Inhibition of drug metabolism by acute ethanol intoxication

Inhibition of Drug Metabolism by Acute Ethanol Intoxication A Hepatic Microsomal Mechanism

EMANUEL RUBIN, M.D. HENRY GANG, Ph.D. PREM S. MISRA, M.D. CHARLES S. LIEBER, M.D. New York, New York

From the Departments of Pathology and Medicine, Mount Sinai School of Medicine of the City University of New York and Section ~f Liver Disease and Nutrition, Bronx Veterans Administration Hospital, New York, New York. This study was supported by U.S. Public Health Service Grants MH 15558, AM 12511 and FR 71. Requests for reprints should be addressed to Dr. Emanuel Rubin, Mount Sinai School of Medicine of the City University of New York, 100th Street and Fifth Avenue, New York, New York 10029. Manuscript received April 20, 1970.

Volume 49, December 1970

The effects of acute ethanol intoxication on drug metabolism were studied in man and rats. Ethanol ingestion significantly retarded the disappearance of pentobarbital and meprobamate from the blood in human volunteer subjects and rats. This was not due to altered compartmentalization, since total body dis. appearance of pentobarbital in rats was also delayed. In vitro, ethanol inhibited the metabolism of meprobamate by rat liver slices, which indicates a direct effect on hepatic drug metabolism. This effect was shown to involve inhibition of hepatic microsomal drug-metabolizing enzymes by ethanol, which was competitive or partially competitive. In addition ethanol decreased the activity of cytochrome P450 reductase, a rate limiting step in the electron transport chain of microsomal drug detoxification. The inhibition of drug metabolism demonstrated in this study may be due to the binding of ethanol to hepatic microsomes and its oxidation at this site. The effects of ethanol ingestion on drug metabolism appear paradoxic. Chronic alcoholics, when not under the direct influence of alcohol, '=' are unusually resistant to the action of drugs such as barbiturates. This greater tolerance has generally been attributed solely to central nervous system adaptation, but more recent studies have suggested that although late tolerance for barbiturates after chronic ethanol ingestion may be due to adaptation of the central nervous system, the early tolerance is probably secondary to accelerated drug metabolism [1]. This is consistent with our demonstration that, in addition to its hepatoxic effects [2-4], chronic ingestion of ethanol induces hepatic microsomal drug-metabolizing enzymes in both rats [5] and man [6], with a concomitant increase in drug clearance from the b~ood [7]. In contrast to chronic ingestion, acute ethanol intoxication leads to increased sensitivity to the effects of barbiturates and tranquilizers [8]; the potentially dangerous consequences of this combination have been extensively documented [9]. Many have previously attributed this potentiation to additive effects of ethanol and drugs on the central nervous system [10]. However, since drugs oxidized by hepatic The terms ethanol and alcohol are used interchangeably in this paper.

801

INHIBITION OF DRUG METABOLISM BY ETHANOL~RUBIN ET AL.

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microsomes inhibit the metabolism of other drugs [11], and since ethanol shares many properties with other drugs [12], including microsomal oxidation [13], we investigated the effects of ethanol on the activities of hepatic microsomal drug-metabolizing enzymes in vitro. We also assessed its action on the rate of drug metabolism in vivo, both in rats and human volunteer subjects. MATERIALS AND M E T H O D S Effects of Ethanol on Drug Metabolism in Vitro. To obtain hepatic microsomes, livers of 200 gm male Sprague-Dawley rats were homogenized in three volumes of 0.25 M sucrose and centrifuged at 9,000 x g for twenty minutes. The supernatant was (~entrifuged at 105,000 x g for one hour. We then measured the activities of aniline [14], pentobarbital [15] and benzpyrene [16] hydroxylases, and aminopyrene [17] and ethylmorphine [17] demethylase activities, with and without ethanol in the incubating medium. Kinetics of these enzymatic reactions were determined by varying the concentrations of substrate and of ethanol. Microsomal NADPH-cytochrome C reductase [18] and cytochrome P450 reductase [18] activities were

