Cyclosporin metabolism by human gastrointestinal mucosal microsomes.

The in vitro metabolism of the immunosuppressant cyclosporin (CsA) by human gastrointestinal mucosal microsomes has been studied. Macroscopically normal intestinal (n = 4) and liver (n = 2) tissue was obtained from kidney transplant donors, and microsomes prepared. Intestinal metabolism was most extensive with duodenal protein (15% conversion to metabolites M1/M17 after 2 h incubation at 37 degrees C; metabolite measurement by h.p.l.c). Western blotting confirmed the presence of P-4503A (enzyme subfamily responsible for CsA metabolism) in duodenum and ileum tissue, but not in colon tissue. The results of this study indicate that the gut wall may play a role in the first-pass metabolism of CsA, and could therefore be a contributory factor to the highly variable oral bioavailability of CsA.     

In-vitro metabolic studies of tacrolimus using precision-cut rat and human liver slices

The objective of this study was to investigate the in-vitro metabolism of tacrolimus in liver slices from rats and humans. [¹⁴C]Tacrolimus (2 or 20 μM) was incubated with precision-cut human and rat liver slices in 12-well plates for up to 12 h. Concentrations of tacrolimus and metabolites were determined by high-performance liquid chromatography (HPLC) radiochromatography. The 13-O-demethylated tacrolimus metabolite (M-I) was the major oxidative metabolite in both rat and human liver slices. The other primary metabolites of tacrolimus (M-II, M-III, and M-IV) were not seen in either species. Unidentified peaks, which eluted early in the HPLC system, were probably due to secondary or conjugated metabolites. The eluate had no pharmacological activity. The finding that M-I was the major tacrolimus metabolite in both human and rat liver slice preparations is consistent with previous studies of rat and human liver microsomes.     

In vitro glucuronidation of D-23129, a new anticonvulsant,by human liver microsomes and liver slices

1. The metabolic profile of D-23129, a new anticonvulsant agent, was studied in vitro using human liver microsomes and fresh liver slices. 2. Oxidative metabolism appeared to be minimal with D-23129. The percent mean total radioactivity not associated with the parent compound recovered from oxidative metabolism studies from three individual liver donors was 0 7% 0 6 SD and was not significantly different from \ [C]-D-23129 incubated with heat inactivated microsomes, mean 0 5% 0 4 SD. 3. Phase II conjugation dominated the metabolism of D-23129 producing two distinct N -glucuronides as the primary metabolites. These metabolites were identified by electrospray ionization LC MS. 4. The apparent K for one of the glucuronide metabolites was determined in human m liver microsome preparations from two individual liver donors to be 131 and 264 mu M respectively. V determined for the same microsomal preparations yielded 48 9 and max 59 9 pmol?min?mg protein.     

Drug-metabolizing activity of human and rat liver,lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo V(max) in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.     

Characterization of rat small intestinal and colon precision-cut slices as an in vitro system for drug metabolism and induction studies.

The aim of this study was to characterize rat small intestinal and colon tissue slices as a tool to study intestinal metabolism and to investigate gradients of drug metabolism along the intestinal tract as well as drug-induced inhibition and induction of biotransformation. Tissue morphology and the intestinal mucus layer remained intact in small intestinal and colon slices during 3 h of incubation, while alkaline phosphatase was retained and the rate of metabolism of three model compounds (7-hydroxycoumarin, 7-ethoxycoumarin, and testosterone) appeared constant. Phase I and phase II metabolic gradients, decreasing from stomach toward colon were shown to be clearly different for the model compounds used. Furthermore, the observed slice activities were similar or even higher compared with the literature data concerning metabolism of in vitro intestinal systems. Preincubation with beta-naphthoflavone for 24 h induced the O-deethylation of 7-ethoxycoumarin from nearly undetectable to 140 pmol/min/mg protein in small intestine (fresh slices, 43 pmol/min/mg protein) and to 100 pmol/min/mg protein in colon slices (fresh slices, undetectable). Ketoconazole inhibited metabolism of testosterone by 40% and that of 7-ethoxycoumarin by 100%. In conclusion, we showed that the intestinal slice model is an excellent model to study drug metabolism in the intestine in vitro, since we found that the viability parameters remain constant and the measured enzyme activities are relevant, sensitive to inhibitors, and inducible. Therefore, it is a promising tool to study intestinal drug metabolism in human intestine in vitro in the future.     

