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The pharmacokinetics of metoclopramide in normal rats and in rats with experimental renal and hepatic… Tam, Yun Kau 1980

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THE PHARMACOKINETICS OF METOCLOPRAMIDE IN NORMAL RATS AND IN RATS WITH EXPERIMENTAL RENAL AND HEPATIC DYSFUNCTION by YUN KAU/VAM B.S'. University of Minnesota, 1975 M . S c , University of Br i t ish Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Division of Pharmaceutics and Biopharmaceutics) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1980 © YUN KAU TAM, 1980 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the 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 ag ree that the L i b r a r y s h a l l make 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 and s t u d y . I f u r t h e r agree 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 may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha 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 not be 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 . Department o f iPX&r-J3ia/'/>,*/-/^d ^CUJZA^^> The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date J t & c . f f f a ABSTRACT Metoelopramide (MCP), 4 amino-5-chloro-2-methoxy-N(2-diethyl -aminoethyl) benzamide, a procainamide derivative, i s a potent a n t i -emetic and gastric m o t i l i t y modifier. C l i n i c a l l y , MCP i s used in gastrointestinal diagnostic examinations, treatment of various types of gastrointestinal disorders and as a pre- and post-opera-t i v e anti-emetic. Very l i t t l e pharmacokinetic information was available prior to this study. This thesis reports the pharmacokinetic study of MCP i n normal rats and in rats with renal and hepatic dysfunction. The Pharmacokinetics of MCP in Normal Rats The pharmacokinetics of MCP have been examined as a function of dose and route of administration. No si g n i f i c a n t first-pass metabolism was seen in urine and plasma studies (F = 0.9). The area under the plasma concentration vs^. time curves (AUC) increased disproportionately while average plasma clearances ( C l j B ) reduced with increasing dose indicating dose-dependent kinetics for MCP. However, the percentage of dose excreted as intact drug and de-ethylated metabolite (DE-MCP) was constant after administration of a range of intravenous doses (35 fold) indicating dose independ-ent k i n e t i c s . The plasma and urine results could not be explained readily by conventional Michaelis-Menten k i n e t i c s . The elimination of MCP was proposed to be blood flow dependent . After a high dose, of MCP (35 mg/kg), the drug may transiently i reduce pe r fus ion to the e l i m i n a t i n g ' o r g a n s ( v i z . , l i v e r , k i dney , e t c . ) * This hypothes is was supported by an i n i t i a l reduc-t i o n and then a subsequent re tu rn to con t ro l l e v e l s of the c l e a r -ance of the blood f low i n d i c a t o r , indocyanine g reen , a f t e r MCP a d m i n i s t r a t i o n . P a r a l l e l to t h i s o b s e r v a t i o n , i t was noted that the plasma h a l f - l i f e o f MCP was i n t i a l l y prolonged (104 ± 14 minutes) and fo l lowed by a resumption of the normal e l i m i n a t i o n h a l f - l i f e (58 ± 7 m inu tes ) . A ques t ion remaining unresolved i s why MCP does not undergo f i r s t - p a s s metabol ism over the dose range s t u d i e d . K i n e t i c s o f MCP i n Hepat ic Impaired Rats The hepat ic impairment caused by carbon t e t r a c h l o r i d e ( C C I 4 ) i nc reased the h a l f - l i f e and AUCs of MCP by approx imate ly 3 f o l d [ t - | /2 : con t ro l = 52 ± 15_min. , t e s t = 170 ±.40 m i n : ; AUC: con t ro l = 290 ± 50 mcg-min/ml , t e s t = 840 ± 200 mcg-min/ml] wh i le the C l j g was d im in ished to a s i m i l a r extent [ con t ro l = 13 ± 1 m l / m i n ; t e s t = 4 .6 ± 1.0 m l /m in ] . The volume of d i s t r i b u t i o n (V^) d id not change s i g n i f i c a n t l y . The renal c lea rance (ClpJ o f MCP was reduced s l i g h t l y probably due to renal damage caused by CC14 [ con t ro l = 2 . 9 m l / m i n ; t e s t = 2 . 3 m l / m i n ] . The reduc t ion of hepa t i c f u n c t i o n caused a s i g n i f i c a n t i nc rease in the percentage o f dose excre ted as i n t a c t drug in u r ine [ con t ro l = 20 ± 2%; t e s t = 47 ± 7%]. However, the DE-MCP f r a c t i o n was una l te red [ con t ro l = 11 ± 3%; t e s t = 9.3 ± 3%]. These r e s u l t s i n d i c a t e that the l i v e r i s a major metabo l i c organ f o r MCP removal but e x t r a -i i hepatic metabolism may also occur. Kinetics of MCP in Renal Impaired Rats The effect of renal dysfunction on the kinetics of MCP has been studied using two surgically [bi lateral ureteral l igat ion (BUL) and two-step 5/6 nephrectomy (TSN)] and a chemically [uranyl nitrate (UN)] induced method providing a Wide range of renal impairment [creatinine clearance: control = 1.7-2.0 ml/ min; test = 0.03- 0.38 ml/min]. Renal damage was the highest in the UN group followed by the BUL and TSN groups. The t-j/2 ° f M C P w a s signif icant ly increased [control = 50-70 min, test = 130-160 min] and Cljg [control = 11-16 ml/min, test = 3.4-7 ml/min] and Clp- [control = 2.0-2.9 ml/min, test = 0.2-0.7 ml/min] decreased while the V d [control = 4.0-4.5 L/kg, test = 3.2-4.4 L/kg] is only s l ight ly decreased in al l models studied. A positive correla-tion was observed between the C l jg , ClR and nonrenal clear-ance with creatinine clearance (r^ >^ 0.97) indicating that the metabolic and renal clearances of MCP are related to renal func-t ion . The findings obtained from the hepatic and renal impairment models indicate that, besides the l i v e r , the kidneys may also play an important role in metabolizing MCP. This may, in part, explain why MCP does not undergo f i rst -pass metabolism in the rat. i i i TABLE OF CONTENTS ABSTRACT LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS LIST OF SCHEMES ACKNOWLEDGEMENTS INTRODUCTION Pharmacodynamic S tud ies G a s t r o i n t e s t i n a l E f f e c t s Mechanism of Ac t i on . C l i n i c a l T r i a l s Symptomatic R e l i e f Radio logy G a s t r o i n t e s t i n a l In tuba t ion Upper G a s t r o i n t e s t i n a l Endoscopy G a s t r o i n t e s t i n a l Symptoms a f t e r Vagotomy In A c i d - p e p s i n Disease G a s t r i c U l c e r Duodenal U l c e r Ref lux Esophag i t i s In Anes thes ia In G a s t r o i n t e s t i n a l D iso rders In Migra ine Side E f f e c t s In f luence on Drug Absorp t ion A n a l g e s i c s D igox in A n t i b i o t i c s Levodopa Ethanol I s o n i a z i d i v Page Drug Disposition and Hepatic Blood Flow 12 Effect of Hepatic Blood Flow on Clearance 13 Indocyanine Green - A-Blood Flow Rate Indicator 1 4 Effects of Enzyme Activi ty on Drug Clearance 15 Clearance and Half Li fe 16 Influence of Route of Administration 16 Renal Failure and Drug Accumulation 18 Pharmacokinetics of MCP 21 Metabolism 25 EXPERIMENTAL 28 Electron-capture gas l iquid chromatographic assay (EC-GLC) for simultaneous determination of MCP and i ts major metabolite De-Ethyl-MCP(DE-MCP) in rat urine. 29 Materials 29 GLC 29 Extraction and Derivative Formation 29 Mass Spectrometry 30 Quantitative Studies 31 Animal Handling 31 Pharmacokinetic Study of MCP in Normal Rats 31 i .v . Administration 31 Oral Administration 32 Urinary Excretion Study 33 Plasma Indocyanine Green Studies 33 Pharmacokinetics of MCP in CCl^ Treated Rats 33 Plasma Level Study 33 Urine Study 35 Biochemical Study 35 Pharmacokinetics of MCP in Chemical and Surgically Induced Renal Dysfunction 36 Surgical Methods 36 Two step 5/6 Nephrectomy 36 Bilateral Ureteral Ligation 36 v: Page Chemical Induction 37 Uranyl Nitrate . 37 Plasma Level and Urine Study 37 Assessment of Renal Function 37 Pharmacokinetic and Stat ist ica l Analyses 38 RESULTS 41 GLC 41 Mass Spectrometry 41 Electron Impact 41 Chemical Ionization 46 Recovery 46 Dose Dependent Kinetics of MCP 46 Urinary Excretion Data 50 ICG Clearance 56 Carbon Tetrachloride Treatment 58 Biochemistry 58 Plasma Study 58 Urine Study 62 Renal Failure 62 Body Weight 62 Biochemistry 62 Plasma and Urine Study 67 DISCUSSION 80 GLC 81 Structural Confirmation of Derivatives 81 81 84 Electron Impact ' Chemical Ionization Removal of Excess of Derivatizing Agent 84 Appl icabi l i ty of the Assay Method 85 The Pharmacokinetics of MCP in Normal Rats 85 Pharmacokinetic Studies in Hepatic and Renal Impaired Rats 92 Carbon Tetrachloride 92 Renal Failure 94 Conclusions 101 REFERENCES "102 vi LIST OF TABLES V Change of body weight with time after induced renal injury. Page I Kinetic parameters obtained from the intravenous data 48 II % of MCP recovered after 48 hr cumulative urine analysis 54 III % of De-ethylated metabolite equivalent to MCP recovered after 48 hr cumulative urine col lect ion. 55 IV Biochemical information after carbon tetrachloride treatment. 59 64 VI The plasma urea nitrogen levels after induction of renal injury. 65 VII The plasma creatinine levels after induction of renal injury 66 VIII The plasma glutamic oxalo-acetic transaminase levels after induction of renal impairment. 68 IX The pharmacokinetic parameters obtained after bi lateral ureteral l igation treatment. 71 X The pharmacokinetic parameters obtained after two-step 5/6 nephrectomy treatment. 72 XI The pharmacokinetic parameters obtained after uranyl nitrate treatment. 73 v i i LIST OF FIGURES : Page 1. The relationship between l iver blood flow and hepatic clearance for drugs with varying extraction ratios (ER). The arrows indicate the range over which l iver blood flow can vary and extraction ratios refer to a normal flow of 1.5 L/min. 15a 2. The relat ionship, according to equation 3, between the in t r ins ic clearance, hepatic extraction and actual hepatic clearance assuming a l iver blood flow of 1.5 L/min. The inset indicates on an expanded scale the relationship at low values of C l in t . 15a 3. Diagram to show the device employed in collecting urine samples from rats without fecal contamination. 34 4. A representative chromatogram obtained from the urine extract. Peaks with retention times 3.04 and 3.59 minutes are the HFB derivative of MCP and DE-MCP respectively. The peak at 7.45 minute is the internal standard, diazepam. 42 5. Calibrations curves of the urine extracts: a. MCP b. DE-MCP. 43 6. Mass spectra of the HFB-derivative of MCP a. electron impact b. chemical ionization 44 7. Mass spectra of the HFB-derivative of DE-MCP a. electron impact b. chemical ionization. 45 8. Representative MCP log plasma concentration vs. time curves after a range (5-25 mg/kg) of i .v . doses. 47 9. A representative log plasma concentration ys^ time curve after a 35 mg/kg dose of MCP ( i - v . ) . The sampling period was extended to 720 minutes. INSERT: The plasma t 1/2 of the intermediate kinetic phase was evaluated using a truncated and more intensive sampling protocol. 49 10. A plot of AUC vs^ . dose to i l lustrate the non-linear nature of MCP kinet ics. 51 vi i i Page 11.. Representative plasma concentration vs. time plots obtained after i .v . ( A ) and oral ( A j administration of MCP (15 mg/kg). 52 12. The AUC comparison between i .v . and oral administration of a 15 mg/kg dose of MCP.(The bars represent the mean ± standard deviation). 53 13. Comparison of ICG clearance 30 minutes and 7 hours after sal ine, 5 mg/kg and 35 mg/kg MCP. (The bars represent the mean± standard deviation). 57 14. Representative plasma MCP concentration vs.time curve after carbon tetrachloride pretreatment ( 0 » control and • , CC14) 60 15. Kinetic parameters obtained for MCP after CCl^ pretreatment (c, control and t , test).(The bars represent the mean± standard deviation). 61 16. Percent of dose recovered as a) intact drug and b) de-ethylated metabolite after CC1* pretreatment (c, control and t , test) . The bars include plus and minus one standard deviation. 63 17. Representative log plasma MCP concentration ys_. time curves obtained after a 15 mg/kg dose of MCP was given to the bi lateral ureteral ligated (BUL), 5/6 two step nephrectomized (TSN) and uranyl nitrate (UN) treated rats ( , control and test ) . 69 18. Area under the plasma versus time curves after BUL, TSN and UN treatments, (c, control and t , test ) . The bars include plus and minus one standard deviation. 74 19. Total body clearance after BUL, TSN and.UN (c, control and t , test) . The bars include plus and minus one standard deviation. 75 20. Half l i f e obtained after BUL, TSN and UN (c, control and t , test ) . The bars include plus and minus one standard deviation. 76 21. Volume of distribution calculated by the area method for BUL, TSN and UN treated animals (c, control and t , test) . The bars include plus and minus one standard deviation. 77 ix Paoe 22. Percent of dose excreted as a) intact drug and b) de-ethylated metabolite after UN pretreatment (c, control and t , test) . The bars include plus and minus one standard deviation. 78 23. Percent of dose excreted as a) intact drug and b) de-ethylated metabolite after TSN pretreatment (c, control and t , test) . The bars include plus and minus one standard deviation. 79 24. Blood/plasma ratios after 15 and 35 mg/kg MCP.(The bars represent the mean± standard deviation). 88 25. A plot of plasma creatinine vs_. the total body clearance of MCP ( • = UN, Y = TSN and 0 = BUL) ( r 2 = 0.76). 98 26. A plot of PUN levels vs_. the total body clearance of MCP (•= UN, •= TSN and 0 = BUL) (r-2=0.71). 99 27. A plot of the average total body ( • , r 2=0.99), non-renal ( B , r 2 = .98) and renal ( T ,r2=.97) clearance of MCP vs^ . the average creatinine clearance. 