Enantioselective carbonyl reduction of eperisone in human liver microsomes

Eperisone, 4-ethyl-2-methyl-3-piperidinopropiophenone, is a centrally acting muscle relaxant widely used to relieve muscle stiffness and back pain. In this study, enantioselectivity for carbonyl reduction of eperisone was investigated in human liver microsomes, and the enzymes involved in the carbonyl reduction were characterised. Carbonyl reduction of eperisone predominantly occurred in microsomal fractions and 11β-hydroxysteroid dehydrogenase type 1(11β-HSD 1) played a major role in this reaction as judged by selective inhibition of the activity by BVT-14225 and KR-66344. The kinetic study with (+)-S- and (-)-R-eperisone showed that the formation of the carbonyl reduced metabolite (M5) from the (-)-R-isomer was more efficient than that from the (-)-S-isomer. As eperisone is a racemic compound with one chiral centre, the carbonyl reduced metabolite of eperisone (M5) may have four possible diastereoisomeric structures. Chiral separation of incubation mixtures of racemic eperisone with human liver microsome revealed that (1S, 2S)-M5 and (1R, 2R)-M5 were generated specifically from (+)-S- and (-)-R-eperisone, respectively. Selective formation of anti-diastereomers was further confirmed by incubation of individual enantiomer with microsomes. Carbonyl reduction of eperisone by microsomal 11β-HSD 1 may significantly contribute to the metabolic disposition of eperisone in human and (-)-R-isomer is preferentially reduced by this enzyme.     

Enantioselective carbonyl reduction of eperisone in human liver microsomes

1. Eperisone, 4-ethyl-2-methyl-3-piperidinopropiophenone, is a centrally acting muscle relaxant widely used to relieve muscle stiffness and back pain. In this study, enantioselectivity for carbonyl reduction of eperisone was investigated in human liver microsomes, and the enzymes involved in the carbonyl reduction were characterised.

2. Carbonyl reduction of eperisone predominantly occurred in microsomal fractions and 11β-hydroxysteroid dehydrogenase type 1(11β-HSD 1) played a major role in this reaction as judged by selective inhibition of the activity by BVT-14225 and KR-66344. The kinetic study with (+)-S- and (?)-R-eperisone showed that the formation of the carbonyl reduced metabolite (M5) from the (?)-R-isomer was more efficient than that from the (?)-S-isomer.

3. As eperisone is a racemic compound with one chiral centre, the carbonyl reduced metabolite of eperisone (M5) may have four possible diastereoisomeric structures. Chiral separation of incubation mixtures of racemic eperisone with human liver microsome revealed that (1S, 2S)-M5 and (1R, 2R)-M5 were generated specifically from (+)-S- and (?)-R-eperisone, respectively. Selective formation of anti-diastereomers was further confirmed by incubation of individual enantiomer with microsomes.

4. Carbonyl reduction of eperisone by microsomal 11β-HSD 1 may significantly contribute to the metabolic disposition of eperisone in human and (?)-R-isomer is preferentially reduced by this enzyme.

     

