in the metabolism of MCP. The levels of one of the metabolites, De-MCP however, were too small (« 5%) in both the treatments to draw any conclusions. 4.2.1.2. CaKbon^tetrachloride (CC14) pretreatment study Carbbnri.tetrachloride (CCl 4 ) , a well known hepatotoxin, has been widely used in experimental animals to produce varying degrees of l iver impairment including necrosis (Gallagher, 1962), fatty in f i l t ra t ion (Slater, 1966) and decreased act iv i ty of microsomal enzymes that catalyze the oxidation of drugs (Neubert and Maibauer, 1959, Kato et a l . , 1962; Dingell and Heimberg, 1968). Carbon tetrachloride impairs oxidative enzymes in l iver microsomes by decreasing the amount of cytochrome P-450 which might be related to an active metabolite (Sasame et a l . , 1968) or free radical formation (Glende, 1972a,b). Hepatotoxicity in rats is evident within 24 hours of CC1 4 treatment (1.0 ml .kg - 1 ) (Hirano et a l . , 1975). However, the toxic effects of C C l d to other organs such as the kidney do not occur until 126 several weeks after the exposure to the toxin CSmetana, 19.39; Moon, 1950; Zimmerman and Norbach, 19801. Therefore, 24 hour CC1 4 pretreated rats were used in the present studies to ensure minimal damage to other organs. The intraperitoneal route of drug administration without ether anaesthesia was used as before to avoid ether toxic i ty . The percentage of Intact MCP recovered from the 48 hour cumulative urine of CCl^ pretreated rats is almost three-fold higher (Fig. 14) than that of normal saline pretreated rats. This observation strongly suggests that the rat l iver is a major organ for the metabolism of MCP in the rat. A similar pattern of MCP recovery was found by Tarn et a l . (1981b). Tarn et a l . (1981b) found no significant difference in the levels of De-MCP in the 48 hour cumulative urine of CCl^ pretreated rats as compared to the normal saline pretreated rats. Based on the unchanged levels of De-MCP in both the treatments, these investigators have speculated the poss ibi l i ty of extra-hepatic metabolism of MCP in the rat. But, in the present studies, the levels of De-MCP in the 48 hour cumulative urine of CCl^ pretreated rats were higher as compared to the normal saline pretreated rats. Uncomplete derivatization of De-MCP (in the absence of a catalyst) by Tarn et a l . (1981b) may explain the observed discrepancy. If the l iver were to be the only metabolic organ for MCP, then one would expect that, during l iver damage, (eg. CCl^,pretreatment), metabolism would be impaired, the extent of which being a function of hepatic injury. This damage should result in higher levels of parent 127 drug being excreted unchanged and lower levels of jnetabolite in the urine of rats with hepatic injury as compared to the normal rats. The observation that the De-MCP levels, in our present study are higher rather than lower in the CCl^ pretreated rat urine as compared to those of normal rats appears inconsistent. This can be rationalized from the in-v i tro metabolism study of MCP and De-MCP. It has been found in our present in-v i tro studies that MCP is metabolised in the rat l iver and one of the metabolites is De-MCP (Fig. 15), which is then further metabolised in the l iver (Fig. 19). Therefore, during CC1 4 pretreatment, the metabolism of both MCP and De-MCP are probably impaired resulting in higher overall levels of both of these compounds in the urine as compared to those in