Biotransformation of lovastatin. II. In vitro metabolism by rat and mouse liver microsomes and involvement of cytochrome P-450 in dehydrogenation of lovastatin

Metabolism of lovastatin, a new cholesterol-lowering drug, by liver microsomes from rats and mice was investigated. Liver microsomes from rats catalyzed biotransformation of lovastatin at a rate of 3 nmol/mg of protein/min, whereas the rate of metabolism was 37% higher with liver microsomes from mice. The profiles of metabolites were similar, but the relative abundance of individual metabolites was species dependent. Hydroxylation at the 6'-position was the principal pathway of lovastatin biotransformation, whereas hydroxylation at the 3"-position of the side chain was a minor pathway. In both species the 6'-beta-hydroxy-lovastatin accounted for half of the total metabolism. Liver microsomes from rats produced 2- to 4-fold higher amounts of the other three metabolites, namely, 6'-exomethylene-, 3"-hydroxy-, and the hydroxy acid form, than mouse liver microsomes. The conversion of lovastatin to the novel 6'-exomethylene metabolite was catalyzed by cytochrome P-450 since it required microsomes and NADPH and was inhibited by SKF-525A, metyrapone, and 2,4,-dichloro-6-phenylphenoxyethylamine (DPEA). Furthermore, neither 6'-beta-hydroxy-lovastatin nor the 6'-hydroxymethyl analogs could be demonstrated to be intermediates in the formation of the 6'-exomethylene metabolite. The hydroxy acid form of lovastatin was not a substrate for liver microsomes from either species.     

Metabolism of lovastatin by rat and human liver microsomes in vitro

The metabolism of lovastatin (Mevacor) was examined using isolated microsomes derived from the livers of normal and phenobarbital-treated rats and from human liver samples. Incubation of lovastatin with rat liver microsomes resulted in the formation of several polar metabolites of lovastatin. The metabolites were isolated by HPLC and identified by NMR and mass spectrometry. One fraction consisted of a 2:1 mixture of 6-hydroxy-lovastatin and the rearrangement product delta 4,5-3-hydroxy lovastatin. Addition of a trace of acid to this mixture resulted in the formation of a single aromatized product, the desacyl-delta 4a,6,8-dehydro analog of lovastatin. Another microsomal metabolite was determined to be the delta 4,8a,1-3-hydroxy-lovastatin derivative. The chromatographic pattern of metabolites produced from lovastatin by human liver microsomes was similar to that obtained with rat liver microsomes. Metabolism of lovastatin by rat liver microsomes was both time and concentration dependent; optimal microsomal metabolism occurred with 0.1 mM lovastatin, whereas higher lovastatin concentrations inhibited the reaction. The open acid form of lovastatin was poorly metabolized by both the rat and the human liver microsomes.     

Felbamate in vitro metabolism by rat liver microsomes.

Incubation of [14C]felbamate at 37 degrees C for 60 min with liver microsomes from untreated Sprague-Dawley rats converted 10% of the drug to the p-hydroxy (6%) and 2-hydroxy (4%) metabolites. With microsomes from phenobarbital-pretreated rats, 21% of the drug was metabolized to the p-hydroxy (7.5%) and 2-hydroxy (13.5%) metabolites. With microsomes from felbamate-pretreated rats, up to 25% of drug was metabolized to the p-hydroxy (5%) and 2-hydroxy (20%) metabolites. In addition, a small amount of the monocarbamate metabolite was also present, but no other metabolites were formed. Coincubations of [14C]phenytoin with felbamate had no effect on the metabolism of phenytoin, whereas the amount of [14C]felbamate metabolized in the presence of phenytoin decreased by 30-38%.     

Lindane metabolism by human and rat liver microsomes

1. Human liver microsomes convert lindane (gamma isomer of 1,2,3,4,5,6-hexachlorocyclohexane) to four major primary metabolites; gamma-1,2,3,4,5,6-hexachlorocyclohex-1-ene (3,6/4,5-HCCH), gamma-1,3,4,5,6-pentachlorocyclohex-1-ene (3,6/4,5-PCCH), beta-1,3,4,5,6-pentachlorocyclohex-1-ene (3,4,6/5-PCCH), and 2,4,6-trichlorophenol (2,4,6-TCP); and two major secondary metabolites; 2,3,4,6-tetrachlorophenol (2,3,4,6-TTCP) and pentachlorobenzene (PCB). 2. Under the same conditions, rat liver microsomes produce 3,6/4,5-HCCH, 2,4,6-TCP and 2,3,4,6-TCCP at rates similar to human liver microsomes. 3,4,6/5-PCCH is produced at much lower rates and 3,6/4,5-PCCH and PCB are not detected when lindane is incubated with rat liver microsomes for up to 30 min. 3. The identity of 3,4,6/5-PCCH, previously not identified as a mammalian metabolite of lindane, is confirmed by column chromatography and g.l.c.-mass spectrometry by comparison with authentic material. 4. It is concluded that there is potentially substantial hepatic metabolism by humans of lindane, a topically used scabicide and pediculicide.     