Isolation and characterization of rabbit liver xenobiotic carbonyl reductases

Xenobiotic carbonyl reductases have been isolated from rabbit liver by ammonium sulfate fractionation and isoelectric focusing. Although these enzymes are very heterogeneous, the above procedures resolve the majority of the reductases in good yield. Most of the carbonyl reduction of oxisuran, 3,7-dimethyl-1-(5-oxyhexyl)-xanthine, metyrapone and daunorubicin (pH6.0) was accomplished by two distinct enzymes of pI 4.84 and 4.98. Other reductases with lesser activities toward these same substrates also occurred at higher pI values. Also resolved were several forms of enzymes that reduced daunorubicin (pH8.5) (previously identified as aldehyde reductase), naloxone and naltrexone (dihydrornorphinone reductases), and the model compounds, p-nitrobenzaldehyde and p-nitroacetophenone. The hydrogen stereospeciflcity of each of the rabbit liver carbonyl reductases, as well as rat liver aldehyde reductase, was determined by reducing the carbonyl substrates with A- and B-labeled [4-³H]NADPH and examining transfer of label to alcohol products and retention of label in the resulting oxidized cofactors. All of the oxisuran, metyrapone and daunorubicin (pH 6.0) reductases displayed B-hydrogen stereospecificity. Some enzymes that reduce 3,7-dimethyl-1-(5-oxyhexyl)-xanthine, p-nitroacetophenone and p-nitrobenzaldehyde were also B-stereospecific while other forms of these same enzymes were A-stereospecific. Only daunorubicin (pH8.5) (rabbit and rat), naloxone and naltrexone reductases were exclusively A-stereospecific. Apparent deuterium isotope effects of A- and B-labeled [4-²H]NADPH with daunorubicin (pH 6.0) reductases, daunorubicin (pH 8.5) reductase and naloxone reductases confirm the above hydrogen stereospecificity assignments. The results confirm the hydrogen specificity of aldehyde reductases as A-stereospecific and the majority of ketone reductases as B-stereospecific. In addition, several significant A-stereospecific ketone reductases appear to represent exceptions to the generalization that enzymes which catalyze the same reaction have the same stereospecificity. Finally, the binding of rat liver aldehyde reductase to NADPH produced a red shift in the cofactor 340 nm absorbance maximum which is opposite to that predicted on the basis of its hydrogen stereospecificity.     

Metabolism of a novel hypnotic, N3-phenacyluridine, and hypnotic and sedative activities of its enantiomer metabolites in mouse

1. The metabolism of N3-phenacyluridine (3-phenacyl-1-beta-D-ribofuranosyluracil), a potent hypnotic nucleoside derivative, was studied in mouse. 2. Of the radioactivity, 65% was excreted in urine within 48 h after intraperitoneal (i.p.) administration of [3H]N3-phenacyluridine. The urinary metabolites N3-phenacyluracil and N3-alpha-hydroxy-beta-phenethyluridine were extracted, isolated and analyzed by mass spectrometry. 3. Racemates of N3-alpha-hydroxy-beta-phenethyluridine were synthesized and both isomers were separated as N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine and N3-(R)-(-)-alpha-hydroxy-beta-phenethyluridine by hplc (CHIRALCEL-OJ column) with retentions of 13.8 and 17.9 min respectively. The reduction process took place with high stereo-selectivity, which gave an alcohol product in the urine with the same retention (17.9 min) as one of the synthetic isomers separated by hplc. 4. One of urinary metabolites was identified as N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine. N3-phenacyluridine was predominantly converted to an alcoholic metabolite of (S)-(+)-configuration. 5. N3-phenacyluracil and uridine were also identified as minor metabolites. 6. The pharmacological effects of the metabolites and related compounds were also evaluated in mouse. N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, but not N3-(R)-(-)-alpha-hydroxy-beta-phenethyluridine, possessed hypnotic activity and potentiated pentobarbital-induced sleeping time with a similar potency to the parent compound, N3-phenacyluridine. N3-alpha-hydroxy-beta-phenethyluridine (racemate) had almost two thirds of the hypnotic activity of N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine. No other metabolites exhibited hypnotic activities. 7. The present study indicates that N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, a major metabolite of N3-phenacyluridine, is an active metabolite and contributes a significant CNS depressant effect.     

Metabolism of a novel hypnotic,N3-phenacyluridine,and hypnotic and sedative activities of its enantiomer metabolites in mouse