normal saline pretreated rats. 4,2.1,3,..';BioaVai 1 abi 1 i ty experiments The results of both in-vivo as well as in-v i tro experiments have clearly shown that the l iver is the primary organ responsible for the MCP metabolism in rats , essentially eliminating the hypothesis of significant extra hepatic metabolism. Based on this hypothesis, b ioavai labi l i ty studies involving urinary excretion patterns, following oral and i . v . administration of various MCP doses, were characterised. These studies were undertaken to explore the possible reason(s) why MCP does not undergo hepatic f i r s t pass metabolism following oral administration over the dose range studied (Tam et a l . , 1981a). In 128 spite of the fact that total body clearance of MCP approaches hepatic blood flow. One of the hypotheses proposed by Tam et a l . (1981a) to explain this phenomenon, was that the concentrations of MCP in the hepatoportal vein during the absorption phase are high enough to saturate the l iver ' s enzyme capacity at the dose range studied (1-35 mg.kg - 1 ) . To test this , the bioavai labi l i ty (F) of MCP was determined at lower doses (0.1, 0.5, 1.0 and 5.0 mg.kg - 1 ) . The 48 hour cumulative urinary excretion data, obtained in our present studies showed significant hepatic first-pass metabolism of MCP at 0.1 and 0.5 mg.kgTV:doses, vXu_ -j resulting in lower bioavai labi l i ty values (Table 6) (F = - T T T T : ) A non-linear, dose dependent trend in the bioavai labi l i ty values of MCP.at different doses was evident. The bioavai labi l i ty of MCP was observed to increase with increasing doses, suggesting that saturation of l i ver enzyme capacity occurs at higher doses of MCP during gastro-intestinal absorption. This may explain why MCP does not undergo significant hepatic f i r s t pass metabolism at doses exceeding 1.0 mg .kg. 1 The phenomenon of dose dependent bioavai labi l i ty has also been exhibited by other high l iver clearance drugs such as propranolol (Suzuki et a l . , 1972, 1974; Evans et a l . , 1973; Shand et a l . , 1973;. Routledge, 1979; Machichan et a l . , 1980; Rikhisia et a l . , 1981) and alprenolol (Ablad et a l . , 1972, 1974). 129 4.3. Prediction of Bioavai1abi1ity 4.3.1. Linear models Rowland (1972) and Gibaldi (1975), u t i l i s ing perfusion and compartmental models respectively, have independently developed similar linear relationships to predict the bioavai labi l i ty of any compound. According to these investigators, the predicted bioavaila-b i l i t y of a compound with high hepatic clearance is expressed by the following equation: fm x DoseT F = 1 1 • v P R E D I C T E D - 1 QL( / c P . d t ) I V where fm is the fraction of the total administered dose metabolised by the l iver (fm = 1 - 1^- ) D o s e I . V . zXu being the amount of intact MCP recovered in 48 hour cumulated urine), Q L is the l iver blood flow rate in the rat (17.2 ml/nrin)*(Boxenbaum, 1980), Dosej y is intravenous dose administered, and (°°| Cp.dt)j y is the area under the plasma concentration versus time curve after i . v . administration of the drug. An estimate of FpREQjcjED f o r M C P » f r o m t n e ^ Mg.Kg"^ i . v . data of Tarn et al.(1981a), was found to be 0.64. The experimental value of F at the same dose, as calculated from the following expression: *(MCP)b1ood , c T 7 7 ,1no, , f w p p T s 1.5 Tarn et a l . (1981a) ^ L H ; p l a s m a 130 co (0 / ' C P - d t W L oo (of C p . d t ) i ; ^ e q u a l t o . 