Drug-metabolizing activity of human and rat liver,lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo V_max in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.     

Metabolism of coumarin and 7-ethoxycoumarin by rat,mouse, guinea pig,Cynomolgus monkey and human precision-cut liver slices

1. The metabolism of 50 μM 7-ethoxycoumarin and 50 μM [3-¹⁴C]coumarin has been studied in precision-cut liver slices from the male Sprague-Dawley rat, female DBA/2 mouse, male Dunkin-Hartley guinea pig, male Cynomolgus monkey and man.

2. In liver slices from all five species 7-ethoxycoumarin was metabolized to 7-hydroxycoumarin (7-HC), which was extensively conjugated with D-glucuronic acid and sulphate. In rat and mouse, 7-HC was preferentially conjugated with sulphate, whereas rates of glucuronidation and sulphation were similar in the other three species.

3. [3-¹⁴C]coumarin was metabolized by liver slices from all five species to various polar products and to metabolite(s) that bound covalently to liver slice proteins. In Cynomolgus monkey and both human subjects studied, 7-HC was the major metabolite that was conjugated with D-glucuronic acid and sulphate, whereas in rat the major metabolites were products of the 3-hydroxylation pathway and unknown metabolites. Major metabolites in mouse liver slices were 7-HC, 3-hydroxylation pathway products and unknown metabolites, and in guinea pig liver slices, 7-HC and unknown metabolites.

4. The metabolism of 7-ethoxycoumarin to free and conjugated 7-HC and [3-¹⁴C] coumarin to total polar products was greater in liver slices from mouse and Cynomolgus monkey than the other three species.

5. With liver slices from all five species there appeared to be little difference in the extent of metabolism of 7-ethoxycoumarin and [3-¹⁴C]coumarin to various products in either a complex tissue culture medium (RPMI 1640 plus foetal calf serum) or a simple balanced salt solution (Earle's balanced salt solution).

6. These results demonstrate that precision-cut liver slices are avaluable in vitro model system for investigating species differences in xenobiotic metabolism. Generally, the observed species differences in coumarin metabolism in vitro agree well with available in vivo data.     

Cyclosporin metabolism by the gastrointestinal mucosa.

The intestinal mucosal metabolism of the immunosuppressant cyclosporin (CsA) has been studied in vitro using the Ussing chamber technique. Histologically normal colon was obtained from six patients undergoing resections. The mucosal sheets were mounted between two perspex chambers. Three hours after addition of [3H]-CsA (0.2 microCi; 10 microM) to the mucosal chamber, more than 90% of the radioactivity was present in that chamber. Metabolite analysis, by high performance liquid chromatography, indicated that 77.6 +/- 9.2% (mean +/- s.d.) of the drug present was CsA, 9.9 +/- 4.4% and 8.7 +/- 4.7% were the oxidative metabolites M17 and M21 respectively (metabolites identified by co-chromatography with authentic standards). Total metabolite production in tissues from the six individuals was variable (10.1-30.6% at 3 h) and increased over the time period of the study. A different pattern of metabolism was obtained from a single sample of gastric mucosa. More than 20% of CsA was metabolised although neither M17 nor M21 were detected. The results of this study suggest that the gut wall is involved in the first pass metabolism of CsA in vivo and that this could be a contributory factor to the poor systemic availability of CsA seen in some patients.     