1. The metabolism of N³-phenacyluridine (3-phenacyl-1- beta-D-ribofuranosyluracil), a potent hypnotic nucleoside derivative, was studied in mouse. 2. Of the radioactivity, 65% was excreted in urine within 48 h after intraperitoneal (i.p.) administration of [³H] N³-phenacyluridine. The urinary metabolites N³-phenacyluracil and N³-alpha-hydroxy-beta-phenethyluridine were extracted, isolated and analyzed by mass spectrometry. 3. Racemates of N³-alpha-hydroxy-beta-phenethyluridine were synthesized and both isomers were separated as N³-(S)-(+)-alpha-hydroxy-beta-phenethyluridine and N³-(R)-(-)-alpha hydroxy-beta-phenethyluridine by hplc (CHIRALCEL-OJ column) with retentions of 13.8 and 17.9 min respectively. The reduction process took place with high stereo-selectivity, which gave an alcohol product in the urine with the same retention (17.9 min) as one of the synthetic isomers separated by hplc. 4. One of urinary metabolites was identified as N³-(S)-(+)-alpha-hydroxy-beta-phenethyluridine. N³-phenacyluridine was predominantly converted to an alcoholic metabolite of (S)-(+)-configuration. 5. N³-phenacyluracil and uridine were also identified as minor metabolites. 6. The pharmacological effects of the metabolites and related compounds were also evaluated in mouse. N³-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, but not N ³-(R)-(-)-alpha hydroxy-beta-phenethyluridine, possessed hypnotic activity and potentiated pentobarbitalinduced sleeping time with a similar potency to the parent compound, N³-phenacyluridine. N³-alpha-hydroxy-beta-phenethyluridine (racemate) had almost two thirds of the hypnotic activity of N³-(S)-(+)- alpha-hydroxy-beta-phenethyluridine. No other metabolites exhibited hypnotic activities. 7. The present study indicates that N³-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, a major metabolite of N³-phenacyluridine, is an active metabolite and contributes a significant CNS depressant effect.     

Reductases for carbonyl compounds in human liver

Two aldehyde reductases with mol. wt 78,000 and 32,000 and one carbonyl reductase with mol. wt 31,000 were purified to homogeneity from human liver cytosol. The high molecular weight aldehyde reductase exhibited properties similar to alcohol dehydrogenase; it had a single subunit of mol. wt 41,000 and a pI value of 10 to 10.5, and showed preference for NADH over NADPH as cofactor and sensitivity to SH-reagents, pyrazole, o-phenanthroline and isobutyramide. The enzyme reduced aliphatic and aromatic aldehydes, alicyclic ketones and alpha-diketones and an optimal pH of 6.0, and oxidized various alcohols with NAD as a cofactor at an optimal pH of 8.8. The identity of the enzyme with alcohol dehydrogenase was established by starch gel electrophoresis and co-purification of the two enzymes. The other enzymes were NADPH-dependent and monomeric reductases; the aldehyde reductase reduced aldehydes, hexonates and alpha-diketones and was sensitive to barbiturates, diphenylhydantoin and valproate, while the carbonyl reductase showed a broad substrate specificity for aldehydes, ketones and quinones and was inhibited by SH-reagent, quercitrin and benzoic acid. The latter enzyme appeared in three multiforms with different charges which occurred in differing ratios in liver specimens. Comparison of kinetic constants for aldehydes among the enzymes indicated that alcohol dehydrogenase is the best reductase with the highest affinity and Kcat values. The enzyme also catalyzed oxidation and reduction of aromatic aldehydes in the presence of NAD at physiological pH of 7.2. Tissue distribution of the three enzymes and variation of their specific activities in human livers were examined.     

Properties of NADPH-dependent carbonyl reductases in rat liver cytosol

Rat liver cytosol was shown previously by us to contain multiple forms of 3 alpha-hydroxysteroid dehydrogenase. Two (F4-II and -III) of the seven forms were purified to homogeneity, and four (F3-II, -III, -IV and F4-I) of them partially purified. One of them (F4-III) has been shown previously to catalyze the reduction of long-chain aliphatic and aromatic aldehydes or aromatic ketones as well as 3-oxosteroids [M. Ikeda et al., Biochem. Pharmac. 30, 1931 (1981)]. The reducing activity of such compounds was examined with the other F4 enzymes, and it was revealed that they also reduce a number of carbonyl compounds described above. In addition, quinones were tested for the first time in this report as substrates for all the F4 enzymes, and among them 9,10-phenanthrenequinone was found to be the best substrate for them, followed by hydrindantin and 2,6-dichlorophenolindophenol, while menadione was a poor substrate. The F4 enzymes did not catalyze the reduction of the oxo group at the 9-position of the prostaglandins of the E and A class with NADPH or NADH. On the basis of this evidence, the identity of ketone reductases (F4-I-III) in the rat liver is proposed to be 3 alpha-hydroxysteroid dehydrogenase, rather than prostaglandin 9-ketoreductase, which was demonstrated to correspond to ketone reductase in human brain [B. Wermuth, J. biol. Chem. 256, 1206 (1981)].     