0 > 9 1 > EXPERIMENTAL = ~ W A S F O U N D T 0 B E Theoretically, the results of • r p R E D I C T r £ D and EXPERIMENTAL c o u l d D e related in three ways: ^ EXPERIMENTAL = ^PREDICTED (B) EXPERIMENTAL * ^PREDICTED (C) EX P E R I M E N T A L - F P R E D I C T E D If the predicted value of F is equal to the experimental one (Case a) , then a l l the forgoing approximations and subsequent equations in the derivation of ^PREDICTED w o u ^ ' 3 e reasonable. If the experimental value of F is less than the predicted value (Case b), then most l i k e l y , incomplete gastrointestinal (G.I.) absorption and/or metabolism is taking place. In a s ituation, where the experimental F value is greater than the predicted one (Case c ) , there is a poss ibi l i ty that the concen-tration of drug in the hepatoportal view during the G.I . absorption stage is high enough to saturate l iver enzyme capacity. The rate of drug metabolism in such a case, would be described by nonlinear (Michaelis-Menten) Kinetics. If the aforementioned assumption is true, 131 then the bioavai labi l i ty of the drug to the systemic circulation becomes a function of dose and the rate of G.I , absdrption [Rowland, 1972]. Our results f a l l into the latter category ^EXPERIMENTAL > FPREDICTED^' i n d i c a t i n 9 t n a t t n e b ioavai labi l i ty of MCP is nonlinear and dose dependent. 4.3.2. Non-1inear bioavai1abi1ity Keller and Scholle (1981), have recently derived an integrated form of the Michael is Menten equation to describe the non-linear hepatic f irst-pass metabolism of an oral ly administered drug: F = 1 Vm KaTD" D.Ka.T. + Ki-10LN 2 + Ln-D.Ka.T+Kme 10Ln(2) Where F is the non linear bioavai labi l i ty of the drug in question and are the Michaelis-Menten constants in mass terms (Mg.Hr -^) and Mg respectively), T =„time interval for portal blood circulat ion (1 minute) Ka = apparent f i r s t order absorption rate constant (Hour -^) and D = oral dose of the drug administered (Mg). It is apparent from the equation that the nonlinear bio-ava i lab i l i ty is a function of dose as well as rate of absorption, as stated in the preceding section. An estimate of Ka for MCP at 15 Mg/Kg 132 dose using a two compartmental open model was found to be 1.9008 hr' •1 Ut i l i s ing the experimentally determined values of F at doses of 0.1 mg and 15 mg.kg - 1 respectively (boundary conditions) the following two equations were obtained: 0.488 = 1 m 1.9008 x 0.025 10Ln(2) + Ln-1.9008 x.O.0167.x 0.025 + Km 1.9008 x 0.0167 x 0.025 + 1024Km (1) AND 0.91 = • 1 m 1.9008 x 3.75 10Ln(2) + Ln-1.9008 x 0.0167.x 3.75 + Km 1.9008 x 0.0167 x 3.75 + 1024Km (2) Rearrangement of these two equations yielded: 41.1 m i n m + \ n 1.9008 x 0.0167 x 0.025 + Km l U L n ^ j + Ln 1 > 9 0 0 8 x 0 > 0 1 6 7 x 0 > 0 2 5 + 1 Q24Km -1.559 10Ln(2) + Ln 1.9008 x 0.0167 x 3.75 + Km 1.9008 x 0.0167 x 3.75 + 1024Km =0 (3) Although Equation (3) has only one unknown parameter, there is no simple method to solve i t . Therefore, a stepwise iteration procedure was used to determine the value of Km. Table 7 represents various iterated values *** of Km, from which the Km value of 0.007 mg was chosen to be the best estimate. Substitit ion of this value of Km, in equation (1) or (2) yielded a Vfn value of 0.225 mg/hr~\These estimates of Km and Vm were used to predict (F) values at different MCP doses. Table 8 shows a comparison of predicted and experimental F values as a function of dose. The 133 41.1 (X) - 1.559 (X 1) = 0 = Y TABLE 7 Km Y 10 + 249.9 1 + 24.99 0.1 + 2.26 0.01 + 0.0669 0.009 + 0.0454 0.0085 + 0.0350 0.008 + 0.0244 0.007 + 0.004*** 0.005 - 0.0362 0.001 - 0.0954 0.0001 - 0.0905 experimental 'F ' values clearly follow a dose dependent pattern which is similar to that of the predicted values. This strongly suggests that the bioavai labi l i ty of MCP is nonlinear. TABLE 8 Dose 0. l mg.kg"1 0. ,5mg.kg - 1 1 .0 mg.kg' 1 5.0 mg.kg - 1 iB.omg. kg"1 PREDICTED F VALUES 0. 