802

determined in microsomes which had been washed in 1.15 per cent potassium chloride to remove hemoglobin. These activities were measured in the presence and absence of ethanol. Rat liver slices were prepared and incubated as described previously [19]. Each incubation flask contained approximately 500 mg of liver tissue in 6 ml of Krebs phosphate buffer, with 0.3 mM meprobamate, including 1 /~c of 14C-meprobamate per flask. The incubations were carried out at 37~ for 120 minutes. Parallel incubations contained 10, 50 and 100 mM ethanol. Polar metabolites of meprobamate were extracted by the method of Hoffman and Ludwig [20], and radioactivity was determined in a liquid scintillation counter. Effects of Ethanol on Drug Metabolism in Rats. Six Sprague-Dawley male rats, weighing about 150 gm, were given ethanol, 5 gm/kg as a 50 per cent solution, by gastric intubation. Six control animals were given an isocaloric amount of glucose. Two hours later, control and ethanol treated animals were given intraperitoneal injections of 14C-pentobarbital, 40 mg/kg. Serial blood samples were obtained from the tail vein for 100 minutes. For determination of plasma pentobarbital concentration, 0.1 cc of blood was added to 1 ml of acetate

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buffer, pH 5.0, containing 0.1 gm of sodium chloride. Unmetabolized pentobarbital was extracted twice with 10 volumes of petroleum ether, containing 1.5 per cent isoamyl alcohol, which was evaporated to dryness under nitrogen. The radioactivity of the residue was counted in a liquid scintillation counter. To measure the effect of ethanol on total body metabolism of pentobarbital, rats were treated with ethanol and pentobarbital as in the previous experiment, and killed in groups of five at intervals of 25, 50, 75 and 100 minutes after injection of pentobarbital. The entire rat was immediately homogenized in 500 ml of acetate buffer, pH 5.0, at O~ in a large Waring blender. The homogenate was filtered through several layers of gauze, an aliquot of 5 ml taken, 0.5 gm sodium chloride added, and unmetabolized pentobarbital extracted and counted by the method described.

Effects of Ethanol on Drug Metabolism in Human Volunteer Subjects, F o u r nonalcoholic volunteer subjects, three men and one woman, ranging in age from twenty-five to thirty-eight years, were studied in the Clinical Research Center of The Mount Sinai Hospital, after informed consent was obtained. After an overnight fast, they were given 6 mg/kg ~4C-pentobarbital orally. Sixteen hours later each was fed 95 per cent ethanol, diluted 1:5 with fruit juice, in a dose of 1 gm/kg. This was followed by 24 gm ethanol every two hours until thirty-four to forty hours after pentobarbital administration. Serial blood samples were analyzed for pentobarbital content by the method described. Blood alcohol concentration was determined by the method of Bonnichsen [21]. One week later the same experiments were performed using meprobamate, 12 to 15 mg/kg, orally. In these studies ethanol ingestion was begun fourteen hours after administration of the drug and extended to twentyfour hours. Plasma meprobamate concentration was measured by the method of Hoffman and Ludwig [20]. During the time periods chosen for studies of pentobarbital and meprobamate metabolism in volunteer subjects, the clearance of these drugs from the blood is semilogarithmic [7]. RESULTS In vitro, ethanol inhibited the activities of aniline and pentobarbital hydroxylases, and the demethylation of aminopyrene and ethylmorphine (Figure 1). Benzpyrene hydroxylase (0.06 mM benzpyrene) was inhibited 27 per cent by 50 mM ethanol. The inhibition of aniline hydroxylase and aminopyrene demethylase was competitive, whereas that of pentobarbital hydroxylase and ethylmorphine demethylase was of a mixed type. Aniline hydroxylase was far more sensitive to inhibition by ethanol than the other systems studied, the Ki for ethanol in this system being 3.0 mM. The Ki for