Comparative studies of the in vitro metabolism and covalent binding of 14C-benzene by liver slices and microsomal fraction of mouse, rat, and human

Metabolism of benzene by the liver has been suggested to play an important role in the hepatotoxicity of benzene. The role of the different benzene metabolites and the causes of species differences in benzene hepatotoxicity are, however, not known. The metabolism and covalent binding of 14C-benzene by liver microsomal fractions and liver slices from rat, mouse, and human subjects have been studied. Rat microsomal fraction formed phenol at a rate of 0.32 nmol/min/mg of protein; mouse microsomal fraction formed phenol at 0.64 nmol/min/mg and hydroquinone at 0.03 nmol/min/mg; and human microsomal fraction formed phenol at 0.46 nmol/min/mg and hydroquinone at 0.07 nmol/min/mg. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver microsomal protein was 29, 113, and 169 pmol/min/mg of protein, respectively. The rates of metabolite formation from benzene by liver slices in nmol/min/g of tissue were: rat, phenol 0.15, hydroquinone 0.26, and phenylsulfate 1.22; mouse: phenol 0.13, hydroquinone 0.29, phenylsulfate 1.37, and phenylglucuronide 1.34; and human: phenol 0.16, hydroquinone 0.27, phenylsulfate 0.83, and phenylglucuronide 0.52. trans,trans-Muconic acid formation was not detected with liver slices of any species. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver slices was 8.2, 79.7, and 27.3 pmol/min/g liver, respectively. There was no correlation between ascorbic acid levels in the human liver slices and covalent binding of 14C-benzene metabolites. The results show that phenol and hydroquinone found in extrahepatic tissues, including bone marrow, of animals exposed to benzene could originate from the liver. There was no evidence for the release of highly reactive benzene metabolites such as trans,trans-muconaldehyde or p-benzoquinone from liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The metabolism of DuP 753, a nonpeptide angiotensin II receptor antagonist, by rat, monkey, and human liver slices.

The in vitro metabolism of DuP 753, a novel nonpeptide angiotensin II receptor antagonist, has been investigated in incubations with liver slice preparations from rats, monkeys and humans. Metabolites were identified by HPLC/MS, FAB/MS, Cl/MS, and/or 1H NMR. In the rat, the primary route of metabolism was oxidative, leading to either monohydroxylated or oxidized (carboxylic acid) metabolites, whereas in monkeys, glucuronidation of the tetrazole moiety predominated. An equal mixture of both oxidized and glucuronic acid-conjugated metabolites was isolated from incubations with human liver slices. All metabolites were tested in an in vitro assay to determine their activity as angiotensin II receptor antagonists. The monohydroxylated products and glucuronic acid conjugates were determined to be much less active than DuP 753. Biotransformation to the carboxylic acid, however, was shown to dramatically increase the activity of this agent. The in vivo duration of action of DuP 753 has been observed to be much longer in the rat than in the monkey. This may be explained, at least in part, by these in vitro metabolism studies. The predominance of glucuronidation observed in incubations with monkey liver slices would yield metabolites with diminished activity and might be expected to shorten the in vivo duration of DuP 753 in that species. The oxidative conversion to the carboxylic acid metabolite, along with the low level of glucuronidation observed in incubations with rat liver slices, may be responsible for the prolonged duration observed in vivo in the rat.     

Metabolism of 4,4'-methylenebis(2-chloroaniline) by canine liver and kidney slices