492 0. 58 0 .64 0.82 0.909 EXPERIMENTAL F VALUES 0. 488 0. 75 0 .777 0.83 0.91 134 5. SUMMARY AND CONCLUSIONS (A) A modified, quantitative gas l iquid chromatographic -electron capture detector technique for the simultaneous analysis of metoclopramide and i t ' s mono-de-ethylated metabolite was developed. The method was based on the acylation of these compounds with heptafluorobutyryl anhydride at 55°C for 60 minutes in the presence of 0.1 Ml of 0.1 M triethylamine catalyst. A 1.9 M x 2 MM ( i .d . ) glass column packed with 3% Si lar - 9CP on Chromosorb - W ( H . P . ) ^ 100-120 mesh size was used. (B) In-vivo experiments involving two-thirds hepatectomy and carbon tetrachloride pretreatment studies suggest that the rat l iver is involved in the metabolism of metoclopramide. (C) In-vitro studies involving incubation of equal amounts of MCP (5 Micromole. M L - 1 ) with various tissues homogenates (viz. l i v e r , kidney and lung) and their 9000g supernatant fractions indicated that: (i) the metabolism of metoclopramide occurs in the 1iver, ( i i ) the mono-de-ethylated metabolite of metoclopramide is further metabolised in the l i v e r , and ( i i i ) the kidney and lung tissues are not involved in the metabolism of metoclopramide. 135 (D) Forty-eight hour cumulative urinary excretion studies "were conducted following oral and intravenous administration of 0.1, 0.5, 1.0 and 5.0 mg.kg - 1 of metoclopramide. The bioavai labi l i ty (F) of metoclopramide was found to be non-linear, i . e . the ' F ' value increased with increasing doses. This observation suggested that metoclopramide undergoes dose-dependent hepatic f irst-pass metabolism in the rat . (E) An attempt to predict the bioavai labi l i ty of MCP, on the basis of a non-linear model was made. 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A . and G i l l e t t e , J .R. : Studies on the destruction of l iver microsomal cytochrome P-450 by carbon tetrachloride administration. Biochem. Pharmacol. 1_7: 1759 (1968). Savio, E. and Pontiggia, P.: Metoclopramide e assorbimento intestinale di isomiazide. Annali di Medicina Scondalo J_3:402 (1965). Schmidt, G . F . ; Angel, T h . ; Bauer, H. and Doenicke, A. : The effect of domperidone and metoclopramide on antraT'motility. Anaesthesist,27:427 (1978). 152 Schulze-Delrieu, K. : Drug Therapy: Metoclopramide. The New Eng. J . Med. July 2:28 (1981). Schuppan, V . D . ; Schmidt, I. and Hel ler , M. : Untersuchungen Zur pharmakokinetik von metoclopramid am menschen. Arzneim. Forsch./ Drug Research. 29 (1):151 (1979). (Eng. summary). Shand, D .G. ; Branch, R .A . ; Evans, G . H . ; Nies, A.S. and Wilkinson, G . : The Disposition of propranolol (VII) The effects of saturable hepatic tissue uptake on drug clearance by the perfused rat l i v e r . Drug Metal. Dispos. 1:679 (1973). 