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ethanol was 76 mM for aminopyrene demethylase, 90 mM for pentobarbital hydroxylase and about 100 mM for ethylmorphine demethylase. Cytochrome P450 reductase activity was inhibited 35 per cent by ethanol. Using a suspension of 1 mg microsomal protein/ml, the control initial velocity was 17.6 nanomoles (nM) cytochrome P450 reduced/minute, compared to 11.3 nM in the presence of 100 mM ethanol. The activity of NADPH-cytochrome C reductase was unaffected by ethanol. Ethanol conspicuously reduced the rate of meprobamate metabolism by rat liver slices; 50 mM ethanol about 40 per cent and 100 mM ethanol about 60 per cent (Figure 2). Acute ethanol administration to rats retarded pentobarbital disappearance from the blood (Figure 3). The plasma half-life of pentobarbital in ethanol-treated rats was 150 minutes, compared to seventy minutes in glucose-treated control animals. Similarly, in rats, acute ethanol administration strikingly reduced the rate of total body disappearance of pentobarbital (Figure 4). In human volunteer subjects, acute ethanol in. gestion, which resulted in peak blood ethanol levels of 50 to 120 m g / l O 0 ml (except for one value at 168 m g / l O 0 ml), slowed the rate of disappearance of drugs from the blood. The plasma half-life of pentobarbital was approximately double

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INHIBITION OF DRUG METABOLISM BY E T H A N O L ~ RUBIN ET AL.

the control value (Figure 5), whereas that of meprobamate was augmented two to fivefold (Figure 6) (p < 0.02).

COMMENTS The data presented here demonstrate that ethanol inhibits drug metabolism at all levels studied. It inhibits the activities of drug metabotizing systems in microsomes, a subcellular level. In turn this is reflected in a reduced rate of drug biotransformation in liver slices, a cellular level. Finally, acute ethanol ingestion results in reduced clearance of drugs from the blood and total body. Thus, in addition to additive effects of ethanol and drugs on the central nervous system [10], delayed hepatic drug metabolism may also play a role in the increased susceptibility of inebriated persons to sedatives and tranquilizers. The mechanism of the inhibition of drug metabolizing enzymes by ethanol is probably related to the interaction of ethanol and hepatic microsomes. The effects of chronic ethanol administration on hepatic microsomes are in many respects similar to those of drugs, exemplified by phenobarbital [12]. Chronic administration of both leads to increases in the liver of endoplasmic reticulum [2,5,11,22,23], activities of drug-metabolizing enzymes [2,6,11], cytochrome P450 [11,24] and cholesterol biosynthesis [25,26]. These effects are generally produced by chronic administration of drugs which are oxidized by hepatic microsomes [11]. It is therefore reasonable to assume that the effects of chronic ethanol ingestion may be related to the hepatic microsomal ethanol-oxidizing system, which has characteristics similar to drugmetabolizing systems [13]. Microsomal metabolism of drugs can be inhibited in vitro and in vivo by other drugs [11,27], presumably owing to competition for oxidation. The observation that inhibition of drug metabolism by ethanol is either completely or partially competitive suggests that a similar mechanism may be involved. Moreover, ethanol binds to hepatic microsomes [28], which is a feature displayed by many drugs [29]. Again, as in the case of drugs, the magnitude of the binding is increased by chronic ethanol consumption [28] and is inhibited by prior exposure of microsomes to other drugs [30]. Furthermore, in vitro, ethanol inhibits to some extent the binding of aniline to cytochrome P450 [28]. It is therefore possible that ethanol-induced inhibition of drug metabolism may involve, at least to some extent, interference with the microsomal binding sites of