100 x ABBREVIATIONS ADP adenosine diphosphate ATP adenosine triphosphate BUL bilateral ureteral l igation (or ligated) CCl^ carbon tetrachloride CI chemical ionization Cl , hepatic clearance C l ' 1 - n t in t r ins ic clearance of free drug Cl^ n^. in t r ins ic clearance of total drug Cl total body clearance TB C l ^ non-renal clearance C l r e n a l clearance CNS central nervous system CTZ chemoreceptor trigger zone DE-MCP de-ethylated metoclopramide E extraction ratio E^ hepatic extraction ratio E^ , renal extraction ratio ECD electron capture detector EI electron impact GFR Glomerular f i l t r a t ion rate GLC gas l iquid chromatography HFB heptafluorobutyryl HFBA heptfluorobutyryl anhydride xi HPLC high performance l iquid chromatography HVA homovanillic acid i .d . internal diameter i.m. intramuscular i .v . intravenous MCP metoclopramide MCP-HCl-HgO metoclopramide monohydrochloride monohydrate MS mass spectrometry (or spectrometer) PUN plasma urea nitrogen kidney blood flow PGOT plasma glutamic oxalo-acetic transaminase t^2 h a l f - l i f e TSN two step 5/6 nephrectomy (or nephrectomized) UN uranyl nitrate ^d(area) volume of distr ibution determined by area method v"d(ss) volume of distribution determined by steady-state method. ± one standard deviation xi i LIST OF SCHEMES Page I. A schematic of metabolites of metoclopramide recovered from rabbit urine. 26 II. The postulated fragmentation pattern of the MCP HFB-derivative. 82 III. The postulated fragmentation pattern of the DE-MCP HFB-derivative. 83 x i i i To Theresa - the dearest person in my l i f e . The thesis is dedicated to her because she has l ived through a l l • the hardship with me. xi v ACKNOWLEDGEMENT The author would l ike to thank Dr. Axel son for providing such a friendly atmosphere during my study. The friendship is going to be an everlasting one. The author is indebted to Dr. W. Godolphin who has assisted us in the biochemical analysis. The author would l ike to express his sincere thanks to Mrs. B. McErlane who has assisted me in surgery, blood sampling and drug analysis. Dr. Price's constructive cr i t ic ism in renal aspect of this project is deeply appreciated. The author also appreciate Mr. R. Kapil and K.W. Rigg's help. This project has been made possible by the support of Medical Research Council and the Kidney Foundation. xv INTRODUCTION Metoclopramide (MCP), 4-amino-5-chloro-2 methoxy-N-(2-diethyl aminoethyl benzamide )(pKa=9.3),was introduced in 1964 (Justin-Besancon and Laville). The pharmacological properties of this new drug were f irst evaluated in the early sixties. Unlike its analog, procainamide, MCP has no significant cardiac effects. 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 Laville, 1964; Magrieson et a l . , 1966; Martin and Scobie, 1967; Eisner, 1968, 1971; James and Humes, 1969; Ramsbottom and Hunt, 1970; Howells et a l . , 1971; Johnson, 1971a, 1973; Kreel, 1973, and Hancock et a l . , 1974). / \ J / C ' h > H,N— ( / v — C — N H — C H 2 — C H 2 — N X C 2 H 5 >CH3 Metoclopramide Pharmacodynamic Studies Gastro-intestinal Effects - Extensive studies in man (Justin-Besancon and Laville, 1964; Magrieson et a l . , 1966; Martin and Scobie, 1967; Eisner, 1968; James and Humes, 1969; Ramsbottom and Hunt, 1970; Howell et a l . , 1971; Johnson, 1971a, 1973; Kreel, 1973; and Hancock et a l . , 1974),dog (Tinker and Cox, 1969,and Jacoby and Brodie, 1967), 2 guinea pig and rat (Marmo et a l . , 1970 and Hay, 1975) have established the effect of MCP on the gastrointestinal t ract . MCP increases the lower esophageal sphincter pressure and the force of per is ta l ic contractions (Heitmann and Mi l ler , 1970; Bremner and Bremner, 1972; and Guelrud, 1974)without any effect on relaxation in man. MCP increases the per is ta l ic strength of the gastric antrum, relaxes the pyloric canal and duodenal cap and increases the synchronization of the gastric antral and duodenal moti l i ty (Johnson, 1973) in humans. Therefore, the gastric emptying time is reduced (James and Humes, 1969). Effects resembling those in the esophagus and stomach after MCP have been observed in the small intestine (Johnson, 1971a, 1973). The transit time through the duodenum and jejunum is reduced in man as a result of the drug induced increased motil i ty (James and Humes, 1969 and Magrieson et al., 1966). No marked effect on colonic motor act iv i ty in vivo is observed ( Eisner, 1971; Banke et a l . , .1972). Mechanism of Action'' - MCP has been postulated to have both peripheral and CNS actions. It has been shown that MCP inhibits emesis in animals to local ly acting emetics such as copper sulfate ( R ) and to centrally acting drugs such as Hydergine and apomorphine (Justin-Besancon and Lav i l l e , 1964). However, the actual pathways are yet uncertain. Esophageal and gastric contractions induced by MCP are 3 blocked by anticholinergic drugs such as atropine and potentiated by cholinergic drugs such as carbachol and methacholine (Jacoby and Brodie, 1967 and Johnson, 1971b). MCP has no anticholinesterase act iv i t ies (Eisner, 1968) and i ts actions are unaffected by ganglion blocking agents such as chlorisondamine. Eisner (1968) has shown that MCP has no action on the isolated human smooth muscles from the body or the antrum of the stomach, however, MCP sensitizes the preparations to acetylcholine. It is postulated that MCP acts via the intramural cholinergic neurons responsible for modifying gastric moti l i ty but not gastric secretion (Eisner, 1968). A recent study showed that MCP part ia l ly and signi f icant ly 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 per ista ls is of guinea-pig ileum was decreased, the effect of adrenaline was potentiated and the effect of theophylline ethylenediamine was not affected by MCP. This specif ic action of MCP was postulated to be due to i ts sensitive blockade, at the post-synaptic s i tes , of the effect of the inhibitory purinergic transmitter (ATP and related nucleotides) released during per is ta ls is (Okwuasabaand Hamilton, 1975). Such action of MCP in antagonizing the action of the in t r ins ic inhibitory 4 mechanism may well be complementary to the documented muscarinic sensit izing action reported by others (Jacoby and Brodie, 1967; Birt ley and Baines, 1973 and Fontaine and Reuse, 1973). In man, the gastric emptying time prolonged by L-dopa was reduced by MCP action on the gastro-intestinal t rac t . This might be partly due to the ant i -dopaminergic effect of MCP (Berkowitz and McCallum, 1980). MCP is reported to raise the threshold of act iv i ty in the chemoreceptor trigger zone (CTZ) and decreases the sensi t iv i ty of visceral nerve which transmits afferent impulses from the gastrointestinal tract to the emetic center in the lateral ret icular formation. Thus, MCP prevents vomiting induced by central emetics (Justin-Besancon and Lav i l l e , 1964 and Klein et a l . , 1968). Cannon (1975) had indicated that drugs which stimulate the CTZ are dopamine-l i ke . The action of MCP in the CTZ is believed to be due to i ts dopamine antagonistic effect . More evidence has been accumulated recently to support this hypothesis. Peringer et a l . , (1975) have observed that after injecting MCP intraperitoneally, MCP has no effect on the dopamine concentrations on the whole brain, but i t increases the homovanillic acid (HVA) (a dopamine metabolite) concentration both in the corpus striatum and in the mesolimbic area. It has been postulated that MCP blocks dopamine receptors (Peringer et a l . , 1975 and Blower, 1975) and as a result MCP causes an increase in f i r ing of the dopaminergic neurons. Hence, the turnover of dopamine is increased (E l l io t t et a l . , 1977). MCP has been shown to stimulate prolactin secretion both in animals (Carlson et a l . ,1977 5 and Jiro et a l . ,1977) as well as in humans (Del i tala et a l . ,1976; Ogihara et a l . , 1977; Sowers et al.,1977; and Huizing et a l . , 1979). This effect is abolished by CB154, a dopaminergic stimulant(Jiro et a l . , 1977). Behavioral studies (Dolphin et a l . , 1975) indicate that MCP resembles pimozide in antagonizing the effect of apomorphine or amphetamine in producing turning behavior in mice with unilateral lesions of the nigrostriatal pathway. It also inhibits the apomorphine induced stereotopy and the reversal of reserpine-induced suppression of locomotor act iv i ty (Jenner et a l . , 1975). It appears that MCP is a relat ively potent antagonist of str iata l dopamine receptors. The spec i f ic i ty of MCP as a dopamine receptor antagonist has also been shown on the peripheral system (Goldberg, et a l . , 1978 and Brodde and Schemuth, 1978). This was indicated by a sh i f t of the dose-response curve for dopamine induced relaxation s igni f icant ly to the right in a concentration dependent manner (Brodde and Schemuth, 1979) when the isolated rabbit mesenteric arteries were studied with MCP. Cl in ical T r i a l s . As a result of the wide c l in ica l implications of i ts properties, MCP has been tested in various conditions of upper gastro-intestinal distress and disease since the 1960's. Symptomatic Relief - MCP is very effective in rel ieving postoperative nausea and vomiting (Trafford, 1967 and Handley, 1967). In a c l in ica l study of 1,500 patients, MCP has been shown to be as effective as phenothiazine but with fewer side effects (Robinson, 1973a) in the treatment of nausea and vomiting. However, pre-operative nausea and vomiting are not s igni f icant ly 6 relieved by MCP (Dundee and Clark, 1973). This is probably due to i ts CNS as well as peripheral ef fect . Radiology - Barium meal examination is usually a time consuming procedure, especially for those patients who have pyloric obstruction which makes the visualization of the duodenum more d i f f i c u l t . MCP, with i ts properties in accelerating gastric emptying by increasing per is ta l t ic act iv i t ies and relaxing the pyloric canal, reduces the radiological examination time signi f icant ly (Howarth et a l . , 1969; James and Melrose, 1969 and Kreel, 1970). Furthermore, this agent is part icularly useful in small-bowel examination (James and Humes, 1969). Gastrointestinal Intubation - MCP when given i,m. (Pirola , 1967) or i .v . (Bolin, 1969) shortens the time required to introduce a biopsy capsule or aspirating catheter through the pylorus into proximal jejunum. This is due to the effect of MCP to relax the pyloric sphincter. Upper Gastrointestinal Endoscopy - Emergency endoscopy for upper gastrointestinal hemorrhage is benefited by giving MCP i .v . This stimulates the passage of blood into the small bowel, cleaning the f i e ld for improved inspection (Bader, 1973), by the action of increasing the strength of per ista ls is of the gastrointestinal t ract . 7 Gastrointestinal symptoms after vagotomy - Post-vagotomy symptoms l ike postprandial vomiting, belching, epigastric d is t ress , and diarrhea are alleviated by MCP in patients up to 2 to 3 years after vagotomy (Stadaas and Aune, 1972) simply due to the acceleration of the gastric emptying after MCP. In Acid-Pepsin Disease -Gastric Ulcer - Impaired gastric emptying time has been implicated in the pathogenesis of benign gastric ulceration, therefore, an agent which enhances gastric emptying may be benef ic ia l . A c l in ica l study (Hoskin, 1973) showed that MCP is almost equivalent to carbenoxolone in treating a s ingle, chronic, lesser-curve gastric ulcer with fewer side effects. However, the results of controlled t r i a ls are not conclusive (Hoskin, 1973). Further well designed studies are awaited to prove the eff icacy of MCP in gastric ulcer treatment. Duodenal Ulcer - MCP is found to be highly effective in preventing relapse of duodenal ulceration, but no beneficial effects are shown with patients who have acute exacerbation of duodenal ulcer (Moshal, 1973). MCP is postulated to exert i ts effect by reducing gastric emptying time. Thus, i t reduces the acid content next to the site of hemorrhage. 8 Reflux Esophagitis - Despite the action of MCP on the lower esophagus (Heitman and Mi l le r , 1970), i t does not improve the condition of patients with severe symptomatic reflux esophagitis (Venables et a l . , 1973). In Anesthesia - MCP signi f icant ly increases gastric emptying in pregnant women during labor thereby reducing the incidence of vomiting and aspiration during emergency anesthesia, resulting in a reduction in the death rate caused by Mendel son's syndrome (McGarry,1971; Howard, 1973 and Howard and Sharp, 1973). Aspiration of stomach contents into the bronchial tree is a major cause of mortality and morbidity in emergency anesthesia. MCP is an effective agent to accelerate gastric emptying in a short time (Davies and Howells, 1973 and Dundee et a l . , 1974). Thus, the incidence of mortality caused by pulmonary aspiration of stomach contents has been reduced. In Gastrointestinal Disorders - MCP has been found to be; effective in di lat ing the pylorus of those patients with pyloric stenosis. This enables the patients to avoid emergency operations and a better radiological evaluation and c l in ica l preparation is obtained before surgery. MCP is s t i l l not the ultimate treatment of pyloric stenosis (Zer and D.intsman, 1975). In Migraine - Some beneficial effects on migraine after MCP treatment are observed (Matts, 1974). However, the mechanism of action of MCP in migraine is not known. It has been suggested that 9 MCP enhances the absorption of analgesics (Nimmo, 1973) with i ts potent anti-emetic properties (Trafford, 1967). MCP reduces the incidence of nausea and vomiting caused by the analgesics used in migraine (Matts, 1974). Side Effects - In a l i terature survey (Robinson, 1973b), a total of 1,023 patients showed an incidence of side effects in 11% of the cases. The most common adverse effects are drowsiness and lassitude (4%), bowel disturbances (1.2%), other untoward effects (4.3%) and dizziness or faintness (0.8%). Also facial dyskinesia (Melmed and Bank, 1975) and tetanus-like motor disorder resembling phenothiazine induced "pseudo-tetanus" (Cochlin, 1974) have been documented. Recently, i t has been reported that hyperprolactinemia was observed in patients treated with MCP (Aono et a l . , 1978).This 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). Also, hyperprolactinemia has been associated with hypogona-dism in man (Thorner et a l . , 1974). 