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Suzuki, T . ; Isozaki, S.; Ohkuma, T. and Ri ki hi sa, T. : Influence of the route of administration on the mean hepatic extraction ratio of propranolol in the rat . J . Pharm. Dyn. _3^ 603 (1980). Tam, Y.K. and Axelson, J . E . : A GLC-ECD assay for metoclopramide in biological f lu ids . J . Pharm. Sc i . 6J_: 1 9 7 3 (1978). Tam, Y . K . ; Axelson, J . E . and Ongley, R. : Modification of metoclopramide GLC assay: application to human biological specimens. J . Pharm. Sc i . 68(10)1254 (1979a). Tam, Y.K. and Axelson, J . E . : Sensitive electron-capture gas-liquid chromatographic assay for the de-ethylated metabolite of metoclopramide. J . Chromatog. 170, 157 (1979b). 154 Tarn, Y . K . ; Axelson, J . E . ; McErlane, B . ; Ongley, R. and Price, J . D . E . : Dose dependent pharmacokinetics of metoclopramide in rat: an effect of hemo perfusion? J . Pharmacol. Exp. Therap. 217^ : (3)764 (1981a). Tarn, Y . K . ; Axelson, J . E . ; McErlane, B . ; Kapi l , R .P . ; Riggs, K.W.; Ongley, R. and Price, J . D . E . : The pharmacokinetics of metoclopramide in rats with experimental renal and hepatic dysfunction. J . Pharmacol. Exp. Therap. 219(1):141 (1981b) Tamagna, E . T . ; Lane, W. and Hershman, J .M. : Effect of chronic metoclopra-mide therapy on serum pituitary hormone concentrations. Hor. Res. Vl_:161 (1979). Tarsy, D . ; Parkes, J .D . and Marsden, C D . : Metoclopramide and pimozide in Parkinson's disease and levodopa-induced dyskinesias. J . Neurol. Neurosurg. Psych. 38:331 (1975). Teng, L . ; Bruce, R.B. and Dunning, L .K. : Metoclopramide metabolism and determination by high pressure l iquid chromatography. J . Pharm. Sc i . 66 (11): 1615 (1977). Thorburn, C.W. and Sowton, E. : The haemodynamic effects of metoclopramide, Postgraduate Medical Journal 49 (suppl. 4): 22 (1973). Tisdale, S.A. and Brinckerhoff, J . H . : Metoclopramide. The New Eng. , J . Med. 305(18):1093 (1981). Trafford, J . A . 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J . 49 (suppl 4):73 (1973). 155 Venho, V.M.K. : Drug absorption from in-s i tu rat small intestine during metoclopramide administration. J . Pharm. Sc i . 68 (4): 517 (1979). Venkateswaren, P.S. and Oho, A . G . : Acute dystonia due to metoclopramide. Br. Med. J . J_V: 178 (1972), Venkataramanan, R.: Ph.D. thesis, UBC, Vane , B . C . , Canada, p.64 (1978). Volans, G.N. : The effect of metoclopramide on the absorption of effervescent aspirin in migraine. B r i t . J . C l i n . Pharmacol. 2_: 57 (1975). Wagner, J . G . : Biopharmaceutics and Relevant pharmacokinetics. Hamilton, 111. Drug Intelligence Publications (1971). Wagner, J . G . ; Welling, P . G . ; Roth, S .B. ; Sakmar, E . ; L e i , K.P. and Walker, J . E . : Relative ava i lab i l i ty of propoxyphene. Int. J . C l i n . Pharmac. 5_:371 (1972). Walle, T. and Ehrsson, H. : Quantitative gas chromatographic determination of picogram quantities of amino and alcoholic compounds by electron capture detection. Part I. Preparation and properties of heptafluorobutyryl derivatives. Acta Pharm. Suecica, 7:389 (1970). Walle, T. and Ehrsson, H. : Quantitative gas chromatographic determination of picogram quantities of amino and alcoholic compounds by electron capture detection. Part II application to plasma and urine samples. Acta Pharm. Suecica, 8:27 (1971). Wandless, I . ; Evans, J . G . and Jackson, M. : Fever associated with metoclopramide-induced dystonia. The Lancet ]_:1255 (1980). Ward, H.W.C. : Metoclopramide and prolactin. Br. Med. J . 