Interaction of famotidine with rat liver microsomes, a study showing less inhibition of drug metabolism than with cimetidine

Interaction of famotidine with rat liver microsomes and its effect on drug metabolism in vitro were studied. Famotidine interacted with liver microsomes obtained from untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated rats to produce characteristic type II spectral changes with peaks at 423-426 nm and troughs at 387-390 nm. The spectral dissociation constants were in the range of 0.84-0.94 mM. Famotidine inhibited aminopyrine N-demethylase activity to a much lesser extent than did cimetidine. The extent of inhibition at a concentration of 5 mM of famotidine was from 12 to 18% for the microsomes from the rats with different pretreatments. In contrast, 5 mM of cimetidine inhibited the activity 80, 59 and 80% in the microsomes from untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated rats, respectively. Both famotidine and cimetidine inhibited aminopyrine N-demethylase in a mixed-type manner for the microsomes from phenobarbital-pretreated rats, with inhibition constants of 4.7 and 0.7 mM, respectively. These results demonstrate that famotidine is an in vitro inhibitor of microsomal drug metabolism in rats but is much less inhibitory than cimetidine.     

In vitro metabolism of perospirone in rat, monkey and human liver microsomes.

In vitro metabolism of perospirone was examined with rat, monkey and human liver S9, human liver microsomes and yeast microsomes expressing human P450, using 14C labeled perospirone. With rat liver S9, the major metabolites were MX9 and ID-11614, produced by cleavage at the butylene chain. However, some butylene non-cleavage and hydration of the cyclohexane ring were found, although limited in extent. Unknown metabolites accounted for about 10% of the total. After incubation for 10 minutes with monkey liver S9, the major metabolites were ID-15036 and MX11, hydrated in the cyclohexane ring. After incubation for 60 minutes, ID-15001, i.e. the butylene chain cleavage type increased. Unknown metabolites accounted for about 20%. After incubation for 10 minutes with human liver S9, the major metabolite was ID-15036, hydrated in the cyclohexane ring. In addition, MX11 and many unknown metabolites were evident. After incubation for 60 minutes, the butylene chain cleavage type and unknown metabolites increased. Individual differences were found in the metabolic reaction rate. With human liver microsomes. MX11, ID-15001 and unknown metabolites were again the major metabolites. With yeast microsomes expressing human P450 subtypes, CYP1A1, 2C8, 2D6, 3A4 were responsible for the metabolism in particular, and CYP3A4 contributes greatly. Therefore it is unlikely that genetic polymorphism will arise a present a problem with regard to the clinical drug. The results demonstrated that the main metabolic pathway in human liver S9 and liver microsomes involve oxidation at cyclohexane, oxidative cleavage of the butylene side chain and S-oxidation. The same was the case in rat and monkey S9, but species differences were found in the proportions of the metabolites produced.     

Inhibition of drug metabolism in human liver microsomes by nizatidine, cimetidine and omeprazole

1. The inhibitory effects of cimetidine, nizatidine and omeprazole on the metabolic activity of CYP2C9, 2C19, 2D6 and 3A were investigated in human liver microsomes. Both cimetidine and omeprazole inhibited each of the CYP subfamily enzymes; in particular, omeprazole extensively inhibited the hydroxylation of S-mephenytoin (CYP2C19, Ki = 7.1 microM). Nizatidine exhibited no inhibition of any of the CYP isoforms examined. 2. Cimetidine inhibited the hydroxylation of tolbutamide but not of diclofenac, whereas omeprazole inhibited the hydroxylation of diclofenac but not that of tolbutamide. The ability to inhibit CYP2C9 varied with incubation time, as measured by the metabolic rate constant for the substrates. Therefore, suitable substrates and incubation times must be selected in inhibition studies examining metabolic clearance and the mechanism of inhibition of these drugs. 3. Nizatidine did not inhibit the metabolism of cisapride, glibenclamide, benidipine and simvastatin. Omeprazole inhibited the metabolism of cisapride (Ki = 0.4 microM), glibenclamide (11.7 microM) and benidipine (6.5 microM), whereas cimetidine inhibited the metabolism of glibenclamide (11.6 microM). To avoid drug-drug interactions, care needs to be taken to select suitable medicines for co-administration with anti-ulcer drugs.     