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 (Falaschi et a l . , 1978). Influence on Drug Absorption - Most drugs are absorbed from the gastro-intestinal tract as the unionized form by passive di f fusion. The formation of the unionized species is pH dependent. It was 10 previously assumed that acidic drugs wil l be absorbed faster in the stomach where the pH in the environment is low (Shore et a l : , ' . 1957). However, Nimmo et al., (1973)showed that the absorption of the low molecular weight and easily di f fusible compounds such as ethanol and weakly acidic drugs such as asp i r in , warfarin and pentobarbital i s , in fact,slower from the stomach. The site where maximal absorption occurs is the upper small intestine due to the high relative surface area of the small intestine as compared to the stomach. With this in mind, i t can be postulated that the time required for a drug to be delivered into the small intestine may affect the rate and/or extent of i ts absorption. It has been shown by Heading et al.,(1973) that the absorption of paracetamol was dependent on the rate of gastric emptying. MCP, an agent which modifies gastric moti l i ty , may influence the absorption of those drugs which are administered concomitantly. Spec i f ica l ly , MCP may affect the peak plasma level attained and the area under the curve of the concentration vs- time curve. ' Analgesics - The effects of MCP for migraine headache are s t i l l uncertain. However, i t has been observed that the absorption of aspirin is delayed during a migraine attack (Volans, 1975). MCP, when administered i.m. immediately before the ingestion of aspi r in , increases the rate of absorption of the latter (Volan, 1975). Therefore, i t is postulated that MCP decreases the gastric emptying time during a migraine attack. This fac i l i ta tes the absorption of the analgesic. Similar observations have been obtained 11 by Heading et a l . , (1973) in the study of paracetamol absorption in man. , Digoxin - MCP decreased the steady-state blood level of digoxin (from 0.72 to 0.46 ng/ml) when administered in multiple doses (tablets) to man. Although there is no direct evidence showing that this is a result of increased gastric moti l i ty , the digoxin blood level is raised by propantheline which has the opposite effect of MCP (Manninen and Apajalahti, 1973). Antibiotics - It has been observed by (Jamali and Axelson, 1977) that MCP enhances the exent of absorption of griseofulvin given in solution while depressing the extent of absorption of griseofulvin given as a suspension dosage form. MCP accelerates the rate of absorption of tetracycline and pivampici l l in in normal subjects as well as convalescent patients (Gothoni et al.,1972). The time to achieve maximum plasma level was signi f icant ly reduced. It was suggested that i t was due to the effects of MCP on the gastrointestinal tract, tor stimulate gastric emptying arid intestinal moti l i ty. Levodopa - The absorption of L-dopa is variable (Mearrick et al..,1974). MCP effectively increases the gastric motil ity resulting in an increase in the rate and extent (3-fold increase) of levodopa absorption when tested in Parkinsonian patients (Mearrick et al.,, 1974) Ethanol - MCP given oral ly or i . v . before the ingestion of a standard dose of ethanol increases the rate of absorption of ethanol (Johnson 1973; Gibbons and Lant, 1975 and Bateman et a l . , 1978). 12 in man. This is due to the effect of MCP on the rate of gastric emptying. Isoniazid - MCP has no apparent effect on the absorption of isoniazid in tuberculosis patients (Savio and Pontiggia, 1965). Drug Disposition and Hepatic Blood Flow A perfusion limited model was f i r s t proposed by Rowland (1973) to describe the effect of changes of physiological factors such as blood flow, enzyme act iv i t ies and protein binding on the hepatic disposition of drugs. This model was later further elaborated by Wilkinson and Shand (1975) and Nies et a l . , (1976). The basic assumptions of this model are: 1) the drug is removed solely by the l iver 2) the removal processes follow f i r s t order kinetics and 3) the hepatic venous drug concentration is in equilibrium with the drug in the l i ver . The hepatic clearance is described by the following equations (1 and 2) C1H = Q.E. (1) C1H =Q. f cv V i n t (2) S + f B C 1 i n t | where C l H is the hepatic clearance, E, the extraction ra t io , Q, the hepatic blood flow rate, f g , the free fraction of drug in the blood and C l ! j n t , the in t r ins ic clearance of the free drug by the enzyme. 13 With an appropriate analysis, one can predict changes in drug disposition induced in three fundamental variables. Effect of Hepatic Blood Flow on Clearance. For this discussion, protein binding is assumed to be constant. The effect of protein binding changes in drug disposition wil l be discussed later , If drug binding to blood is constant, fg can be incorporated into the intr insic clearance term, Cl • t > which is the in t r ins ic ab i l i ty of the l iver to clear the drug from the blood. Then equation 1 and 2 become: C1H = Q. E. = Q. C 1 i n t Q + C 1 i n t (3) For drugs with low int r ins ic clearance when compared to hepatic blood flow, C1H depends on the int r ins ic clearance, C l i n t (equation 4). C1H ~~ C l i n t (4) On the other hand, i f C l i n t is much greater than the hepatic blood flow Q, then, the hepatic elimination would be sensitive to changes in flow, Q (equation 5). C1 H * Q (5) Since the hepatic blood flow rate is not in f in i te ly variable and i t is within certain physiological l imits (0.5 - 2.L/min in man),any alteration in hepatic blood flow rate would not be expected to change hepatic clearance by more" than 4 fold (Wilkinson and Shand,.1975). -14-Th e wide variation seen in drug extraction ratios depends upon the differences in in t r ins ic clearance for these drugs. From equation 3 i t can be deduced that i f the C l i n t is equal to blood flow, the extraction ratio is equal to 0.5. A plot of hepatic clearance vs_. hepatic blood flow (within the physiological l imits) shows that when Cl^ n ^. is greater than Q, the hepatic clearance would tend to be dependent on flow (E>0.5) whereas the opposite is true when C l ^ n t is less than flow rate (E<0.5). (Fig. 1). Indocyanine Green (ICG) - A Blood Flow Rate Indicator The tricarbocyanine dye, ICG, has been introduced into c l in ica l medicine by Fox et a l . , (1957) for measuring cardiac output. Since then, a number of investigators have used this agent to study hepatic blood flow rate. The physiochemical properties, toxicity and distribution of ICG has been extensively reviewed and studied by Paumgartner (1975).ICG (molecular weight = 775) is rapidly and completely bound to plasma proteins. ICG is rapidly distributed and retained in the vascular system. Hepatic uptake and b i l iary excretion are the routes of elimination. (90-100%:ofa dose) Enterohepatic cycling has been found to be minimal. The dye is relat ively non-toxic (L.D.^Q = 50 -: 80 mg/kg). Distribution to and elimination by the other organs such as the kidneys has not been measurable. Therefore, this dye seems to have a l l the required qual i t ies of being a cardiac output and hepatic blood flow rate indicator. It has been shown that the ICG clearance at lower dose levels is a good indicator -15-of the hepatic blood flow and cardiac output (<_.0.-64 ymoles/lOOg body weight in rats) . However, at higher dose levels (>_ 1.3 ymoles/100 gm body weight in rats) , the hepatic uptake process become saturated and the removal of ICG from blood does not ref lect hapatic blood flow rate. In this thesis, a low dose of ICG (5 mg/kg) has been used to measure the changes in the cardiac output and the hepatic blood flow after various treatments. Effects of Enxyme Act iv i ty on Drug Clearance - Changes of metabolic and b i l ia ry excretory processes wi l l be reflected in C l . . ^ values. Figure 2 i l lustrates the effect of changes in Cl^ .^ on the actual hepatic clearance of a drug. Such changes are the largest when ^ i n t ^ s s m a ^ ' A change of Cl • ^ from 167 ml/min to 375 ml/min results in a change in the extraction rat io from 0.1 to 0.2 at average l iver blood flow rate (1.5 L/min) and a doubling of hapatic clearance. A change in Cln- t from 6 to 13.5 L/min would only result in a change of E from 0.8 to 0.9, an increaseof 12.5% in the drug clearance at the normal blood flow (1.5 L/min.). Therefore, i t can be summarized that drugs with a high in t r ins ic clearance such as propranolol (Nies et al. ,1973), propoxyphene (Nies et a l . , 1976), lidocaine (Branch et a l . , 1973), to name a few, have high extraction ratios and the elimination of these drugs is very sensitive to flow change and least sensitive to enzyme act iv i ty changes. Drugs such as tolbutamide (Nies et a l . , 1976) and antipyrine (Branch et a l . , 1974) which 15 a a. LU <_> < CE O I 2 5 H 2.<H 05H 0 0.5 1.0 1.5 2.0 2 5 LIVER BLOOD FLOW, L I T E R / M I N Fig. 1. The relationship between l iver blood flow and hepatic clearance for drugs with varying extraction ratios (ER). The arrows indicate the range over which l iver blood flow can vary and extraction ratios refer to a normal flow of 1.5 L/min. too-, 75 50 25 I.S i I ".0 K>5 p S u x 50 100 150 INTRINSIC METABOLIC CLEARANCE. Ilttr/min Fig. 2. The relationship, according to equation 3 , between the in t r ins ic clearance, hepatic extraction and actual hepatic clearance assuming a l i ver blood flow of 1.5 L/min. The inset indicates on an expanded scale the relationship at low values of Clint-16 have low extraction ratios are very sensitive to changes in enzyme act iv i t ies but insensitive to changes in flow, Clearance and Half L i fe . The total clearance of a drug is expressed by equation 6 where Cl is the systemic - 0-693 . . . - 1 / 2 " c\~ • V d ( 6 ) clearance, ty2> i s the half l i f e of a drug and V^, the volume of distr ibution of the drug. It is noted in equation 3 that the clearance term reflects the disposition of a drug which is determined by the physiological parameters such as enzyme ac t iv i ty , blood flow and protein binding,only. C1H is not affected by any other terms which are not related to the removal processes. On the other hand, the h a l f - l i f e of a drug could be related to changes not only due to clearance but also distributional changes. Therefore, half-l i f e when compared to clearance is not the best index to ref lect drug elimination. Influence of Route of Administration- When a drug is given intravenously, the ava i lab i l i t y , F, is assumed to be 1. However, when a drug is given oral ly (assuming complete absorption and no prehepatic elimination),•the ava i lab i l i ty is related to the extraction ratio in equation 7. F = l - E (7) 17 C l s a f t e r an o ra l dose can be expressed by Equat ion 8 , FD C 1 s = A 0 f = Q £ <8> o where D Q , i s the o ra l dose and AUCQ i s the area under the plasma concen t ra t i on yj^. t ime curve a f t e r an o r a l dose. S u b s t i t u t i n g equat ion 7 i n to 8 , the f o l l o w i n g r e l a t i o n s h i p i s obta ined (equat ion 9 ) : ^ - = 3L_ = r l (9) AUCQ 1-E L l i n t Th is i n t e r e s t i n g r e l a t i o n s h i p i n d i c a t e s tha t the AUCQ and, hence, the average drug concen t ra t i on i s independent o f f low rega rd less o f whether the drug has a high or low i n t r i n s i c c l e a r a n c e . Changes in C 1 i n t w i l 1 g i v e a r e c i P r o c a l change i n the AUCQ which i n tu rn r e f l e c t s the changes i n F. For drugs w i th low i n t r i n s i c c l e a r a n c e , the f r a c t i o n o f an o ra l dose F reach ing the sys temic c i r c u l a t i o n i s high whereas the oppos i te i s t rue f o r drugs which have a high i n t r i n s i c c lea rance (Equat ion 7 ) . MCP has been i n d i r e c t l y shown to undergo s i g n i f i c a n t f i r s t pass metabol ism both i n man (Sc'hupan e t a l . , 1979; G ra f f ne r e t a /1 . , 1979 and Bateman e t a l . , 1980a)and i n r a b b i t s (Bakke and Segura , 1976). I t i s t he re fo re pos tu la ted tha t the e l i m i n a t i o n of MCP may be b lood f low ra te l i m i t e d i n the r a t . In a d d i t i o n , an impairment o f the hepa t i c f u n c t i o n may s i g n i f i c a n t l y a l t e r MCP d i s p o s i t i o n . Par t o f t h i s t h e s i s i s devoted to the study of these e l i m i n a t i o n c h a r a c t e r i s t i c s o f MCP i n r a t . 