3_:169 (1974). Weeks, J .R. and Davis, J . D . : Chronic intravenous cannulas for rats. J . Appl. Physiol. ]_9: 540 (1964). Wilkinson, G. and Shand, D. : A Physiological approach to hepatic drug clearance. C l i n . Pharmac. Ther. ^8:4 (1975). Wing, L . M . H . ; Meffin, P . J . ; Grygiel , J . J . and Smith, K . J . The effect of metoclopramide and atropine on the absorption of oral ly administered mexiletine. Br. J . C l i n . Pharmacol. 9_:505 (1980). Zimmerman, S.W. and Norbach, D.H. : Nephrotoxic effects of long term carbontetrachloride administration in rats. Arch. Pathol. Lab. Med. J04:94 (1980). 156 7, APPENDIX 7.1. Indications In Canada, MCP fs approved for use in a l l the the available dosage forms (viz. injectables, tablets and syrups) as an adjunct in the management of delayed gastric emptying associated with subacute and chronic gastri t is and sequelae of surgical operations such as vagotomy and pyloroplasty (A.H. Robins, 1981). In such indications where there is delayed gastric emptying, MCP may relieve symptoms such as nausea, vomiting, bloating and epigastric distress. It is also used in fac i l i ta t ing small bowel intubation. Until now, the Food and Drug Administration (FDA) of the United States approved only the use of parenteral MCP for diagnostic intubation and radiographic examination of the proximal gut (Schulze-Delrieu, 1981). More recently (Federal Register December 30th, 1980) however, FDA has approved the use of MCP tablets for the re l i e f of symptoms associated with acute and chronic gastric motor fai lure due to diabetic gastroparesis (Ponte and Nappi, 1981; Tisdale et a l . , 1981). 7.2. Contraindications MCP should not be given to patients receiving monoamine oxidase inhibitors , t r i c y c l i c anti-depressants or sympathomimetic amines until the safety of these combinations has been established 157 (Pinder et a l . , 1976), Also, because of possible aggravation of extrapyramidal symptoms, MCP should not be used in conjunction with phenothiazines, butyrophenones or thioxanthines. The drug should not be given to patients with per i toni t i s , abdominal abscess, pheochromocytoma, gastrointestinal hemorrage or mechanical obstruction/perforation (Ponte and Nappi, 1981). The safety of MCP use during pregancy so far has not been established. Drugs with atropine-!ike actions antagonise the gastrointestinal effects of MCP, and they should not be administered concurrently (Pinder et a l . , 1976). Conversely, acetylcholine - l ike drugs may potentiate the effects of metoclopramide. 7.3. Precautions MCP should be used cautiously in patients with epilepsy, extra-pyramidal symptoms and in children (Ponte and Nappi, 1981) Intravenous injections should be given over a period of 1-2 minutes. Rapid administration has been associated with an intense transient feeling of anxiety and restlessness followed by drowsiness (Robins, 1981). The effect of MCP on stomach evacuation and intestinal act iv i ty results in faster absorption of certain drugs which are absorbed from the small intestine (Table 9). Metoclopramide on the other hand, may l imit absorption of slowly absorbable drugs 158 (Jamali and Axelson, 1977) and those absorbed from the stomach, for example digoxin (Manninen and Apajalahti , 1973). MCP reduces absorption of cimetidine (Grugler et a l . , 1981). However, the mechanism for reduced absorption of cimetidine by MCP is not clear. Drugs for which absorption is apparently not affected by concomittant administration of MCP are: Isoniazid (Savio and Pontiggia, 1965; Venho, 1979) and Quinidine (Venho, 1979); and Propranolol (Charles, et a l . , 1980). 