Dehydrogenation of indanol by rabbit liver 3-hydroxyhexobarbital dehydrogenase.

1. Among the several enzyme activities in rabbit liver cytosol able to dehydrogenate 1-indanol, only the main activity was not separable from 3-hydroxyhexobarbital dehydrogenase during purification including polyacrylamide gel disc electrophoresis. 2. Results of mixed substrate method indicated that the same enzyme catalyses the dehydrogenation of 1-indanol and 3-hydroxyhexobarbital. The ratio between the two dehydrogenation activities was almost constant as the enzyme underwent thermal inactivation. The Ki values of p-chloromercuribenzoate, the Km values for NAD+, and the Km values for NADP+ were very similar for the two dehydrogenations. These results lead to the conclusion that the same enzyme catalyses the dehydrogenation of 3-hydroxyhexobarbital and 1-indanol. 3. 1-Tetralol, 1-acenaphthenol, 9-fluorenol, thiochroman-4-ol and 4-chromanol also served as substrate of the enzyme, but 2-indanol, 2-tetralol, and trans- and cis-indan-1,2-diol were not oxidized. 4. Reversibility of the reaction was also confirmed using 1-indanone as substrate.     

A further study of the pharmacokinetics of gitoxin in rabbit isolated liver: clearance of 3H-gitoxin

Hepatic clearance of 3H-gitoxin was studied in the rabbit using an isolated perfused liver technique with an emulsion of a perfluorocarbon. The liposoluble material in the perfusion medium was extracted with dichloromethane, and gitoxin was assayed in the extract by high performance liquid chromatography. Pharmacokinetic parameters were estimated for the liposoluble (dichloromethane soluble) material in the water phase obtained by centrifugation of the emulsion, for the liposoluble material and unchanged gitoxin in the total emulsion. Distribution and elimination half-lives of the liposoluble fraction in the water phase, were estimated to be 0.47 and 4.80 hours respectively, Vd to be 148 ml.g-1 and intrinsic clearance to be 1.16 ml.min-1.g-1; these parameters were compared with those of a previous study with unlabelled gitoxin. Distribution and elimination half-lives of the liposoluble compounds in the emulsion were estimated to be 0.48 and 4.62 hours, Vd to be 47 ml.g-1 and intrinsic clearance to be 1.07 ml.min-1.g-1; these data were compared with those of the liposoluble compounds in the water phase. Distribution and elimination half-lives of unchanged gitoxin in the emulsion were estimated to be 0.22 and 0.70 hour, Vd to be 59 ml.g-1 and intrinsic clearance to be 11.4 ml.min-1.g-1; these data were compared with those of the liposoluble compounds in the emulsion. The subcellular distribution of gitoxin and its metabolites in the liver indicated that 79% of the radioactivity was found in the soluble fraction, no significant binding occurring in the mitochondrial and microsomal fractions.     

Inhibition of rabbit heart carbonyl reductase by fatty acids.

The inhibition of rabbit heart carbonyl reductase (RHCR) by fatty acids was examined using 4-benzoylpyridine (4BP) as a substrate. The inhibitory potency of saturated fatty acids increased with elongation in the carbon chain from caprylic acid to myristic acid, but decreased with further elongation. Myristic acid with 14 carbon atoms most strongly inhibited RHCR. All of the unsaturated fatty acids tested strongly inhibited RHCR; the cis-isomers were more potent inhibitors than the corresponding trans-isomers. The methyl esters and alcohols, which lack a carboxyl group, derived from fatty acids did not exert a significant inhibitory effect on RHCR. These results indicate that the existence of a proper length of carbon chain, double bond(s), and a carboxyl group in a fatty acid molecule is important for RHCR inhibition. We also propose the possibility that myristic acid at low concentrations inhibits the reduction of 4BP by interacting with a binding site other than the coenzyme- and substrate-binding sites of RHCR.     