18 Renal Failure and Drug Accumulation When renal function is diminished, through acute or chronic renal disease, those drugs which are predominantly eliminated via the kidney tend to be retained in the body and may accumulate to toxic levels with multiple dosing (Welling and Craig, 1976, and Levy, 1977). Some drugs are cleared by the kidneys as the unchanged form while others may undergo extensive metabolism and subsequent urinary excretion, thus, i f a drug is excreted largely in the urine (e.g. MCP - 84% of dose) regardless whether the drug is excreted as intact drug or metabolized and then excreted, i ts overall pharmacokinetic prof i le is par t ia l ly a function of the integrity of renal function. Besides the risk of toxic accumulation of drugs and/or toxic metabolites in renal insuff ic iency, certain physiological and anatomical changes also alter the pharmacokinetic parameters of drugs which can make dosage modification even more d i f f i c u l t . The volume of distr ibution of drugs such as digoxin (Reuning et.al . , 1973), cephalexin, colistimethate, and insulin (Gibaldi and Perr ier , 1972), have been reported to decrease due to the change of distr ibution characteristics of these drugs in renal impairment. These authors noted a signif icant increase in half-l i f e for the drugs examined,which was accompanied by a change in volume of distr ibution (Gibaldi and Perrier, 1972 and Reuning et a l . , 1973). It was further suggested that a given drug concentration in the serum of patients with renal impairment may produce signi f icant ly less intense c l in ica l or toxicological reponse than the same 1.9 concentration in normal subjects i f the site of action is within the tissue compartment (Gibaldi and Perrier, 1972). The influence of disease states on protein binding has recently been recognized (Craig et a l . , 1976). The presence of uremia has been associated with a signif icant reduction in the protein binding of a number of drugs, in particular organic acids (Reidenberg et a l . , 1971; Shoeman and Azarnoff, 1972; Andreasen et a l . , 1973; Reidenberg and Affrime, 1973i and Craig et a l . , 1974). and a concomitant increase in .apparent distribution volume ( Reidenberg, 1971; Fischer, 1972; Craig et a l . , 1973, and Craig et a l . , 1974). The sera from uremic subjects had lower albumin concentrations than normal. Furthermore, the decreased plasma protein binding in uremia could not be accounted for by hypoalbuminemia alone (Craig et a l . , 1976). This may be due to the increase in the level of endogenous inhibitors which compete for the binding sites in renal impairment (Dromgoole, 1973; Andreassen and Jacobsen, 1974; Craig and Wagnild, 1974; and Sjoholm et a l . , 1976) and/or the binding quality of the protein may change in uremia (Shoeman and Azarnoff, 1972 and Boobis, 1977). The binding of most basic drugs (desmethylimipramine, quinidine, dapsone, trimethoprim, propranolol and tubocurarine) studied to date is normal in uremic patients (Reidenberg et a l . , , 1971; Craig and Kunin, 1973 and Ghoneim, 1973 and Reidenberg, 1977). The drugs triamterene and diazepam have proven to be exceptions to 20 this observation, however, (Reidenberg et a l . , 1971 and Reidenberg and Affrime, 1973). Alteration of the erythrocyte concentration can also perturb the volume of distr ibution of some drugs. For example, gentamicin binding to red blood ce l ls is decreased in anemia and as a consequence plasma concentrations are elevated relative to subjects with normal haemoglobin levels (Riff and Jackson, 1976). Since the hematocrit is often from 17-20% in renal impairment, the presence of anemia may further al ter the volume of distr ibution of some drugs. Physiological changes such as edema and dehydration can alter the volume of distribution of some drugs, as has been shown for the sulphonamides or thiocyanate. The effect of other factors such as drug receptor sensi t iv i t ies in diseased cases have been reviewed (Fabre and Balant, 1976). The effect of changes in protein binding may alter the distr ibution and elimination pattern of a drug. A typical example would be phenytoin. Letteri et a l . , (1971) observed more rapid elimination of phenytoin in patients with renal fa i lure than in normal subjects. This was confirmed by Odar - Cederlof and Borga (1974) who proved that the rapid elimination of phenytoin in patients with renal dysfunction was due to rapid metabolic clearance of the drug. The increase in the metabolic clearance could be partly explained by an increase in the free circulat ing concentration of phenytoin available for elimination. MCP has been shown to moderately bind to bovine, rat and rabbit sera (<20%) (Pognini and Dicarlo, 1972). Therefore, a change in protein binding in renal fai lure is unlikely to 21 alter the kinetics of MCP. The influence of kidney disease on drug metabolism has been reviewed (Reidenberg, 1974 and Reidenberg, 1978 ). Drug metabolism by oxidative pathways appears to be normal in uremic patients. The pentobarbital elimination h a l f - l i f e was shortened in dialyzed patients who are not on dia lysis (Reidenberg 1978). This was shown to be due to a reduction in volume of d istr ibut ion; the actual metabolic clearance of pentobarbital was unaltered. Highly protein bound drugs which have a low in t r ins ic clearance may have their elimination rates increased because of decreased protein binding and increased clearance of free drug (Wilkinson and Shand, 1975). Antipyrine (characterized by a low int r ins ic clearance and weak plasma protein binding) has been shown to have accelerated metabolism in patients with renal dysfunction. This cannot be accounted for by the reduction in plasma protein binding (Reidenberg, 1978). Oxidative drug metabolism was found to decrease in uremic rats (Leber and Schutterle, 1973). There appeared to be a species difference in the oxidative metabolism of drugs in uremia (Reidenberg, 1978)-.; Pharmacokinetics of MCP - Despite the signif icant number of papers published on MCP, very l i t t l e information pertaining to i ts pharmacokinetics was available prior to the in i t ia t ion of the present study. The general complaint was the lack of a highly sensitive assay. Tunon et a l . , (1974) in a study using rats, showed that MCP followed bi-exponential elimination kinetics after an i .v . dose (10 mg/kg). The h a l f - l i f e of the drug reported was extremely short 22 (13 minutes). A spectrophotometric technique was employed to analyse . the blood samples taken from the same rat. In samples after 16 minutes, MCP could not be detected due to the lack of sensit iv i ty of the method. The h a l f - l i f e calculated was almost certainly erroneous due to the use of truncated data. Bakke et a l . , (1976)observed that MCP followed f i r s t order elimination = 20 minutes) in rats. The h a l f - l i f e of MCP after oral administration was prolonged but the mechanism was unrecognised. By comparing the area under the plasma concentration versus time curve (AUC), i t appeared that the ava i lab i l i ty of the drug administered oral ly was only about a tenth of an equivalent i .v . dose. With no evidence of incomplete absorption, f i rst -pass metabolism was postulated. These resul ts , however, would have to be re-examined by using a more sensitive assay method because each datum obtained by Bakke et a l . , (1976) was obtained by sacr i f ic ing individual rats , and the standard deviation (probably due to the inter- individual variations) observed was very high. The TLC-photodensitometry method was capable of detecting 0.25 mcg/ml of MCP provided 1 ml of plasma was used. That is to say the capabil i ty of this assay is limited to detect lower nanogram levels of MCP. Continuous sampling in small animals such as rats was prohibited because comparatively large volumes of plasma samples (1 ml) were required. A colorimetric technique was used by Arita et al.,(1970a) to study the metabolites of MCP. The sensi t iv i ty of this method was only comparable to the TLC-photodensitometry technique but not superior. A GLC-FID assay (Kaempe, 1974) was employed by forensic investigators to isolate 23 and identify MCP qual i tat ively. The sensi t iv i ty of this method was not reported. Recently, a HPLC assay of MCP was reported (Teng et al. ,1977). The lowest detectable concentration of this assay is about 5 ng/ml provided 5 ml of plasma is being analysed. Again, this assay was not found to be sensitive enough to be used to analyse small volume samples. In view of the available technology to analyse MCP, the development of a highly sensitive assay was of prime importance to fac i l i ta te the study of the pharmacokinetics of this drug. A highly sensitive GLC-electron capture detection (ECD) assay has recently been reported by our laboratory (Tarn and Axel son, 1978). This enabled the quantitation of minute amounts of MCP (detection l imit ~ 20 peg) in serial blood samples (0.1-0.2 ml). Subsequent to this development, a modified assay for human plasma and urine analysis has also been reported (Tarn and Axelson, 1979). In the past year, a HPLC assay (Graffner et al., 1979) and a GC-mass spectrometric assay (Bateman et a l . ,1978) have appeared in the l i terature for MCP quantitation in human biological samples. The sensit iv i ty of these assays,although not superior to our methods, have been found to be adequate for quantitation of MCP in human plasma samples. The appl icabi l i ty of these methods has been shown by using these techniques in studying the kinetics of MCP in normal human volunteers (Bateman et a l . , 1979;,. Graffner et a l . , 1979-and Bateman et a l . , 1980a). 24 The kinetics of MCP in man have been found to be dependent on the route of administration (Graffner et al 1979, Bateman et al. H , 1980a). Substantial f i r s t pass metabolism has been reported by these authors. The high var iab i l i ty observed in the effect of route of administration study may in part, be due to the tablet dosage form employed (Bateman, et a l , , 1980a). The bioavai labi l i ty calculated by Graffner et a l , (1979) was achieved by comparing the area under the plasma concentration vs_. time curve (AUC) of an unequal intravenous ( i .v . ) dose (10 mg) and oral doses (20 mg), though no evidence of non-linear kinetics was provided (Graffner,et a l . ,1979). Although linear kinetics were proposed by Teng et al.,(1977) for MCP, a bioavai labi l i ty study was not reported. A more rigorous method such as comparing the AUCs after identical i .v . and oral (solution) dose is required. A 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. Preliminary data suggest signif icant f i rst -pass metabolism. A preliminary study has been reported describing the pharmacokinetics of MCP in patients with renal impairment (Bateman et a l . , 1980b). The h a l f - l i f e of MCP has been observed to increase 4 fold whereas the renal and total body clearance were diminished to a similar extent in uremic patients. This observation was unexpected since MCP is extensively metabolized (approximately 20% of an administered dose is excreted intact) . 25 The underlying mechanism has not been elucidated nor has any hypotheses been put forward to attempt explanation of this observation. Since conjugation is one of the major metabolic pathways in man, (40% of dose) whether the unexpected alteration in kinetics is due to an increase in b i l i a ry excretion of MCP conjugates which may be hydrolyzed and reabsorbed from G.I. tract remains to be examined. This proposal is analogous to the one proposed by Levy (1979) for D i f lun isa l . In fact , enterohepatic cycling has been reported for MCP in one of the studies (Donatell i , 1971). Protocols have been designed to study the kinetics of MCP in patients with renal dysfunction and are currently underway in our laboratory. Metabolism - MCP is extensively metabolized in a l l animal models studied (Ar i ta et a l . , 1970b, Bakke et a l . , 1976, and Cowan et a l . , 1976). Conjugation is the major metabolic pathway in man (Teng et a l . , 1977, Bateman et a l . , 1978, 1980a and Tarn et a l . , 1979), and rabbit (Arita et a l . , 1970b). De-ethylation is also dominant in the rabbit (Arita et a l . , 1970b) and the rat (Teng et a l . , 1977). An in vitro study (Beckett and Hiuzing, 1975) showed that MCP had eight metabolites. Later, Cowan et a l . , (1976) was able to identify four additional metabolites (scheme I). These metabolites exist in very minute quantities (<1% of a dose). In this thesis, a GLC-ECD assay is reported to simultaneously . quantitate MCP and the de-ethylated metabolite (DE-MCP) in rat urine (Tarn and Axelson, 1979b). The pharmacokinetics of MCP has been studied as a function of dose and route of CK OXIDIZED ( / \ \ - C - N H - ( C H 2 ) - N / C 2 H 5 2 \ C , H , H N — ^ N H - ( C H 2 ) - l / C 2 H 5 N O C H 3 IX S,0 _ N O C H , C , H 5 VII t CK O / C 2 H {/ \ y . C - N H - ( C H ^ - ? i ^ C 2 H 5 C H 3 C 6 H 9 0 6 V L " X C , H 5 CK H , N - / / ^ > - C — N H — C H 2 C H 2 - N X C 2 H 5 CK H,NY/ V II / - C - N H - C H 2 C H 2 - N \ H N O C H , C , H 2 n 5 H 2 N - ( / \ V i - N H - C H 2 - C H 2 O H ^ O C H , IV IV) CK H2NV/ V Q / H - C — N H - C H 2 C H 2 — N ^ Cl, C , H 5 x O H H,N—V \ ) - C - N H - C H 2 C H 2 - N VI III Scheme I. A schematic of metabolites of metoclopramide recovered from rabbit urine. 27 administration in the rat. Finally, the kinetics of MCP in renal and hepatic impaired rats have also been reported in this thesis. 28 EXPERIMENTAL 29 ELECTRON-CAPTURE GAS LIQUID CHROMATOGRAPHIC ASSAY (EC-GLC) FOR  SIMULTANEOUS DETERMINATION OF MCP AND ITS MAJOR METABOLITE  DE-ETHYL-MCP (DE-MCP) IN RAT URINE Materials 4-Amino-5-chloro-2-methoxy-N-(2-ethylaminoethyl) benzamide (DE-MCP) and 4-amino-5-chloro-2-methoxy-N-(2-diethyl aminoethyl) benzamide monohydrochloride monohydrate (MCP-HC1 ^ O ) (Lot no.9207) were supplied by A.H. Robins Co. (Richmond, Va . , U.S.A. ) . Heptafluorobutyric anhydride (HFBA) was purchased from Pierce (Rockford, 111. U.S.A. ) . Diazepam was supplied by Hoffmann-La Roche (Montreal, Canada). One .normal sodium hydroxide and 4% ammonium hydroxide were prepared from BDH and ACS reagent grade materials respectively. Chloroform (d is t i l l ed in glass) was obtained from Caledon (Georgetown, Ontario, Canada). GLC A Hewlett-Packard gas chromatograph (model 5840) equipped with a 6 3 N i ECD and a glass column (1.2 m x 2 mm I.D.) containing 3% of OV-225 D coated on Supelcoport (100-120 mesh) was used. The operating conditions for routine analysis were: injection temperature, 250°; oven temperature 235°; detector temperature, 350°; carr ier gas [argon-methane (19:1, v/v)] flow-rate, 40 ml/min. Extraction and Derivative Formation To 1 ml of blank urine containing DE-MCP and MCP-HC1-H20 was added 1 ml of 1 N NaOH (pH % 13) and 6 ml of chloroform, and the mixture was shaken on a horizontal shaker for 20 min to extract the DE-MCP. 30 After centrifugation, 5 ml of the organic phase was transferred to a 15-ml centrifuge tube, and the contents were dried under a gentle stream of nitrogen. The residue was reconstituted with 1 ml of internal-standard solution (1 Mg /ml of diazepam in benzene) and 20 yl of heptafluorobutyricanhydride (HFBA) were added. After thorough mixing (vortex-type mixer), the reaction mixture was incubated at 55° for 20 min, then allowed to cool , and the excess of derivatizing agent was removed by hydrolysis with 0.5 ml of water and neutralization with 0. 