159 TABLE 9 List of drugs where rate of gastro' intestinal absorption is increased in the presence of metoclopramide Tetracycline Acetaminophen Ethanol Levodopa Aspirin Griseoful vin Mexiletine Amitript;! ine, Diazepam Nimmo, 1973; Gothoni et a l . , 1973 Heading et a l . , 1973; Nimmo, 1973 Johnson, 1973; Gibbons and Lant, 1975; Bateman et a l . , 1978. Mearrick et a l . , 1974. Volan, 1975 Jamali and Axelson, 1977 Wing et a l . , 1980 Prescription Guide, 1981 Proprietary 160 7.4. Treatment of Overdosage and Toxic Effects Management of overdosage and toxic effects consists of emptying the gastric contents (v i z . , aspiration and lavage), close observation and supportive therapy (Pinder e t . a l . 1976). The extra-pyramidal reactions can be controlled by antiparkinson agents such as benzhexol (Kiloh, 1973; Extra Pharmacoepoeia, 1977) or other anticholinergic drugs, v i z , benztropine (Venkateswaren and Otto, 1972) 7.5. Dosage and Administration (A.H. Robins, Canada, 1981). Note: Total daily dosage must not exceed 0.5 Mg.Kg body weight (as per Health Protection Branch, (HPB, Ottawa). Adults: Tablets: h to 1 tablet (5 - 10 Mg) 3 or 4 times a day before meals and at bedtime. Syrup : 5 to 10 Ml(5-10 Mg) 3 or 4 times a day before meals and.at bedtime. Ampoules:When parenteral administration is required; one ampoule (10 Mg) IM or IV (slowly) 2 or 3 times a day, i f necessary Children: (5 to 14 years): Syrup : 2.5 to 5 Ml (.2.5-5 Mg) 3 times a day before meals. For small bowel intubation: Adults: One ampoule (10 Mg) injected slowly IV, preferably at the time when the t ip of the tube reaches the pyloric region. Children: Single dose of 0.1 Mg.Kg slowly IV. 161 7,6, Preparations (E,P, 77; B.P, 80.; C P . S , 81; P,P,G. 81) MCP Injection: A s ter i le solution of MCP, HC1 tn water for Injections. An Injection containing the equivalent of 10 Mg of anhydrous MCP.HC1 in 2 Ml is usually available. MCP Tablets: Tablets containing, in each, the equivalent of 10 Mg of anhydrous MCP.HC1 are usually available. MCP Syrup: Syrup containing 5 Mg. MCP.HC1 in each 5 Ml is also available. Proprietary Names: M a x o l o n ^ , Primperan, Metamide (All Austr. P.P.G. 81); Donopon-GP, Metoclol, Moriperan (all Jap. , D.P. 77); Maxeran (Canada E.P. 77); Paspertin (Ger., E.P. 77), Reglan (Canada and U . S . A . , C.P.S. 80); Reliveran, Cerucal, E u c i l , Plasi l (M.I. 76). 162 7.7. First-Pass Effect: Non linear concept comprising an expl ic i t solution of integrated Michael is-Menten equation. The mathematical description of f i r s t pass effect usually is based on f i r s t order compartment or clearance models. F irs t order models are only val id under the condition Cp << Km A model that is independent of the above condition wil l be deduced from the Michaelis-Menten equation SCHEME OF FIRST PASS EFFECT GUT LIVER SYSTEMIC CIRCULATION M JI Amount given as oral dose(D) = Amount in Gut(G)+Amount Metabolised + amount reaching circulation (B) Keller and Scholle (1981) 163 As absorption of a dose (D) from G,I.,T. ts, a f i r s t order process, . ' . K..S therefore, the amount in the gut (G) diminishes in an exponential fashion with a constant absorption rate (Ka): G = D. e"10*'* The (-) sign changes to a (+) sign since the decrease of the Gl amount (-dG/dt) Is the increase of