4,4'-Methylenebis(2-chloroaniline) (MBOCA) metabolism in canine liver and kidney slices was investigated using HPLC to separate the metabolites. Liver slices metabolized 5-10% of the 14C-MBOCA in 60 min and produced seven metabolites resolved by HPLC. The major metabolite, representing approximately 80% of the metabolism, was 2-amino-5-[(4-amino-3-chlorophenyl)methyl]-3-chlorophenyl hydrogen sulfate, previously identified as the major urinary metabolite in dogs. An MBOCA-glucoside was identified by mild acid hydrolysis, which released MBOCA and glucose. An O-glucuronide was characterized as labile to beta-glucuronidase, stabile to arylsulfatase, and mild acid. It was formed in increased amounts when 2,6-dichloro-4-nitrophenol (DCNP) was added to the incubation. Two other glucuronide metabolites were labile to mild acid and beta-glucuronidase, stabile to arylsulfatase, and were formed in decreased amounts in the presence of D-(+)-galactosamine (D-gal) and p-nitrophenyl sulfate (PNPS). Renal cortical slices metabolized 3-5% of the 14C-MBOCA in 90 min, producing six metabolites. Based on retention time and lability to hydrolysis, three of these, the MBOCA-glucoside, a glucuronide, and 2-amino-5-[(4-amino-3-chlorophenyl)methyl]-3-chlorophenyl hydrogen sulfate were also found as kidney metabolites. One additional sulfur-containing metabolite was labile to mild acid and arylsulfatase. The major kidney metabolite represented 25-40% of the metabolism and was unaffected by mild acid, beta-glucuronidase, arylsulfatase, DCNP, and D-gal. Covalent binding in liver slices was 20-27 pmol/mg of wet weight/60 min and in kidney was 9-13 pmol/mg of wet weight/90 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biotransformation of halothane in guinea pig liver slices

The biotransformation of halothane was studied using liver slices. Precision-cut Hartley male guinea pig liver slices (1 cm diameter; 250-300 microns thick) were incubated in sealed roller vials containing supplemented Krebs-Henseleit buffer at 37 degrees C under different O2 tensions (2.5, 21, and 95%). After a 1-hr preincubation, halothane was vaporized in the vial producing a 1.9 mM medium concentration. Halothane metabolites (Br-, trifluoroacetic acid, F-) were measured at 2, 4, and 6 hr. Viability of the incubated slices was verified by determining intracellular K+ content and levels of cytochrome P-450, which were maintained under 95% O2 atmosphere but decreased with lower O2 tensions (2.5%). The highest fluoride production was 300 +/- 22 pmol/mg slice weight/6 hr at low O2 tension (2.5%). Defluorination decreased with increasing O2 tension to undetectable levels under 95% O2. Production of the oxidative metabolite, trifluoroacetic acid, was highest at 95% O2 (2.35 +/- 0.17 nmol/mg slice weight/6 hr). Trifluoroacetic acid production decreased with decreasing O2 tension. Br- production was the highest at 21% O2 (1.8 +/- 0.13 nmol/mg slice weight/6 hr). Production of Br- was not dependent on the O2 tension. The guinea pig slices are capable of biotransforming halothane (oxidative/reductive); therefore, this in vitro system appears suitable for studying the biotransformation of halothane.     

Inhibition of zaleplon metabolism by cimetidine in the human liver: in vitro studies with subcellular fractions and precision-cut liver slices

1. The effect of cimetidine on the metabolism of zaleplon (ZAL) in human liver subcellular fractions and precision-cut liver slices was investigated. 2. ZAL was metabolized to a number of products including 5-oxo-ZAL (M2), which is known to be formed by aldehyde oxidase, N-desethyl-ZAL (DZAL), which is known to be formed by CYP3A forms, and N-desethyl-5-oxo-ZAL (M1). 3. Human liver microsomes catalysed the NADPH-dependent metabolism of ZAL to DZAL. Kinetic analysis of three microsomal preparations revealed mean (+/-SEM) S(50) and V(max) of 310 +/- 24 micro M and 920 +/- 274 pmol/min/mg protein, respectively. 4. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (+/-SEM), K(m) and V(max) of 124 +/- 14 micro M and 564 +/- 143 pmol/min/mg protein, respectively. 5. Cimetidine inhibited ZAL metabolism to DZAL in liver microsomes and to M2 in the liver cytosol. With a ZAL substrate concentration of 62 micro M, the calculated mean (+/-SEM, n = 3) IC50 were 596 +/- 103 and 231 +/- 23 micro M for DZAL and M2 formation, respectively. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver cytosol with a mean (+/-SEM, n = 3) K(i) of 155 +/- 16 micro M. 6. Freshly cut human liver slices metabolized ZAL to a number of products including 1, M2 and DZAL. 7. Cimetidine inhibited ZAL metabolism in liver slices to M1 and M2, but not to DZAL. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver slices with an average (n = 2 preparations) K(i) of 506 micro M. 8. The results demonstrate that cimetidine can inhibit both the CYP3A and aldehyde oxidase pathways of ZAL metabolism in the human liver. Cimetidine appears to be a more potent inhibitor of aldehyde oxidase than of CYP3A forms and hence in vivo is likely to have a more marked effect on ZAL metabolism to M2 than on DZAL formation. 9. The results also demonstrate that precision-cut liver slices may be a useful model system for in vitro drug-interaction studies.     