5 ml of 4% ammonium hydroxide solution. Mass Spectrometry (MS) Electron impact (EI). A Varian Mat-Ill GLC-EI-mass spectrometer was used to study the heptafluorobutyryl (HFB) derivative of DE-MCP and MCP. The following conditions were used: for GLC, the injection and oven temperatures were 250° and 230°, respectively, the carrier-gas (helium) flow-rate was 20 ml/min, and a 1.8-m. x 2-mm 1. D. glass column packed with 3% of 0V-17 coated on Chromosorb W (80-100 mesh) was used. For MS, the ionization energy was 70 eV, the electron-multipl ier voltage was 2 kV, the analyzer temperature was 250°and the separator-oven temperature was 200°. Chemical ionization (Cl) . A Finnigan GLC-CI mass spectrometer (model 4000) was employed to identify the molecular ion of the HFB derivatives. The GLC conditions used were as described for routine analysis except that the glass column (0.6 m x 2 mm I.D.) contained 3% of 0V-101 coated on Chromosorb W (80-100 mesh), and methane was used as 31 a carr ier and reagent gas (flow-rate 40 ml/min). The separator oven temperature was 250°. Quantitative Studies A 1-yl portion of the HFB derivative solution was injected into the Hewlett Packard Model 5840 reporting GLC-ECD equipped with an automatic sampler. Quantitative estimation of DE-MCP in the urine samples was accomplished by plotting the area ratios (derivative to internal standard) against the concentration of DE-MCP. ANIMAL HANDLING Male Wistar rats weighing 200-300 g were used in a l l of the studies. Al l newly received animals were held in isolation and allowed to acclimatize to the surroundings of the animal care f a c i l i t i e s before handling. The rats were fed with the standard Purina rat chow and tap water was allowed ad libitum. A le f t jugular vein cannula (Weeks & Davis, 1964) was implanted in'the animals involved in plasma level and creatinine clearance studies. A two to three day post-surgery recovery period ensured complete recovery of the animals prior to drug administration. After the surgery and during the studies, the rats were housed individually in stainless steel metabolism cages (9.8in x 7 in x 7 in) . PHARMACOKINETIC STUDY OF MCP IN NORMAL RATS I.V. Administration - Isotonic solutions of metoclopramide monohydrochloride monohydrate (MCP.HCl.H?0) (supplied by A.H. Robins, 32 Lot 9207) equivalent to 5, 15, 25 and 35 mg/kg MCP base were prepared by adding appropriate amounts of sodium chloride to bring the osmolality of the solutions to 280 mOsm/kg of H 20. The MCP solution was injected into the jugular vein via the cannula and 0.1-0.2 ml blood samples were taken at 0, 1, 2, 4, 6, 10, 15, 20, 35, 45, 60, 90, 120, 150, 180, 210, 240, 300, 360, and 420 minutes after MCP administration through the jugular vein. The study of the transient non-linear elimination characteristics was carried out in a group of rats which received a 35 mg/kg i .v . MCP dose. The blood sampling schedule was 0, 1, 5, 10, 20, 40, 60, 120, 180, 240, 350, 360, 420, 450, 480, 510, 540, 570, 600, 630, 660, 690 and 720 minutes after the administra-tion of MCP. After each blood sample, the cannula was flushed with 0. 1 ml of heparin solution (20 units/ml) to prevent blood clot formation within the cannula. The blood samples were immediately centrifuged and the plasma separated and stored at -20°C until analyzed. Oral Administration - An aqueous solution of MCP.HC1.h^O equivalent to 15 mg/kg MCP base in 0.5 ml solution was prepared. MCP was introduced to the rat by oral intubation after l ight ether anesthesia. Approximately 0.1-0.2 ml of blood-was collected through the jugular vein via the cannula at 0, 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240, 300, 360 and 420 minutes after drug administration. The cannula and the blood samples were treated as described in the previous section. This experiment was performed simultaneously with another group of rats which received an identical 1. v. dose of MCP. 33 Urinary Excretion Study - The urinary excretion study was carried out following intraperitoneal ( i . p. ) , oral and i . v . ( t a i l vein) administration of 1, 5, 15, 25 and 35 mg/kg of MCP equivalent to free base. Urine samples free of any fecal contaminants from rats housed in individual metabolism cages (Fig.3) were collected at 24 h intervals up to 72 h. The samples were diluted and aliquots were stored at -20°C. until analyzed. Plasma Indocyanine Green (ICG) Studies - ICG (Hynson, Wescott and Dunning, Baltimore, Md.) solution for intravenous injection was always freshly prepared in the supplied solvent. The ICG solution (0.5 mg/kg, 0.2 ml) was injected via the jugular vein cannula 30 minutes after sal ine, 5 mg/kg and 35 mg/kg MCP pretreatment. The same protocol was employed in another study except ICG was administered 720 minutes after saline or 35 mg/kg MCP pretreatment. Exactly 0.3 ml of blood was taken via the cannula 1, 3, 5, 7, 10 and 15 minutes after the dye administration. The blood samples were individually transferred to 15 ml centrifuge tubes containing 1 ml of 2% human serum albumin in normal sal ine. The samples were immediately centrifuged and the plasma samples were analysed within 3 hours after col lect ion. ICG in the plasma was analysed by the UV spectrophotometric method described by Caesar et a l . , (1961). PHARMACOKINETICS OF MCP IN CC14 TREATED RATS Plasma Level Study - The animals were randomly separated into two groups. The control group received 0.5 ml normal saline and the experimental group received a dose of 1.2 ml/kg carbon 34 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. 35 tetrachloride (CC14) oral ly one day prior to MCP administration. An i .v . dose of MCP hydrochloride salt equivalent to 15 mg/kg MCP was administered to rats under l ight ether anesthesia (between 9-10 am). Approximately 0.1-0.2 ml of blood was taken 1, 3, 6, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 360, 420 and 480 minutes after MCP administration. The blood samples were transferred into ( R ) heparinized Carraway tubes v ,centrifuged and the plasma was separated and stored as described in the previous section. Urine Study The rats were randomly separated into two groups. The control and experimental group received a dose of 0.5 ml saline or 1.2 ml/kg CCl^, respectively, one day prior to drug administration. An i .v . dose of MCP hydrocholoride salt equivalent to 15 mg/kg MCP was administered via the ta i l vein to test and control animals under l ight ether anesthesia. Each rat was individually housed in a stainless steel metabolism cage which permitted col lect ion of urine free of faecal contamination (Fig. 3). Food and water were allowed ad libitum. Cumulative urine samples were taken every 24 hrs up to 48 hrs, and the samples were diluted to 100 ml with water and aliquots were frozen until assayed. Biochemical Study The extent of renal and hepatic impairment by CCl^ was determined by measuring the creatinine clearance, plasma urea nitrogen (PUN) and plasma glutamic oxalo-acetic transaminase (PG0T) levels . This was accomplished by taking 1.5.ml of blood from the saline and CCl^ pretreated animals at the end of a 24 hour urine col lect ion. The plasma GOT (aspartate amino transferase E.C. -2611) and creatinine were analyzed on a fast centrifugal analyzer (Electro-Nucleonics (GEMSAEC). The plasma GOT was analyzed using Henry's (1960) modification of the Karmen method (1955). 36 The creatinine levels were determined using the alkaline picrate method. The plasma urea nitrogen was measured using the urease/glutamate dehydrogenase reaction on a Du Pont Automated Cl in ical Analyzer. The precision (coefficient of variation) for creatinine plasma GOT and urea nitrogen was 5% at 2.3 mg/dL, 3% at 14 IU/L and 4% at 20 mg/dL respectively. These biochemical evaluations were performed by the Division of Cl in ica l Chemistry at the Vancouver General Hospital. PHARMACOKINETICS OF MCP IN CHEMICALLY AND  SURGICALLY INDUCED RENAL DYSFUNCTION Surgical Methods Two step 5/6 nephrectomy (TSN) was performed in the following • manner: an incision was made about 1 cm le f t of the spinal cord and extended 2-2 1/2 cm from the rib cage. The le f t kidney was exposed and dissected free of the surrounding t issues. Two ligatures were placed t ightly around the two poles of the kidney with 2-0 s i lk so that the kidney was sectioned into thirds. The two poles were removed and bleeding was observed to be minimal. The remaining one third of the kidney was returned to the peritoneum after cessation of bleeding. The right kidney was completely removed and a jugular vein cannula implanted one week later . Two-step sham operated rats which were handled identical ly served as controls for the TSN rats. MCP administration was performed 24 hrs after the removal of the right kidney. Bilateral ureteral l igation (BUL) was performed after exposing the kidney in the same manner as for the TSN. Two ligatures were tied around each ureter and were then cut between the l igatures. A group of sham operated rats which were handled ident ical ly served as BUL controls. MCP administration was performed no less than 24 hrs after surgery. 37 Chemical Induction Uranyl nitrate (UN), 5 mg/kg (in 0.5 ml sa l ine) , was administered through the ta i l vein 6 days prior to MCP administration. Saline treated animals which served as controls were injected with 0.5 ml of saline intravenously. Plasma Level and Urine Study - MCP (15 mg/kg) was administered intravenously to a l l the animals between 0900-1000 hrs. Blood samples (0.1-0.2 ml) were taken via the jugular vein cannula, 1 ,2,4,6,10,15,20,30,45,60,90,120,150,180,210,240,270,300,360 and 420 minutes after MCP administration. The blood samples were centrifuged and the plasma immediately separated and stored at -20° until analyzed. Cumulative 24 and 48 hr urine samples were collected after MCP administration,as previously described. The samples were diluted to 100 ml with d i s t i l l e d water and an aliquot was stored at -20°C until analyzed. Assessment of Renal Function 1 To establish the renal function of the animals which underwent surgical or chemical induction of renal impairment, one ml of blood was taken via the jugular vein cannula on day 0, 1, 2, 4 and 6 from the control and UN, BUL and TSN treated animals. After centrifugation, a 0.5 ml plasma sample was collected and the 38 plasma creatinine, plasma urea nitrogen and PGOT were analyzed by the Cl in ical Chemistry Division of the Vancouver General Hospital to assess the progression of renal damage. A 1 ml sample of blood was taken from the animals after MCP treatment and the physiological function was assessed by measuring the PUN, plasma creatinine and PGOT for a l l three models (TSN, BUL and UN). Creatinine clearance was estimated by measuring the amount of creatinine excreted in 24 hr in the urine and the plasma creatinine concentration at the time of urine col lect ion. PHARMACOKINETIC AND STATISTICAL ANALYSES Ini t ia l estimates of the pharmacokinetic parameters for MCP after an i .v . or oral dose were obtained by computer analysis using the program, AUTOAN (Sedman and Wagner 1976). These estimates were later f i t ted using the program NONLIN (Metzler et a l . , 1974). Al l the data points were weighted equal to the reciprocal of the concentrations. The AUCs were calculated using equation 10. AUCQ = AUC* + AUC^ (10) where t represents the time when the last sample was taken. The f i r s t term AUC^ was calculated using the trapezoidal rule and the second term was calculated using equation 11. AUC^ = 1.44 C t t 1 / 2 (11) 39 where is the concentration of the last sample and t - ^ is the plasma h a l f - l i f e of MCP. In most instances the AUC obtained from 0-t was over 95% of the total AUC, therefore the error in AUC estimation arising from an inaccurate estimation of t - ^ 1 S minimal. Analysis of variance was employed to s ta t i s t i ca 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 the analyses. i 40 RESULTS 41 RESULTS GLC A representative chromatogram obtained from the extract of rat urine is shown in Fig. 4; no endogenous interference was observed in chromatograms from blank urine samples. Peaks with retention times at 3;04 and 3.59 min were the HFB derivatives of DE-MCP and MCP, respectively. Linearity was observed in the range studied (0.30-1.50 ug /m l MCP and 0.40-1.85 ug/ml DE-MCP in urine). Quantitation of MCP and DE-MCP was accomplished by analysing a serial di lution of known concentrations of MCP and DE-MCP in the urine extract (Fig.5 ). Regression analysis showed that the best-f i t l ines through the data points were described by: y =0.911 x +0.011 for MCP ^ y =1.5 x + 0.039 for DE-MCP with r = 0.999 in both cases. The HFB derivatives were shown to be stable for at least 24h at ambient temperature. MS - The identity of the GC peaks were confirmed by using GC mass-spectrometric methods. EI. The fragmentation pattern of the HFB derivative of DE-MCP is similar to that of the HFB derivative of MCP. For MCP, the base peak is at m/e 86 and the other most abundant peaks are at 99, 380 and 423. (Fig.6 ). The base peak for DE-MCP is at m/e 380 and the most intense peaks are at m/e 396, 409 and 422. (Fig.7 ). 42 O » 0 5 CO 1 0 Fig. 4. A representative chromatogram obtained from the urine extract. The retention times at 3.04, 3.59 and 7.45 minutes were the HFB derivative of MCP and DE-MCP and diazepam respectively. co UJ DC CONCENTRATION OF METOCLOPRAMIDE [ug/ml] CONCENTRATION OF DE-MCP [ug/ml] Fig. 5. Calibrations curves of the urine extracts: a. ,MCP. b. DE-MCP 44 Plasma extract after metoclopramide admin. # 3 8 - 3 2 100 >420 X 5 100 10 1\ i In If i Irfl11 Aift ilvn i F4'M n i i i 50 100 150 200 I I I I I I I I 250 Cl \ J H ~ \ v>—C — N H — | ~ ° X ) C H 3 / C 2 H 5 C H , — C H , — N VH 5 M .W. = 495 10 270 ' ' ' '4)0 100 10 'i 1 1 M 11 i i 11 i M i i i i ] 11 / /—i i 11 11 11 i i i I i 11 11 i ' n 11 20 50 100 400 4M) ! m/e Fig. 6. Mass spectra of the HFB-derivative of MCP a. electron impact b. chemical ionization T T " >bo FT" 45 —llOO Z UJ > LU tc (a) H^^4-NM-CH,CH1-4,H' 300 m/e (W V4 «SeJMH-C.,H,] I! Fig. 3. Mass spectra of the derivative of D E - M C P : (a) E l ; (b) C l . Fig. 7. Mass spectra of the HFB-derivative of DE-MCP a. electron impact b. chemical ionization 46 C L The (M+1)peak f o r the HFB d e r i v a t i v e of MCP i s a t m/e 486 whereas the ( M + 1 ) peak f o r the HFB d e r i v a t i v e o f DE-MCP i s a t m/e 636. Recovery The e x t r a c t i o n e f f i c i e n c y was eva lua ted by us ing a s tandard curve prepared a f t e r d i s s o l v i n g au then t i c MCP and DE-MCP d i r e c t l y i n ch lo ro fo rm. The average recove r i es from u r ine e x t r a c t s f o r MCP and DE-MCP were 84±6% .