Purification and characterization of oxidoreductases-catalyzing carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human liver cytosol

1. Four enzymes were purified to homogeneity from human liver cytosol and were demonstrated to be responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). 2. Carbonyl reductase (EC 1.1.1.184), a member of the short-chain dehydrogenase reductase (SDR) superfamily, was compared with three isoenzymes of the aldo-keto reductase (AKR) superfamily in terms of enzyme kinetics, co-substrate dependence and inhibition pattern. 3. AKR1C1, 1C2 and 1C4, previously designated as dihydrodiol dehydrogenases (DD1, DD2 and DD4), showed lower K_m (0.2, 0.3 and 0.8?mm respectively) than did carbonyl reductase (7 mM), whereas carbonyl reductase exhibited the highest enzyme efficiency (V_max/K_m) for NNK. Multiplication of enzyme efficiencies with the relative quantities of individual enzymes in cytosol resulted in a rough estimate of their contributions to total alcohol metabolite formation. These were ~ 60% for carbonyl reductase, 20% each for AKR1C1 and 1C2, and 1% for AKR1C4. 4. Except for AKR1C4, the enzymes had a strong preference for NADPH over NADH, and the highest activities were measured with an NADPH-regenerating system. Carbonyl reductase activity was extensively inhibited by menadione, rutin and quercitrin, whereas medroxyprogesterone acetate, phenolphthalein and flufenamic acid were potent inhibitors of AKR1C1, 1C2 and 1C4. 5. In conclusion, cytosolic members of the SDR and AKR superfamilies contribute to reductive NNK detoxification in human liver, the enzymes responsible being carbonyl reductase and aldoketo reductases of the AKR1C subfamily.     

Purification and characterization of oxidoreductases-catalyzing carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human liver cytosol

1. Four enzymes were purified to homogeneity from human liver cytosol and were demonstrated to be responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). 2. Carbonyl reductase (EC 1.1.1.184), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, was compared with three isoenzymes of the aldo-keto reductase (AKR) superfamily in terms of enzyme kinetics, co-substrate dependence and inhibition pattern. 3. AKR1C1, 1C2 and 1C4, previously designated as dihydrodiol dehydrogenases (DD1, DD2 and DD4), showed lower K(m) (0.2, 0.3 and 0.8 mM respectively) than did carbonyl reductase (7 mM), whereas carbonyl reductase exhibited the highest enzyme efficiency (Vmax/K(m)) for NNK. Multiplication of enzyme efficiencies with the relative quantities of individual enzymes in cytosol resulted in a rough estimate of their contributions to total alcohol metabolite formation. These were approximately 60% for carbonyl reductase, 20% each for AKR1C1 and 1C2, and 1% for AKR1C4. 4. Except for AKR1C4, the enzymes had a strong preference for NADPH over NADH, and the highest activities were measured with an NADPH-regenerating system. Carbonyl reductase activity was extensively inhibited by menadione, rutin and quercitrin, whereas medroxyprogesterone acetate, phenolphthalein and flufenamic acid were potent inhibitors of AKR1C1, 1C2 and 1C4. 5. In conclusion, cytosolic members of the SDR and AKR superfamilies contribute to reductive NNK detoxification in human liver, the enzymes responsible being carbonyl reductase and aldoketo reductases of the AKRIC subfamily.     

6Beta-hydroxy-3-epidigitoxigenin--the major metabolite of digitoxigenin by rabbit liver homogenates

³H-digitoxigenin was metabolized in vitro by rabbit liver homogenates. The major metabolite resulting from this biotransformation was isolated and identified as 6β- hydroxy-3-epidigitoxigenin. Approximately 50 per cent of the substrate was converted to this metabolite after 1 hr of incubation. The previously identified 5β-hydroxydigitoxigenin. 3-dehydrodigitoxigenin and 3-epidigitoxigenin were also present as metabolites. This is the first confirmation of the hydroxylation of digitoxigenin at C₆.     