In Vitro metabolism of ciclesonide in human lung and liver precision-cut tissue slices

Ciclesonide is a new-generation inhaled corticosteroid developed to treat the inflammation associated with persistent asthma. In order to identify the properties of ciclesonide responsible for anti-inflammatory activity, ciclesonide metabolism was investigated in human lung and liver precision-cut tissue slices. Three human lung and three human liver tissue slices were incubated with 25 microM [14C]-ciclesonide for 2, 6 and 24 h. Cellular viability was assessed using adenosine 5'-triphosphate content and protein synthesis in lung slices and adenosine 5'-triphosphate content and potassium retention in liver slices. Ciclesonide and ciclesonide metabolites were analysed in tissue samples using high-performance liquid chromatography with ultraviolet and radiochemical detection. Metabolite identity was confirmed using mass spectrometry. In lung slices, the inactive parent compound, ciclesonide, was initially converted to the active metabolite, desisobutyryl-ciclesonide, and subsequently converted to fatty acid conjugates. The reversible formation of fatty acid conjugates was a major pathway of ciclesonide metabolism in human lung slices. The primary conjugate was identified as desisobutyryl-ciclesonide oleate. Ciclesonide was metabolized to at least five polar metabolites in the liver. Dihydroxylated desisobutyryl-ciclesonide was the major polar metabolite in liver slices. Activation and fatty acid esterification in the lung followed by rapid inactivation in the liver may explain the improved safety profile and prolonged anti-inflammatory activity of ciclesonide.     

Liver slices in dynamic organ culture. II. An in vitro cellular technique for the study of integrated drug metabolism using human tissue

1. Precision cut human liver slices in dynamic organ culture have been used to study the integrated metabolism of 7-ethoxycoumarin and the conjugation of 7-hydroxycoumarin. 2. The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was monitored for 6 h. For both substrates there was a time-dependent increase in metabolites present in the incubation medium. The low levels of free 7-hydroxycoumarin found in the medium when 7-ethoxycoumarin was the substrate suggests good coupling of phase I and phase II metabolism. 3. With suitable incubation conditions, i.e. change of medium containing new substrate every 2 h, the metabolism of both 7-ethoxycoumarin and 7-hydroxycoumarin by human liver slices was found to proceed at similar rates for up to 24 h. This was demonstrated using five separate human liver preparations. 4. Human liver slices also metabolized mono-chlorobenzene and o-, m- and p-dichlorobenzene to aqueous soluble metabolites. There was a time-dependent increase in the appearance of aqueous soluble metabolites present in the incubation medium. Metabolites were not retained by the liver slices. 5. A cold-storage transit buffer has been described and used to maintain the levels of drug metabolism in both rat and human tissue for periods of up to 6 h. 6. The use of human liver slices in dynamic organ culture as a suitable method for the direct assessment of integrated hepatic drug metabolism is proposed.     