(n=5)_ and 86±5% (n=5) r e s p e c t i v e l y . Dose Dependent K i n e t i c s o f MCP - The plasma l e v e l s o f MCP a f t e r low i . v . doses (<_ 25 mg/kg MCP) d e c l i n e d i n a manner best desc r i bed by a b i exponen t i a l equat ion (F igu re 8) (equat ion 12 ) : Cp = A e " a t + Be ~ 3 t (12) where Cp i s the plasma concen t ra t i on a t t ime t . A and B are the i n t e r c e p t va lues f o r the a and 3 phases, r e s p e c t i v e l y . The cons tan ts a and g desc r i be the s lope o f the i n i t i a l d i s t r i b u t i v e phase and the te rmina l e l i m i n a t i o n phase, r e s p e c t i v e l y . The pharmacok inet ic parameters ob ta ined from these data are shown i n Table I. Plasma MCP l e v e l s a f t e r high i . v . doses (35 mg/kg) became t r i - p h a s i c ( F i g . 9 ) . Fo l l ow ing the d i s t r i b u t i v e phase, the f i r s t l og l i n e a r p o r t i o n has an average h a l f - l i f e o f 104 ± 14 minutes. Approx imate ly 400 minutes a f t e r MCP a d m i n i s t r a t i o n , the plasma l e v e l s assumed an average h a l f - l i f e (58 ± 7 minutes) which was s i m i l a r to the t-j ob ta ined a f t e r low doses ( t - j^ g = 5 8 ± 1 0 m i n u t e s ) . Th is 47 Fig. 8. Representative MCP Log plasma concentration ys_.time curves after a range (5-25 mg/kg) of i.v. doses. 48 TABLE I Kinetic parameters obtained after i .v . doses Dose (mg/kg) 5 15 25 e (min ) _ 1 0.015 ± 0.003 a 0.0124 ± 0.003 0 .0095 ± 0.0016b t 1 / 2 ( m i n ) 47 ± 9 58 ± 10 72 ± 13 b AUC 110 ± 45 300 ± 26 616 ± 200 (mcg.min/ml) V d(area) 3.8 ± 1.0 4.6 ± 0.6 4.4 ± 0.9 (L/kg) V d s s (L /kg ) 3.3 ± 1.0 4.1 ± 0.6 4.0 ± 0.8 Cljg(m1/min) 12.0 ± 3.5 13.0 ± 1.0 9.78 ± 2.6 ClR(ml/min) 2.91 2.59 2.3 n 6 6 6 a) mean ± standard deviation b) one way AN0VA showed significant difference (p > 0.05) 49 Fig. 9. A representative log plasma concentration vs^  time curve after a 35 mg/kg dose of MCP (I .V.) . The sampling period was extended to 720 minutes. INSERT: The plasma t 1/2 of the intermediate kinetic phase was evaluated using a truncated and more intensive sampling protocol. 50 phenomenon clearly indicates that MCP's disposition kinetics are dose-dependent. The non-linear nature of MCP kinetics was further i l lustrated by the disproportionate increase in AUC with dose at high dose levels (_> 25 mg/kg) (Fig.10). The inspection of a typical plasma concentration vs_ time curve (figure 11) obtained after^oral administration shows that MCP (15 mg/kg) is rapidly absorbed. The time required to achieve maximum plasma concentration is between 30-60 minutes. This agrees closely to previous l i terature values (Bakke and Segura, 1976; Hucker, et a l . , 1968). The AUC values obtained after a 15 mg/kg oral dose are insigni f icant ly different from the AUC values obtained after an identical i .v . dose . (Fig.12). The ava i lab i l i ty (F) after an oral dose has been calculated using equation 13. F = ava i lab i l i ty = ^ A U C ^ oral. (13) T A U C J — " F is equal to 0.91 indicating that f i rst -pass metabolism does not occur at this dose level . Urinary Excretion Data - . A study of the cumulative excretion products in 48 h rat urine after MCP administration (K35 mg/kg) after i . v . , i .p . or oral dosing, showed no signif icant differences (p<0.05) in the percentage of intact drug and metabolite recovered (p<0.05). (Table II and III). There was no signif icant difference.in the excretion of intact drug (Table II) and the de-ethylated metabolite (Table III) over a 35 fold dose range given 51 1500 ^ 1000 c E i m o J 8 T o O < 500 _L 10 20 30 40 Dose (mg/kg) Fig. 10. A plot of AUC vs_ dose to i l lust ra te the non-linear nature of the MCP kinetics. 52 Fig. 11. Representative plasma concentration ys_. time plots obtained after i.v. ( A ) and oral ( • ) administration of MCP (15 mg/kg). 53 350 h 300 h E: E i o E 200 h 8 T O O < 100 i.v oral MCP DOSES, 15mg/kg Fig. 12. The AUC comparison between i .v . and oral administration of a 15 mg/kg dose of MCP. (The bars represent the mean ± one standard deviation) 54 TABLE II % of MCP recovered after 48 hr cumulative urine analysis. Dose (mg/kg) i v. i •P oral 1 21.6 ± 5.1 a ( 4 ) b 23.6 + 4.2 (4) 21.9 ± 6.3 (6) 5 25.1 ± 4.6 (6) 23.9 + 7.6 (6) 20.1 ±8.5 (6) 15 21.0 ± 3.5 (6) 23.2 + 5.6 (5) 24.4 ± 1.2 (6) 25 24.9 ± 3.3 (7) 27.3 + 2.7 (5) 26.4 ± 3.9 (6) 35 26.1 ± 3.6 (6) 25.6 + 5.5 (6) 28.0 ± 2.1 (4) a) mean ± standard deviation-fa) (n), number of animals 55 TABLE III % of De-ethylated metabolite equivalent to MCP recovered after 48 hrs cumulative urine c o l l e c t i o n . % metabolite recovered Dose (mg/kg) i . v. i .p. oral 1 7.1 + 1.3a ( 4 ) b 7.8 ± 1.5 (4) 6.3 ± 1.1 (6) 5 7.0 + 2.9 (6) 8.4 ± 2.0 (6) 8.8 ± 1.8 (6) 15 7.3 + 1.6 (6) 8.6 ± 1.4 (5) 11.1 ± 2.2 (6) 25 10.0 + 1.6 (7) 12.8 ± 1.8 (5) 12.5 ± 1.1 (6) 35 8.6 + 1.5 (6) 9.4 ± 2.3 (6) 12.1 ± 3.0 (4) a) mean ± standard deviation b) (n), number of animals 56 by hepatic (oral , i .p. ) and systemic ( i .v . ) routes of administration. This observation would normally tend to rule out dose-dependent kinetics and/or f i rst-pass metabolism part icularly in the presence of confirmatory plasma data showing equivalent AUC values (15 mg/kg, i .v . vs. oral) (Fig. 12).Dose-dependency would be noted i f the percentage of dose excreted as the intact drug and/or corresponding metabolite(s) changed with an increase in MCP dose. Had MCP been affected by f i rst -pass metabolism, the administration of MCP by a hepatic route of administration would have yielded less excretion of intact drug than that seen after a systemic route of administration. The apparent lack of f i rst -pass metabolism is confirmed after inspection of plasma data at the 15 mg/kg dose level ( i .v . vs oral) (Fig.12) showing identical AUC values. Similar ly , the lack of difference in excretion (Tables II, III) after the oral and intraperi -toneal routes tends to rule out s igni f icant gut metabolism of MCP. It is clear, however, that the urine data showing apparent l inear dose-independent pharmacokinetics for MCP is in conf l ic t with the observed dose-dependent nature of the plasma data. ICG Clearance - 7 ' F ig. 13 shows that the plasma ICG clearance 30 minutes after a 5 mg/kg MCP i .v . dose is s igni f icant ly higher than the plasma clearance of ICG (0.5 mg/kg i .v . ) 30 minutes after a 35 mg/kg i .v . dose (p<0.05). There is no signi f icant difference between the ICG clearances of the control and the 5 mg/kg MCP pretreated group (p<0.05). It would appear that the apparent dose dependent kinetics of MCP are due to a reduction in blood flow . through the major eliminating organ(s). Further evidence for this 50 O) I 40 D30 minutes after treatment •7 hours after treatment S 301 o D u 201 O u • • I u I • • • • 10 c Saline 5 mg/kg MCP 35 mg/kg MCP 35 mg/kg MCP Fig. 13. Comparison of ICG clearance .30 minutes and 7 hours after sal ine, 5 mg/kg and 35 mg/kg MCP. (The bars represent mean ± standard deviation) 58 hypothesis is given by the return of ICG clearance to control levels seven hours after the administered 35 mg/kg i .v . dose of MCP (Fig.13) with a concomitant resumption of l inear kinetics for MCP (35 mg/kg dose level) (Fig. 9) after the transient nonlinear phase. CARBON TETRACHLORIDE TREATMENT Biochemistry. The administration of CCl^ resulted in extensive l i ve r cel l necrosis as indicated by a large increase in the PGOT levels (Table IV). However, the plasma creatinine and PUN level are insigni f icant ly changed (Table IV) when compared to the control. The creatinine clearance was signi f icant ly reduced by CCl^ treatment indicating some damage of kidney tissue (Table IV). Plasma Study. The plasma levels of both the control and the experimental groups follows a bi-exponential decline (Fig.14). The CC1^ pretreated group has a higher plasma MCP level and the terminal elimination phase was delayed when compared to the controls (Fig.14). The pharmacokinetic parameters obtained are summarized in Fig.15. The h a l f - l i f e of MCP was increased by approximately .3 fold and the area under the plasma concentration curve (AUC) was also increased to a similar extent. The volume of distribution calculated by the area (Vd^ ) and the steady -state method (Vd,..) was not s igni f icant ly area ss different between the control and the CCl^ treated group (Fig.15). Therefore, the prolongation of t y 2 w a s due t 0 the reduction in the total body clearance. 59 Table IV B iochemica l In format ion a f t e r Carbon T e t r a c h l o r i d e Treatment Plasma c r e a t i n i n e (mg/dL) C r e a t i n i n e C learance (ml/min) PUN (mg/dL) RGOT ( IU /L ) Cont ro l 0 . 5 ± . 2 ( 6 ) * 2 .0± .5 (6 ) 16±2 (6) 65±30 (6) Treated 0.71±.16(7) 0.77±.26(6) 18±2 (7) 2170±1640 (7) * ( ) number o f animals 60 Fig.: 14. Representative plasma MCP concentration vs. time curve after carbon tetrachloride pretreatment J~<0 , control and • , c c i 4 ) . 300 200 100 Fig. 15. Kinetic parameters obtained for MCP after CC1, pretreatment (c,control and t , test) The bars represent the.mean ± standard deviation. 62 Urine Study. The 24 hr cumulative urinary excretion of intact drug as the percentage of dose increased two-fold af ter .CCl^ treatment when compared to the saline treated animals. (Fig. 16a). The 24 hr cumulative excretion of the de-ethylated metabolite was constant between the two groups. (Fig. 16b). RENAL FAILURE -Body Weight - Table V shows that the body weight of the rats pretreated with uranyl nitrate decreased with time during the period of experimentation whereas the weight of the controls was s l ight ly increased. This was partly due to the loss of appetite and wasting of muscle mass resulting from renal insufficiency (Voegtlin and Hodge, 1949a and Chamutin and Fer r is , 1932). The weight of the animals after TSN was s l igh t ly , but not s igni f icant ly decreased on day 1. On subsequent days, the weight of the TSN group was observed to be constant (Table V). The weight of the sham operated rats fluctuated between 260-270 g during experimentation indicating insignif icant change (p < 0.05)in body weight (Table V). The BUL treated group showed no signif icant weight loss (p < 0.05) (Table V). Biochemistry - The PUN and plasma creatinine levels steadily increased to about 20 and 6 times, respectively, above normal, 6 days after uranyl nitrate treatment (Table VI and VII). The creatinine clearance was signi f icant ly decreased in the test animals (From 1.7±0.4(n=6) to 0.38±0.11(n=6) ml/min for TSN and 2.0±0.5(n=6) to 0.07±0.03 (n=6) ml/min for UN). There was no signif icant differences in the PGOT levels between the control bJO I-I Q OS CCI4 M C P 5Q 401 201 c t £ 101 o 05 +-> C U 5/3 < -a cu % u X w cu o Q D e - M C P CTi C O c t Fig..16. Percent of dose recovered as a) intact drug and b) de-ethylated metabolite after CC14 pretreatment (c, control and t, test). The bars represent the mean ± standard deviation. 64 TABLE V Body Weight Pattern Group Day 0 Day 1 Day 2 Day 4 Day 6 BUL control 247±15 249±7 (6) * (6) BUL 254±17 253±17 (7) (7) TSN control 270±14 270+12 260±15 270±18 260±14 (5) (5) (5) (5) (5) TSN 270±2 255±7 245±3 244±8 243±3 (5) (5) (5) (5) (5) UN control 241+11 244±12 - 243±24 258±21 (8) (8) (8) (8) UN 236±21 212±20 211±20 199+21 190±26 (10) (8) (8) (8) (8) ( )* number of animals • 65 TABLE VI PUN (mg%) Group Day 0 Day 1 Day 2 Day 4 Day 6 BUL control - 16±6 (5)* BUL - 117±11 (7) TSN Control 18±2 18±2 20±2 18±2 16±2 (5) (5) (5) (3) (5) TSN 24±1 ' 60±2 87±17 40±2 78±20 (4) (5) (5) (5) (5) UN Control 15±4 17±4 14±2 16±3 16±2 (7) (7) (3) (6) (7) UN 18±4 51±27 92±40 130±107 300±89 (7) (5) (4) (6) (6) ( )* number of animals 66 TABLE VII Plasma Creatinine Levels (mg/dL) Group Day 0 Day 1 Day 2 Day 4 Day 6 BUL-control - 0 ,54±.23 (5 ) * BUL - 2.8±.9 -(7) TSN Control 0 .53±.08 0.44±.13 0.43±.07 0.6±.3 0.5±.3 (5) (5) (5) (3) (5) TSN 0 .65±.06 1.4±.4 1.6±.4 1.6±.6 1.5±.3 (§) (5) (5) (5) (5) UN-control 0.5±.2 0.38±.l 0.53±.06 0.74±.3 0.51 + .15 (6) (6) (3) (6) (6) UN 0.64±.2 1.2±.7 1.8±2.0 3.3±1.8 5.4±2.5 (7) (5) (4) (6) (6) ( )* number of animals 67 and the renal impaired rats on the day of drug administration (Table VIII). This suggests that signif icant hepatic cel l necrosis was unlikely as a result of UN treatment. The plasma creatinine and PUN levels were s igni f icant ly elevated in the BUL group as compared to the sham operated controls (Table VI and VII). None of the BUL rats lived more than 48 hrs. Similar to the UN group the PGOT levels were not signi f icant ly different from each other (Table VIII). Plasma creatinine and PUN increased the least in the TSN group as compared to the two step sham operated rats (Table VI and VII). In addit ion, creatinine clearance was signi f icant ly reduced for the TSN and UN rats further substantiating the loss of renal function. No apparent difference in cel l necrosis was observed between the TSN and the control group as indicated by the PGOT levels (Table VIII). Plasma Study and Urine Study - F ig . 