Volume 49, December 1970

drugs. The flow of electrons for drug oxidation from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the hemoprotein cytochrome P450 is mediated, at least in one step, by a flavoprotein. Ethanol has no effect on the first step, that is transfer of electrons to the flavoprotein, as shown by the unimpaired activity of NADPH-cytochrome C reductase. However, it does inhibit the reduction of cytochrome P450, which is generally considered the rate limiting step in drug oxidation [18]. This involves the second step, that is, transfer of electrons from the flavoprotein to the hemoprotein. Thus another possible mechanism for inhibition of drug metabolism by ethanol is interference with electron transfer. The Ki for the inhibition of aniline hydroxylase is much smaller than that for the other systems, i.e., ethanol is a far more potent inhibitor of the former. This is perhaps related to the fact that aniline binds to microsomes with a type 2 spectral change [29], which is of the same general configuration as the modified type 2 spectrum of ethanol binding [28]. The other drugs studied, whose metabolism is not as strongly inhibited by ethanol as that of aniline, bind with a different spectral change, the so-called type 1 spectrum [29]. Although ethanol interferes with the binding of aniline, a type 2 binder, it has no effect on the type 1 binding of hexobarbital [28]. Conversely, type 2 binding substances, such as pyrazole or aniline, inhibit ethanol binding to microsomes [30], whereas type i binders, such as hexobarbital (unpublished data), have no effect on ethanol binding. Moreover, type 2 binding drugs inhibit the reduction of cytochrome P450, whereas type 1 binding drugs accelerate this electron transfer [18]. These findings, together with the fact that chronic ethanol ingestion induces aniline hydroxylase far more than other enzymes [5], suggest that the interaction of ethanol with microsomes is closest to that of drugs which display a type 2 binding spectrum. In conclusion, ethanol inhibits hepatic microsomal drug-metabolizing enzymes, which in turn results in decreased hepatic drug metabolism; as a consequence, the clearance of drugs from the body is retarded. This explains, in part, the heightened sensitivity of inebriated persons to the effects of certain drugs. The mechanism of ethanol-induced inhibition of drug-metabolizing enzymes may be related to the interaction of ethanol and hepatic microsomes, exemplified by microsomal ethanol oxidation and the binding of ethanol to microsomal hemoprotein.

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INHIBITION OF DRUG METABOLISM BY ETHANOL--RUBIN ET AL.

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distribution of the components of mixed-function oxidase between the rough and the smooth endoplasmatic reticulum of liver cells, eiochem J 110: 407, 1968. Gigon PL, Gram TE, Gillette JR: Studies on the rate of reduction of hepatic microsomal cytochrome P450 by reduced nicotinamide adenine dinucleotide phosphate: effect of drug substrates. Molec Pharmaco] 5: 109, 1969. Lieber CS, Schmid R: The effect of ethanol on fatty acid metabolism: stimulation of hepatic fatty acid synthesis in vitro. J Clin Invest 40: 394, 1961. Hoffman AJ, Ludwig BJ: An improved colorimetric method for the determination of meprobamate in biological fluids. J Amer Pharm Ass 48: 740, 1959. Bonnichsen R: Ethanol determination with alcohol dehydrogenase and DPN, Methods of Enzymatic Analysis (Bergmeyer HU, ed), New York Academic Press, 1963, 285. Rubin E, Lieber CS: Early fine structural changes in the human liver induced by alcohol. Gastroenterology 52: 1, 1967. Iseri OA, Lieber CS, Gottlieb LS: The ultrastructure of fatty liver induced by prolonged ethanol ingestion. Amer J Path 48: 535, 1966. Rubin E, Bacchin P, Gang H, Lieber CS: Induction and inhibition of microsomal and mitochondrial enzymes by ethanol. Lab Invest 22: 569, 1970. Lieber CS, DeCarli LM: Effects of ethanol on cholesterol metabolism. Clin Res 12: 274, 1964. Jones AL, Armstrong DT: Increased cholesterol biosynthesis following phenobarbital induced hypertrophy of agranular endoplasmic reticulurn in liver. Proc Soc Exp Biol Med 119: 1136, 1965. Stitzel RE, Tephly TR, Mannering GJ: Inhibition of drug metabolism. VI. Inhibition of hexobarbital metabolism in the isolated perfused liver of the rat. Molec Pharmacol 4: 15, 1968. Rubin E, Lieber CS, Alvares A, Levin W, Kuntzman R: Interaction of ethanol and microsomal heine protein. Its effect on human drug metabolism. Amer J Path 59: 55a, 1970. Schenkman JB, Remmer H, Estabrook RW: Special studies of drug interaction with hepatic microsomal cytochrome. Molec Pharmacol 3: 113, 1967. Rubin E, Gang H, Lieber CS: Interaction of pyrazole and hepatic microsomes. Fed Proc 29: 275, 1970.

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