Comparison of human and rat metabolism of molinate in liver microsomes and slices.

Molinate undergoes oxidative metabolism forming either ring-hydroxylated metabolites or molinate sulfoxide. Our previous studies strongly implicated the sulfoxidation pathway in molinate-induced testicular toxicity. The present study compares the metabolic capability of rat and human liver microsomes and slices to form either nontoxic ring-hydroxylated metabolites of molinate or the toxic metabolites derived from the sulfoxidation of molinate. Km and Vmax values indicate that sulfoxidation would be the preferred high-dose pathway whereas hydroxylation would predominate at low dose levels in both species. Examination of phase II metabolism of molinate in liver slices reveals greater detoxification of molinate sulfoxide by glutathione conjugation in humans with rats forming less conjugate. Oxidative metabolism of molinate in both rats and humans appears to be mediated by cytochrome P-450 and not flavin monooxygenases as indicated by the use of metabolic inhibitors. Overall, the metabolism of molinate would be via the nontoxic hydroxylation pathway in both species at low doses whereas at high doses, where sulfoxidation would predominate, the human is more capable than the rat to detoxify via glutathione conjugation.     

Innovative methods to study human intestinal drug metabolism in vitro: precision-cut slices compared with ussing chamber preparations.

Predictive in vitro methods to investigate drug metabolism in the human intestine using intact tissue are of high importance. Therefore, we studied the metabolic activity of human small intestinal and colon slices and compared it with the metabolic activity of the same human intestinal segments using the Ussing chamber technique. The metabolic activity was evaluated using substrates to both phase I and phase II reactions: testosterone, 7-hydroxycoumarin (7HC), and a mixture of cytochrome P450 (P450) substrates (midazolam, diclofenac, coumarin, and bufuralol). In slices of human proximal jejunum, the metabolic activity of several P450-mediated and conjugation reactions remained constant up to4hof incubation. In the colon slices, conjugation rates were virtually equal to those in small intestine, whereas P450-mediated conversions occurred much slower. In both organs, morphological evaluation and ATP content implied tissue integrity within this period. P450 conversions using the Ussing chamber technique showed that the metabolic rate (sum of metabolites measured in apical, basolateral, and tissue compartments) was constant up to 3 h. For 7HC conjugations, the metabolic rate remained constant up to 4 h. The distribution of the metabolites in the compartments differed between the substrates. Overall, metabolic rates were surprisingly similar in both techniques and appear similar to or even higher than in liver. In conclusion, this study shows that both human intestinal precision-cut slices and Ussing chamber preparations provide useful tools for in vitro biotransformation studies.     

Inhibition of zaleplon metabolism by cimetidine in the human liver: in vitro studies with subcellular fractions and precision-cut liver slices

1. The effect of cimetidine on the metabolism of zaleplon (ZAL) in human liver subcellular fractions and precision-cut liver slices was investigated. 2. ZAL was metabolized to a number of products including 5-oxo-ZAL (M2), which is known to be formed by aldehyde oxidase, N-desethyl-ZAL (DZAL), which is known to be formed by CYP3A forms, and N-desethyl-5-oxo-ZAL (M1). 3. Human liver microsomes catalysed the NADPH-dependent metabolism of ZAL to DZAL. Kinetic analysis of three microsomal preparations revealed mean (± SEM) S₅₀ and V_max of 310 ± 24 µM and 920 ± 274 pmol/min/mg protein, respectively. 4. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (± SEM), K_m and V_max of 124 ± 14 µM and 564 ± 143 pmol/min/mg protein, respectively. 5. Cimetidine inhibited ZAL metabolism to DZAL in liver microsomes and to M2 in the liver cytosol. With a ZAL substrate concentration of 62 µM, the calculated mean (± SEM, n = 3) IC₅₀ were 596 ± 103 and 231 ± 23 µM for DZAL and M2 formation, respectively. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver cytosol with a mean (± SEM, n = 3) K_i of 155 ± 16 µM. 6. Freshly cut human liver slices metabolized ZAL to a number of products including 1, M2 and DZAL. 7. Cimetidine inhibited ZAL metabolism in liver slices to M1 and M2, but not to DZAL. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver slices with an average (n = 2 preparations) K_i of 506 µM. 8. The results demonstrate that cimetidine can inhibit both the CYP3A and aldehyde oxidase pathways of ZAL metabolism in the human liver. Cimetidine appears to be a more potent inhibitor of aldehyde oxidase than of CYP3A forms and hence in vivo is likely to have a more marked effect on ZAL metabolism to M2 than on DZAL formation. 9. The results also demonstrate that precision-cut liver slices may be a useful model system for in vitro drug-interaction studies.     