17 clearly shows that there is a signif icant decrease in the slope of a log plasma concen-tration vs^ time curve indicating that the elimination h a l f - l i f e of MCP is prolonged in a l l the renal fai lure models studied. A l l of the plasma MCP concentration vs_. time curves can be described by a bi-exponential equation with the following general equation: C p = Ae ~ a t + Be ~ e t (12) where C is the plasma concentration, a the distribution rate constant, 3 the rate constant of the terminal elimination phase and A and B are the intercept values for the a and B phases, 68 TABLE VIII ftSQt (IU/L) Group Day 0 Day 1 Day 2 Day 4 Day 6 BUL control 73±20 (5)* BUL 86±11 (7) TSN control 61±4 (5) 69±n (5) 66±14 (5) 52±9 (3) 70±25 (5) TSN 51±10 (4) 67±20 (5) 67±28 (5) 40±2 (4) 45±6 (5) UN control 68±14 (7) 46±14 (7) 51±7 (3) 51±18 (6) 65±26 (7) UN 63±20 (7) 56±18 (5) 90±18 (4) 104±36 (6) 58±10 (6) ( )* number of animals S 110 a U s u C O u u OS S ctj bJD O BUL j i-1 2 3 10 TSN 10 1 2 3 4 "2 Time. xlO min UN Fig. 17. Representative log plasma MCP concentration vs. time curves obtained after a 15 mg/kg dose of MCP was given to the bi lateral ureteral l igated (BUL), 5/6 two step nephrectomized (TSN) and uranyl nitrate (UN) rats ( , control and , test). 70 respectively. Table IX:, X arid XI summarize the pharmacokinetic parameters obtained from the plasma and urine studies. The area under the plasma MCP concentration vs_.time curves (AUC) was signi f icant ly increased when the renal impaired rats were compared to the control rats (Fig.17). The UN pretreated group had the highest increase in AUC(> 3 times) whereas the AUC increased approximately 2 times in the TSN and the BUL groups when compared to the control animals(Fig.l8). This ref lects the extent of renal fa i lure induced by the uranium compound. Similar ly, the total body clearance was signi f icant ly reduced in surgical ly or chemically induced renal impaired rats (Fig.1.9) with the h a l f - l i f e showing a corresponding increase (Fig.20). The volume of distribution calculated by the area method (Vd = v, ) and steady state method (Vd,.) was only s l ight ly area ss reduced when the renal impaired rats were compared to the controls (Fig.21). It has been observed in our laboratory that the 48 hr cumulative urine levels decreased more than 4 times in the UN rats when compared to control (Fig.22a) while the TSN group shows a s l ight reduction in total cumulative MCP excretion (Fig.23a). A similar pattern has been reported in the 48 hr cumulative excretion of DE-MCP in both the UN and TSN groups (Fig,22band 23b). The reduction in the urinary excretion of MCP and i ts de-ethylated metabolite (DE-MCP) does not ref lect the extent of change in the pharmacokinetic parameters obtained from plasma and studies in renal impaired rats as compared to the controls. 71 TABLE IX Bilateral Ureteral Ligation e (min - 1 ) tjjmin) AUC O ->• 0 0 (mcg-min/ml) Cljg (ml/min) V d (L/kg) d(area) V d (L/kg) ss Controls (Sham) 0.0138±0.0022(6): 50±8(6) 250±60(6) 16±5(6) 4 . 3 ± . 7 ( 6 ) 3 . 9 ± 0 . 8 ( 6 ) BUL 0. 00609+0.0023(6) 128±46(6) 600±190(6) 7 ± 3 ( 6 ) 4.4±1.4(6) 4 . 1 + 1 . 3 ( 6 ) ( )* number of animals 72 TABLE X Two Step Nephrectomy (TSN) 6(min" ) ta: (min) AUC o -> 0 0 (mcg-min/ml) C l j B (ml/min) C1R (ml/min) V a r e a ) ^ ) V d (L/kg) ss Controls (Sham) 0.00981±.001(6) 70±8(6) 350±40(6) 11±2(6) 2.0 4 .5±1 .0 (6 ) 3 .9±0.6(6) TSN 0.0058±.0013(7) 130±36(7) 670±100(7) 5.50±8(7) 0.72 4±1(7) 3 .8±0.7(7) ( )* number of animals 73 TABLE XI Uranyl Nitrate Controls (Sham) U N B(min _ 1) t (min) AUG o •> 0 0 (mcg-min/ml) Cljg(ml/min) C1 R (ml/min) (area) (L/kg) V d (L/kg) ss 0.0116±0.003(6)* 60±10(6) 330±50(6) 12±2(6) 2.9 4.0±.8(6) 3.6±0.5(6) 0.00484±0.0014(6) 160±50(6) 1100±50.0(6) 3.4±1.6(6) 0.2 3.2±0.7(6) 3.0±0.7(6) '. ( )* number of animals bJD 8 BUL AUC T S N U N | bJD u £ t—I X U D < -p. C T C T C T Fig. 18. Area under the plasma versus time curves after BUL, TSN and UN treatments (c, control and t, test) . The bars include plus and minus one standard deviation. T O T A L B O D Y C L E A R A N C E B U L T S N U N CQ H U 1 5 1 0 CJ1 C T C T C T Fig. 19. Total body clearance after BUL, TSN and UN(c, control and t , test). The bars include plus and minus one standard deviation. 150 100 50 BUL T V 2 TSN UN ri C T C T C T Fig. 20. Half l i f e obtained after BUL, TSN and UN (c, control and t , t e s t ) . The bars include plus and minus one standard deviation. B U L T S N U N 1.5 1.0 0 .5 C T C T C T 21. Volume of distribution calculated by the area method for BUL, TSN and UN treated animals (c, control and t, test). The bars include plus and minus one standard deviation. M C P 3 C H a.) U N , D e - M C P 154 b.) 2o^ bJD Q o O jo OS •l-> c/) < <U +-> Cb o X U J o Q 9^ 10-5H c t c t g. 22. Percent of dose excreted as a) intact drug and b) metabolite after UN pretreatment (c, control and t , te s t ) . The bars inc l plus and minus one deviation. T S N M C P D e - M C P a. 154 b. 204 bJQ Q CU 05 1CH c t eu I 101 •«-> C U s < •l-> <v u X UJ CU o Q c t Fig. 23. Percent of dose excreted as a) intact drug and b) metabolite after TSN pretreatment (c, control and t, test) . The bars include plus and minus one standard deviation. 80 DISCUSSION 81 GLC Inspection of the chromatogram for the derivatives of MCP and DE-MCP shows that base-line resolution between the derivatives of the MCP and DE-MCP peaks was not attained when a 3% OV-225 column was used. Hiowever, the electronic-integration method provided consistent results,which permitted simultaneous quantitation of both the MCP and DE-MCP peaks. The minimal detectable amount of the HFB derivatives is the low picogram range. It has been observed that the sensi t iv i ty of the HFB derivative of DE-MCP was about twice as sensitive as that of the MCP. Structural Confirmation of Derivatives EI The fragmentation patterns of the derivatized free base and urine extracted from metoclopramide dosed animals were found to be identical by GLC-mass spectrometry, indicating that the derivatives of MCP and DE-MCP were being analyzed from the urine samples. Like i ts procaine analogs (Cowan et a l . , 1976)the derivative of MCP cleaved at the amine bond (m/e 423), as well as the carbonyl-amide bond (m/e 99 and 380). The base peak, m/e 86, was a result of the cleavage at the carbon-carbon bond beta to the amine nitrogen (Scheme II). The mass spectrum of the derivative of DE-MCP is similar to that of MCP except the base peak is at m/e 380 and the most abundant peaks occurred at 396, 409 and 422. The postulated fragmentation pattern is shown in Scheme III. Since the molecular ions of both of the derivatives are not readily ident i f ied , CI-MS was employed to monitor the molecular ion. 82 C H 2 N ( C 2 H 5 ) 2 C H C H J N ^ J H ^ m/e 86 m/e 99 CH3N(C2H5)2 m/e 87 Scheme II. The postulated HFB-derivative fragmentation pattern of the of MCP. 83 Scheme III. The postulated fragmentation pattern of the HFB-derivative of DE-MCP. 84 CI_ Since electron impact mass spectrometry was not conclusive, chemical-ionization mass spectrometry was employed to reveal the molecular ion. From the chemical-ionization mass spectrum (Fig 7). a very intense m/e 496 peak, which corresponded to the (MH)+ peak, for the derivative of MCP was observed. The other two peaks, m/e 478 and 446, were postulated to be (MH - water) + and (MH - water -methyl alcohol) 4", respectively (Scheme II). The base peak was observed to be at m/e 636 which corresponds to the removal of the ethylene group at the terminal nitrogen. The [MH]+ peak at m/e 664 suggested a disubstituted HFB derivative of MCP was formed. (Scheme III). Removal of Excess of Derivatizing Agent The presence of trace quantities of HFBA in the derivative solution has been reported to cause spurious peaks and broad solvent fronts. Therefore, development of a method which would remove the excess reagent without diminishing the response of the derivative was necessary. Two methods were suggested by Walle and Ehrsson (1970): one involved drying of the reaction mixture by a gentle stream of nitrogen after incubation, and the other involved hydrolysis of excess of HFBA with water and neutralization with aqueous ammonia. Tarn and Axelson (1978) have recently reported that the former method decreased the response of the derivative of MCP by at least 67%. Similar results were observed for the derivative of DE-MCP; this 85 was probably due to the vo la t i l i t y of the derivative when the former method was employed. Appl icabi l i ty of the Assay Method A highly sensitive GLC-ECD assay has recently been developed to quantitate minute amount of MCP ( : 20 peg) in a small volume of rat plasma (Tarn and Axelson, 1978). This assay has been modified to simultaneously quantitate;the HFB derivatives of MCP and DE-MCP in rat urine (Tarn and Axelson, 1979). This later method is presently reported in this thesis. Both methods have been respectively shown to suitably quantitate MCP in plasma and MCP and DE-MCP in urine. Therefore, they were adopted for the subsequent pharmacokinetic studies. THE PHARMACOKINETICS OF MCP IN NORMAL RATS The pharmacokinetics of MCP were characterized by studying a wide range of single intravenous doses (1-35 mg/kg). The plasma data revealed that at low doses (<_ 15 mg/kg) the kinetics of MCP could adequately be described by a f i r s t order process (Fig. 8). However, at higher dose levels (_> 25 mg/kg), MCP exhibits dist inct non-linear characterist ics. When the dose was increased from 5 to 15 mg/kg, the average AUC values increased proportionately with dose and the hal f - l ives were insigni f icant ly different from each other (Table I). The total 86 body and renal clearance values were constant over the 5-15 mg/kg dose range (Table I). These are typical characteristics of a l inear system. After a 25 mg/kg dose of MCP, a sl ight decrease in the slope of the terminal 3 phase was observed (Fig. 8). This was accompanied by a dose-dependent increase in h a l f - l i f e and a dis-proportionate increase in AUC. Both the total body and renal clearance declined when compared to the low dose levels (5 and -15 mg/kg);(Table I). Evaluation of the 35 mg/kg plasma curves revealed that the non-linear nature of MCP-kinetics became more prominent. (Fig.9) . The t r i -phasic plasma curves exhibited an intermediate phase with an associated h a l f - l i f e of 104 ± 14 minutes (Fig. 9 !, insert ) . Approximately 400 minutes after MCP administration, the plasma MCP curve after a 35 mg/kg dose demonstrated a decline which was parallel to that seen after 5 and 15 mg/kg of MCP. The nonlinear kinetics of MCP are, perhaps, a result of a transient alteration in drug disposition due to saturation of in t r ins ic mechanisms involved in either absorption, distribution or elimination. In this instance, nonlinear absorption could be ruled out since MCP was given i .v . for the dose dependency study. Inspection of the urinary excretion products should reveal whether or not the mechanisms of MCP dose dependency are caused by changes in the in t r ins ic mechanisms responsible for drug elimination. Saturation of any major metabolic pathways would normally result in an increase in the percentage of intact drug recovered in the urine. Similar ly, saturation of any active renal excretory pathways for 87 intact drug would be expected to increase the metabolite(s) levels accumulated in urine. Since the renal clearance of MCP (2.9 ml/min) is higher than the creatinine clearance in rat (2.0 ml/min, measured in our laboratory) active tubular excretion of MCP is suggested. However, the percentage of dose excreted as intact drug is independent of dose suggesting that saturation of an active excretory process is unlikely (Table II). Similar observation was made with one of the many metabolites (Table III). This suggests that the non-linear nature of MCP kinetics may not be explained by conventional means using Michaelis-Menten kinetics. Since only 30-40% of the dose (comprising intact drug and the de-ethylated metabolite) could be accounted for in the urine, changes in the kinetics of the other metabolic pathways might also explain the observed nonlinearity of MCP at or above 25 mg/kg. Unfortunately the present study lacks suff ic ient information to prove or disprove this hypothesis. Since the total plasma concentration (free and bound MCP) has been measured one might speculate that, at high doses, the protein binding or the distribution equilibrium between the red blood cel ls and plasma was changed,therefore causing the apparent conf l ic t between the plasma and urine data. The blood/plasma ratios (B/P) observed after 15 and 35 mg/kg doses are insigni f icant ly different from each other (p< 0.05) (Figure 24). This rules out the possib i l i ty of a distributional change between the plasma and red c e l l s . The non-linear kinetics of MCP absorption, distr ibution and elimination are not adequately described by conventional Michaelis-Menten 88 3-0 h Q_ O o o < rx < Q o o _1 GO 2 0 h 10 h 15 mg/kg MCP 35 mg/kg MCP Fig. 24. Blood/Plasma ratios after 15 and 35 mg/kg MCP. (5-150 minutes after MCP administration). The bars represent the mean ± standard deviation. 