The site of reduction of sulphinpyrazone in the rabbit

1. Comparison of oral and i.v. administration of sulphinpyrazone (10 mg/kg) to rabbits showed that the oral route was associated with an incomplete bioavailability and a six-fold greater formation of the active sulphide metabolite.

2. The bile was an important route of elimination of unchanged sulphinpyrazone in rabbits (18% of an i.v. dose in four hours). Only small amounts of the sulphide appeared in the bile after i.v. administration.

3. Pretreatment with oral antibiotics decreased the area under the plasma concentration-time curve (AUC) for the sulphide but increased that of the parent drug. Excretion of the p-hydroxysulphide metabolite in urine was decreased 30-fold by antibiotic treatment.

4. The contents of the caecum showed the greatest capacity for sulphinpyrazone reduction in vitro. The liver possessed a slight ability to reduce sulphinpyrazone in vitro under anaerobic, but not aerobic, conditions.

5. The gut bacteria are the main site of reduction of sulphinpyrazone to the active sulphide metabolite in the rabbit.

6. These findings contrast with those obtained for sulindac which was reduced extensively under both aerobic and anaerobic conditions by rabbit-liver soluble fraction in vitro. The sulphide metabolites of both sulphinpyrazone and sulindac were oxidized to the parent drug by rabbit-liver microsomes.     

Human carbonyl reduction pathways and a strategy for their study in vitro

Carbonyl reduction plays a significant role in physiological processes throughout the body. Although much is known about endogenous carbonyl metabolism, much less is known about the roles of carbonyl-reducing enzymes in xenobiotic metabolism. Multiple pathways exist in humans for metabolizing carbonyl moieties of xenobiotics to their corresponding alcohols, readying these molecules for subsequent conjugation and/or excretion. When exploring carbonyl reduction clearance pathways for a drug development candidate, it is possible to assess the relative contributions of these enzymes due to their differences in subcellular locations, cofactor dependence, and inhibitor profiles. In addition, the contributions of these enzymes may be explored by varying incubation conditions, such as pH. Presently, individual isoforms of carbonyl-reducing enzymes are not widely available, either in recombinant or purified form. However, it is possible to study carbonyl reduction clearance pathways from simple experiments with commercially available reagents. This article provides an overview of carbonyl-reducing enzymes, including some kinetic data for substrates and inhibitors. In addition, an experimental strategy for the study of these enzymes in vitro is presented.     

Comparative studies on the N-oxidation of aniline and N,N-dimethylaniline by rabbit liver microsomes.

1. Rate studies of N-oxidation of aniline and N,N-dimethylaniline by rabbit liver microsomal preparations were performed at different pH values. The apparent pKs of the free functional groups were 7-2 and 6-9, respectively, at 26 degrees. The apparent heats of ionization of these groups varied from 26-8 to 31-8 kJ mol-1. 2. Photo-oxidation of the microsomal mixed function oxidase resulted in rapid loss of N-oxygenating activity. The enzyme was markedly protected from inactivation by the presence of aniline or N,N-dimethylaniline. The apparent KD values for protection were close to the Km and KS values for the individual arylamines. The pH profiles of the initial rates of photo-inactivation resembled the titration curves of groups with an apparent pKa between 6-0 and 6-2. 3. The N-oxidase was strongly inhibited by diethyl pyrocarbonate at pH 6-2. 3. The N-oxidase was strongly inhibited by diethyl pyrocarbonate at pH 6-0. Catalytic capacity was partially restored by treatment with neutral hydroxylamine. Pyridine protected the enzyme from acylation. 4. A close relationship exists between the N-hydroxylation of aniline and the N-oxide formation from N,N-dimethylaniline with respect to sensitivity to photo-oxidation, reactivity to protective substrates and susceptibility to carbethoxylation.