the absorbed amount (+dG/dt) which is further considered: dG _ i, n a -Ka.t dt " K a - D - e -At every time interval in the circulation of portal blood [T = 1 Min . ] , a fractional amount (M) is absorbed and transported into the l i ver . The actual',amount i s : Metabolised by l iver enzymes, and this metabolism follows Michaelis-Menten kinetics. The Michaelis-Menten constants (Vm and Km) will be decribed here in mass rate terms: dM = Vm . M  d t Km + M 1 6 4 The d i f f e r e n c e between the absorbed a,n.d the njetabol i.sed amount i s the r a t e at which the drug enters the systemic c i r c u l a t i o n (dB/dt ) ; d B . = dG dM_ dt dt " dt The In tegra ted r a t e r e v e a l s the s y s t e m l c a l l y a v a i l a b l e amount (B) rt B = dB dt . dt The r e l a t i o n between the s y s t e m l c a l l y a v a i l a b l e (B) and the a p p l i e d amount (D) expresses the b i o a v a i l a b i l i t y f a c t o r ( F ) : rt p _ ' B_ o r D " dB dt dt D A s , dB = dG_ dM dt dt ~ dt o r , dB _ „ a n - K a . t Vm.M -nr = Ka.D.e -a z Km+M As, H = | ! dB _ i/_ n « - K a . t _ ^m ' 4x- . T , J X - = Ka.D.e - dt dt K m + f . T dG n - K a « t A s , • = Ka.D.e dG by s u b s t i t u t i n g the value of i n the above equat ion 165 d B v pi - K a . t Vrn.Ka,D.T,e dt = K a - u - e -Km + Ka .D .T .e •Ka.t - K a . t I n t e g r a t i n g between time =• t-| and r h dt d t " r t , K a . D . e " K a > t . d t VrTi. Ka .D .T .e ' •Ka.t Km + Ka .D .T .e ' •Ka.t dt Now, f r2 K a . D . e " K a , t . d t - D . e - K a . t • D . e " K a t 2 - ( - D . e - K a t l ) D.e - K a t i _ D e _ - K a . t 2 166 And. r t 2 Vifi . K a . D . T . e " 1 ^ ' 1 dt Km + Ka.D.T.e -Ka.t Now, as differential of denominator, i . e . d {Km + Ka.D.T.e" 1^-*} _ n v 2 _ -Ka.t 3t D.Ka .T.e the above expression can be written in the following form, dA A LnA + C Integration constant which drops out by cancellation when limits/boundary area is defined Therefore, , t 2 Vm.Ka.D.T.e -Ka.t t^  Km + Ka.D.T.e" 1 * 3 -* ^ W . T . e " ^ -Vm Ka t Km + Ka.D.T.e' -Ka.t 1 Vm Ka Ln (Km + Ka .D;T. -e"K a , t ) 167 -Vm Ka Ln [Km + K a . D . T . e " ^ ' ^ ) - LnCKm + K a . D . T . e ~K a t l ) Taking the (-) sign in and as LNa - LNb = Ln | -.,the above expression is equal to: Vm Ka Ln Km + Ka.D.T.e •Ka.t] Km + Ka.D.T.e - K a . t 2 V S In Ka" L n Km . e ^ 1 + Ka.D.T.e-^'*! . e 1^* 1 1 e K a t 2 Katp -Ka.tp Kat^ Km .e + Ka.D.T.e \ e e K a-V Vm Ka Ln Kat, Km . e + Ka.D.T. . _ J Kat 2 L n -Km . e + Ka.D.T. e Kat, Ka.t, •+• Ln.e Vm_ Ka Kat, DKaT + Km . e Kat 2-Kat-| + Ln Katr DKaT + Km .,e r t 2 rt. dB dt dt = r t . K a . D . e . " ^ - * .dt Vm.Ka.D.T.e. •Ka.t . d t Km + Ka.D.T.e -Ka.t 168 4 -Ka.t, •Kat, B = D.e D.E Vm Ka Ka.t-, Ka , t 2 - Kat1 + Ln DKaT + Km.e Ka.t, DKaT + Km.e -Ka.t 1 •Ka.t, - e Vm D Ka r t n —r . . , J I DKaT+Km.e z2~z] • Ka L n Kat, DKaT+Kme ' Unfortunately, this integral shows no convergency, therefore the i n i t i a l (t-j) and final (t,,) conditions must be defined, t 1 = 0 , t 2 = 10 Ln2/Ka [Ln2 = 0.693] The final condition ( t 2 ) is given by the time [ten half- l ives of absorption = 10Ln2/Ka], where > 99% of the applied amount [D] has already been absorbed from the gut. Therefore, the f i r s t pass effect (F) or the bioavai labi l i ty of a metabolised drug can be described as a nonlinear process: „-Ka.lO Ln(2)/Ka = 0.001 F = 1 - Vm_ DK 10 Ln2 + Ln D.Ka.T + Km D.Ka.T + Km.e 10Ln(2) 

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