89 theory, since none of these processes showed clear perturbation at high dose levels of MCP. Therefore, a more rigorous assessment of plasma and urine data was required to explain the anomalous observations. Since MCP is extensively metabolized (Teng, et a l . , 1977) i t is reasonable to assume that the non-renal clearance of this drug approximates i ts metabolic clearance. After a low dose of MCP (15 mg/kg) the non-renal plasma clearance of MCP approaches the hepatic blood flow (QH) of 10-15 ml/min in a 200-300 g rat (Roth and Rubin, 1976). Physiologically,the blood clearance rather than the plasma clearance should be used since distribution between red blood cel ls and plasma is very rapid (Rowland, 1972) and the drug within the red cel ls is available for elimination. The mean blood clearance of MCP calculated from the plasma data is 9.3 ml/min. If one assumes that the non-renal clearance is the hepatic clearance then the extraction ratio (E) calculated using the equation developed by Rowland (1973) (equation 1) C1H = Q.E. (1) is rather high (0.52-0.80). Interestingly, the renal clearance of MCP (2.9 ml/min) approaches renal blood flow (4-9 ml/min) (Sophasan et a l . , 1979). Thus, i t could be postulated that the hepatic and renal elimination of MCP should be blood flow-rate dependent. After high doses (>25 mg/kg), MCP may transiently reduce the cardiac output which, tn turn,reduces the blood flow to the eliminating organ(s) thereby, reducing i ts own clearance. Once 90 the blood level of MCP f a l l s to the range where i t may no longer e l i c i t haemodynamic changes, the blood flow should return to normal. It is clearly shown (Fig. 9) that the plasma MCP h a l f - l i f e returns to normal, approximately 7 hrs. after drug administration. This is further substantiated by the i n i t i a l reduction and then a subsequent return to control levels of ICG clearance after a high dose of MCP (Fig.13). The effect of diminished blood flow on the elimination of MCP can be more readily understood after examination of equation 14. Equation 14 shows that the total body clearance (C1 T B) is C 1 T B = ¥ K + %h OA) equal to the renal (Q^E^) and the non-renal (hepatic), (Q^E^) clearance where and are kidney and hepatic blood flow, respectively. E^ and E^ denote the extraction ratios of kidney and the l iver , respect ively . From equation 6, i t can be inferred that i f the blood flow to the eliminating organs is affected to a similar extent and i f the extraction ratios are high in a l l the eliminating organs, a reduction of the average clearance after a high dose of MCP will proportionally reduce the metabolic and excretory pathways. Therefore, the cumulative amount of intact MCP and metabolite excreted into urine should remain unaltered at time in f in i ty . Examination of the cumulative excretion of intact drug and de-ethylated metabolite (Tables II and III) shows identical percentage excretion at a l l dose leve ls , or no dose-dependency, 91 further supporting this hypothesis. This hypothesis is also supported by the decline of the average total body and renal clearance after a high dose (35 mg/kg) of MCP [C1 T B : from 12.0 ± 3.5 ml/min at 5 mg/kg to 6.73 ± 2.1 ml/min at 35 mg/kg], [C1R: from 2.9 ml/min at 5 mg/kg to 1.64 min at 35 mg/kg]. According to the perfusion model (Rowland, 1973) a drug having a high extraction ratio wi l l undergo f i rst -pass metabolism. However, this does not appear to be the case for MCP. The AUC after a 15 mg/kg oral dose of MCP is not s igni f icant ly different from the AUC obtained after an equal i .v : dose (Fig. 12). The urine data supports the plasma data on this point since there is no apparent difference in the percentage excretion of intact drug and measured metabolite after the hepatic and systemic routes of administration (Table II and III). This clearly shows that MCP does not undergo f i rst -pass metabolism at this dose leve l . What remains untested and therefore unproven is whether or not MCP undergoes f i rst -pass metabolism in rat at doses less than 1 mg/kg. In view of the inferred, extraction ratio (0.52-0.80) one might hypothesize that MCP could exhibit a threshold dose for saturation of f i rst-pass metabolism at or lower than 1 mg/kg, similar to that seen for propranolol (Suzuki et a l . , 1972, 1974; Evans et a l . , 1973; Routledge, 1979). Unfortunately, this hypothesis cannot be confirmed or denied by the present assay capabi l i t ies , in the existing animal model, due to analytical detection l imi ts . Another untested hypothesis would be that extrahepatic metabolism may play an important role in MCP elimination. This also could possibly 92 explain why MCP does not undergo f irst-pass metabolism. Cowan et a l . , (1976) have identi f ied a number of metabolites of MCP using rat l iver s l i c e s , in v i t ro. Ingrand et a l . , .(1970) reported that the total radioactivity was concentrated in the l i v e r , gastro-intestinal tract and the brain shortly after an intramuscular 14 and intragastric dose of C-MCP. These observations suggests that the l i ver may be one of the major eliminating (or binding) organs for this drug. The hypothesis that extra-hepatic metabolism may also play a signif icant role in MCP elimination cannot be readily ruled out with presently available information. Preliminary studies in our laboratory indicate that the rat kidney may eliminate MCP by metabolism as well as excretion. However, further study of this observation is required. PHARMACOKINETIC STUDIES IN HEPATIC AND  RENAL IMPAIRED RATS Carbon Tetrachloride. It is well known that the administration of carbon tetrachloride leads to impairment of l iver function, 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 ( Kato et -aTr, 1962 and Dingell and Heimberg, 1968). The toxic effects of CCl^ are not unique to hepatic injury; in fact , mortality can depend on the fate of organs other than the l iver ( D r i l l , 1952, 1958). Gyorgy et a l . , (1946) revealed that CCl* caused 93 more serious renal lesions in male rats than in females. Although the toxic effects of CCl^ are not speci f ic to the l i v e r , the onset of extensive lesions in other organs such as the kidney do not occur until several days after the exposure to this toxin (Smetana, 1939; Moon, 1950). As shown in Table IV,the PUN and plasma creatinine remained unchanged after CCl^ treatment, however, creatinine clearance was signi f icant ly reduced implying renal injury. The plasma MCP data after CCl^ treatment suggest that the decrease in metabolic act iv i t ies due to hepatic injury does decrease the elimination rate of MCP. This is indicated by a greater than two-fold increase in AUC and h a l f - l i f e , and a similar decrease in the total body clearance (Fig. V<5). The percentage of dose excreted in 24 hours has signi f icant ly increased,indicating a reduction in metabolic act iv i ty after CCl^ (Fig. 16a)... It has been postulated that MCP removal by the excretory mechanisms of the kidney involves tubular excretion since renal clearance of MCP (2.9 ml/min) was higher than the glomerular f i l t ra t ion rate (GFR) as measured by creatinine clearance (2.0 ml/min). The renal clearance of MCP after CCl^ was s l ight ly decreased (2.9 ml/min for saline vs_. 2.25 ml/min for CCl^ treated animals). This implies that the reduction of GFR did not markedly reduce the overall elimination of MCP by either GFR or tubular excretion, or both. We were unable to account for a l l the administered dose as intact drug in the urine despite extensive damage of the l iver . This observation may be due to the incomplete 94 destruction of the microsomal enzymes in this organ. Very interestingly, the percentage of dose excreted as the de-ethylated metabolite of MCP was not altered after CCl^ treatment (Fig. 16b). If the l iver were to be solely responsible for this oxidative metabolic pathway and i f the amount of cytochrome P-450 were reduced in the l i ver by this treatment (Sasame et a l . , 1968), one would expect the level of the de-ethylated metabolite produced (DE-MCP) to decrease. Therefore, this suggests that extra-hepatic metabolism may, in part, be responsible for the formation of this metabolite. Renal Failure - The three renal fai lure models employed have been shown to produce renal insufficiency as indicated by the signif icant increase in the PUN and plasma creatinine levels (Tables VI and VII). The decrease in creatinine clearance in the animals also signif ied the loss of renal function. As indicated by the PGOT levels , l iver cel l necrosis was apparently minimal in the BUL and TSN models, while the UN animals showed a progressive increase in PGOT up to day 4 and a return to control levels on the day of the experiment. The body weight of the UN treated animals decreased gradually whereas a signif icant weight loss has been observed for TSN rats in the f i r s t two days, (Table V). This is probably due to the loss of appetite and muscle wasting resulting from renal insufficiency (Chanutin and Ferr is , 1932;Voegtlin and Hodge, 1949a). The weight of the BUL rats did not change signi f icant ly 95 on the f i r s t day. However, these animals did not l ive long enough to show any signif icant changes in body weight. The degree of renal impairment as measured by creatinine clearance, PUN and plasma creatinine was the highest with the UN treatment followed by the BUL and TSN. As previously reported (Voegtlin and Hodge, 1949b and Giacomini, 1979), the time course to achieve the highest degree of renal impairment after a single dose of UN was around 4-6 days. This observation has been confirmed by using PUN, plasma creatinine and creatinine clearance as the renal function indicators (Table VI and VII). The PUN, plasma creatinine and creatinine clearance have indicated that the condition of renal dysfunction has been stable during the six day test period for the 5/6 TSN group (Table VI and VII). Signif icant reduction of the renal function after TSN was achieved within the f i r s t day. With the BUL rats, the degree of renal impairment developed rapidly and no animal was observed to l ive more than 48 hrs after the surgery confirming the results reported by Giacomini (1979). Based on the biochemical information col lected, i t was decided to administer MCP on the day when renal impairment was observed to be s igni f icant . Therefore, the drug administration time for the UN rats was on the 6th day whereas MCP was administered to the BUL and TSN animals on the f i r s t day after the surgery was performed. The plasma levels achieved were signi f icant ly higher in the 96 renal impaired rats produced by a l l the treatment when compared to the controls (Fig. 17). Calculation of the pharmacokinetic parameters for MCP in renal fai lure shows that the h a l f - l i f e and AUC values were increased by at least 2-fold while the total body clearance and renal clearance were reduced by the same order of magnitude (Tables IX, X and XI). Although there was a 4-fold reduction in the urinary excretion of the intact drug into the urine after UN treatment (Fig.22) and to a lesser extent after TSN (Fig. 23) treatment, this reduction could not account for such a dramatic alteration in the plasma kinetics of MCP since the percentage of intact drug recovered in the urine of the control rats ranged from 20-25%. A complete shutdown of the excretory function of the kidney, assuming the function of the other eliminating organs such as l iver is unchanged, would be expected to cause no more than 20-30% change in the plasma pharmacokinetic parameters. It is now known that the hepatic elimination of some drugs by acetylation, reduction and,in some instances*ester hydrolysis is affected by the state of renal insufficiency (Reidenburg, 1978). Hogan et a l . , (1979) have shown that the important oxidative pathways for drug transformation in the l i v e r , e.g. the hepatic microsomal N-demethylation of aminopyrine and ethyl morphine, were s igni f icant ly reduced in rats with renal fa i lure . This may be one of the causes for the signif icant reduction of the de-ethylated metabolite (DE-MCP) levels in the urine. However, this hypothesis seems unlikely because results of the present hepatic fai lure study using carbon tetrachloride as an hepatotoxin showed that the level of DE-MCP in urine did not 97 drop s igni f icant ly . This would seem to indicate that reduction of hepatic function does not have an effect on the DE-MCP metabolite levels in urine. The reduction of this metabolite level (DE-MCP) may result from the decrease in the excretory function of the kidney tissues and/or reduction of the formation of the metabolite in the kidney during renal insuff iciency. The observation of unexpectedly large alteration in the kinetic parameters in renal fai lure and the insignif icant change in the urine level of DE-MCP in hepatic injury leadsus to postulate that extrahepatic metabolism may play an important role in MCP removal in the rat. A negative correlation was observed when the C 1 T B was plotted against the plasma creatinine and PUN levels (r =0.76, r2=0.71) (Fig. 25 and 26). A positive correlation (r2>0.97) was observed when the average total body, renal and nonrenal clearance were individually plotted against the creatinine clearance (Fig. 27). These findings clearly indicate that the metabolic removal of MCP is dependent upon the renal function. Together with the observations in hepatic dysfunction these finding further substantiate the hypotheses of extrahepatic metabolism for MCP. Further experimentation,such as isolated kidney and l iver perfusion studies,are required to confirm the present hypotheses. If these observations are further confirmed, one may explain in part why MCP does not undergo f i rst -pass metabolism after a hepato-portal route of administration (oral or i .p. ) in the rat. Since the same order of magnitude of changes in the kinetic parameters of MCP was observed both in man (Bateman, 1980b) and in the rat (the present study), the underlying mechanism of this MCP, C 1 T R (ml/min) Fig. 25. A Plot of plasma creatinine vs. the total body clearance of MCP(B=UN, T =TSN and 0 =BUL) (r 2 = 0.76). 500 =0 4001 bJD s 300 z P-i 200 100 o o 0 • A • A 1± *0 • A. 0 5 10 MCP, C1 T B ml/min 15 Fig. 26. A plot of PUN levels vs. the total body clearance of ( B=UN, T=TSNand 0=BUL) (r2=0.71). 15 Fig. 27. A plot of the average total body ( • , r =0.99), non-renal ( B,r2=.98) and renal ( • ,r2=.97) clearance of MCP j / s ^ the average creatinine clearance. 101 unexpected observation in man may prove to be somewhat s imi lar . Investigation of MCP pharmacokinetics in humans is currently underway. Cone!usions The GLC assay has been shown to be highly sensitive and suitable for the analysis of MCP and DE-MCP in rat urine. The disposition of MCP in normal rats has been found to be sensitive to perfusion changes and dose dependent. This unique characterist ic was proposed to be due to a sel f reduction in the perfusion to the eliminating organs after a high dose of MCP (*25 mg/kg). Although the extraction ratio of MCP was postulated to be high, presystemic clearance after hepato-portal administration ( i . p . and oral) was not s igni f icant . Studies using rats with renal and hepatic dysfunction has indicated delay of MCP elimination. 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