Metabolism and covalent binding of [14C]toluene by human and rat liver microsomal fractions and liver slices

The in vitro metabolism of [14C]toluene by liver microsomes and liver slices from male Fischer F344 rats and human subjects has been compared. Rat liver microsomes produced only benzyl alcohol from toluene. Liver microsomes from human subjects metabolized toluene to benzyl alcohol, benzaldehyde, and benzoic acid. Liver microsomes from one human donor also produced p-cresol and o-cresol. The overall rate of toluene metabolism by human liver microsomes was 9-fold greater than by rat liver microsomes. Human liver microsomal metabolism of benzyl alcohol to benzaldehyde required NADPH and was inhibited by carbon monoxide and high pH (pH 10). but was not inhibited by ADP-ribose or sodium azide. These results suggest that cytochrome P-450, rather than alcohol dehydrogenase, was responsible for the metabolism of benzyl alcohol to benzaldehyde. Human and rat liver slices metabolized toluene to hippuric acid and benzoic acid. The overall rate of toluene metabolism by human liver slices was 1.3-fold greater than by rat liver slices. Cresols and cresol conjugates were not detected in human or rat liver slice incubations. Covalent binding of [14C]toluene to human liver microsomes and slices was 21-fold and 4-fold greater than to the comparable rat liver preparations. Covalent binding did not occur in the absence of NADPH, was significantly decreased by coincubation with cysteine, glutathione, or superoxide dismutase, and was unaffected by coincubation with lysine. Protease and ribonuclease digestion decreased the amount of toluene covalently bound to human liver microsomes by 78% and 27% respectively. Acid washing of human liver microsomes had no effect on covalent binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidative metabolism of 14C-pyridine by human and rat tissue subcellular fractions

1. The oxidative metabolism of 14C-pyridine by human and rat microsomal fractions has been studied. Metabolites were separated by h.p.l.c. employing continuous radioactivity monitoring of the column effluent. 2. Human liver microsomal fractions incubated with an NADPH-generating system and oxygen formed pyridine N-oxide (PNO) at an average rate of 275 pmol/min per mg protein, 2-pyridone (2PO) at 207 pmol/min per mg and 4-pyridone (4PO) at 154 pmol/min per mg. One human subject formed 3-hydroxypyridine N-oxide in addition to the other metabolites. 3. Human kidney microsomal fractions formed PNO, 2PO and 4PO at rates similar to those with human liver microsomal fractions, whereas human lung microsomal fractions formed the metabolites at less than half the rate. 4. Metabolism of pyridine by human liver microsomal fractions was inhibited 54% by nitrogen, 34% by 80% carbon monoxide-20% oxygen, and 20% by metyrapone. 2-Diethylaminoethyl-2,2-diphenylvalerate HCl (SKF-525A) did not inhibit pyridine metabolism. 5. Liver microsomal fractions from non-induced rats metabolized pyridine to PNO at a rate of 19 pmol/min per mg protein, 2PO at 17 pmol/min per mg and 4PO at 61 pmol/min per mg. 6. There was no pyridine metabolism by human or rat tissue cytosolic fractions incubated under the same conditions.