On the sulphoxidation of cimetidine and etintidine by rat and human liver microsomes

1. Sulphoxidation of cimetidine and etintidine was investigated by in vitro assays with liver microsomes from untreated 5,6-benzoflavone- and phenobarbital-pretreated rats as well as with human liver microsomes. The formation rate of cimetidine sulphoxide and etintidine sulphoxide with liver microsomes of normal or pretreated rats reached to 1.1 and 0.9 nmol/min mg microsomal protein, respectively. 2. Inhibition experiments with carbon monoxide and n-octylamine indicated that this sulphoxidation is catalyzed by cytochrome(s) P-450, whereas flavin-containing monooxygenase and/or non-enzymatic reactions (via peroxides) seems not to be involved: no inhibition was observed by methimazole, N,N-dimethylaniline, preheating or glutathione and EDTA. 3. With human liver microsomes the cytochrome P-450-dependent sulphoxidation accounted for no more than 40% of the total oxidation.     

On the sulphoxidation of cimetidine and etintidine by rat and human liver microsomes

1. Sulphoxidation of cimetidine and etintidine was investigated by in vitro assays with liver microsomes from untreated 5,6-benzoflavone- and phenobarbital-pretreated rats as well as with human liver microsomes. The formation rate of cimetidine sulphoxide and etintidine sulphoxide with liver microsomes of normal or pretreated rats reached to 1.1 and 0.9 nmol/min mg microsomal protein, respectively.

2. Inhibition experiments with carbon monoxide and n-octylamine indicated that this sulphoxidation is catalyzed by cytochrome(s) P-450, whereas flavin-containing monooxygenase and/or non-enzymatic reactions (via peroxides) seems not to be involved: no inhibition was observed by methimazole, N,N-dimethylaniline, preheating or glutathione and EDTA.

3. With human liver microsomes the cytochrome P-450-dependent sulphoxidation accounted for no more than 40% of the total oxidation.     

Biotransformation of tranylcypromine in rat liver microsomes

Metabolism of tranylcypromine (TCP) in rat liver microsomes was studiedin vitro using fortified microsomal preparations. As well as unlabeled TCP, two deuterium labeled analogs, TCP-phenyl-d₅ and TCP-cyclopropyl-d₂ were used and GC/MS employed for the analysis. It was found that TCP was converted chemically to hydrocinnamaldehyde which was then metabolized to cinnamaldehyde and hydrocinnamyl alcohol. Schiff bases of TCP with hydrocinnamaldehyde and acetaldehyde were detected and possibility of the metabolic formation of N-ethylideneTCP was proposed. In addition, acetophenone (benzoylacetic acid), benzaldehyde, benzoic acid, and benzyl alcohol were detected as the metabolites. Chemical decomposition studies suggested that parts of the oxidized products might be derived by air oxidation processes. A potential metabolite assumed to be N-ethylidene-1,2-dihydroxy-3-phenylpropanamine oxide was also detected.     

In vitro metabolism study of combretastatin A-4 in rat and human liver microsomes.

The phase I biotransformation of combretastatin A-4 (CA-4) 1, a potent tubulin polymerization inhibitor with antivascular and antitumoral properties, was studied using rat and human liver subcellular fractions. The metabolites were separated by high-performance liquid chromatography and detected with simultaneous UV and electrospray ionization (ESI) mass spectrometry. The assignment of metabolite structures was based on ESI-tandem mass spectrometry experiments, and it was confirmed by comparison with reference samples obtained by synthesis. O-Demethylation and aromatic hydroxylation are the two major phase I biotransformation pathways, the latter being regioselective for phenyl ring B of 1. Indeed, incubation with rat and human microsomal fractions led to the formation of a number of metabolites, eight of which were identified. The regioselectivity of microsomal oxidation was also demonstrated by the lack of metabolites arising from stilbenic double bond epoxidation. Alongside the oxidative metabolism, Z-E isomerization during in vitro study was also observed, contributing to the complexity of the metabolite pattern. Moreover, when 1 was incubated with a cytosolic fraction, metabolites were not observed. Aromatic hydroxylation at the C-6' of phenyl ring B and isomerization led to the formation of M1 and M2 metabolites, which were further oxidized to the corresponding para-quinone (M7 and M8) species whose role in pharmacodynamic activity is unknown. Metabolites M4 and M5, arising from O-demethylation of phenyl ring B, did not form the ortho-quinones. O-Demethylation of phenyl ring A formed the metabolite M3 with a complete isomerization of the stilbenic double bond.     

In vitro metabolism of clindamycin in human liver and intestinal microsomes.

Incubations with human liver and gut microsomes revealed that the antibiotic, clindamycin, is primarily oxidized to form clindamycin sulfoxide. In this report, evidence is presented that the S-oxidation of clindamycin is primarily mediated by CYP3A. This conclusion is based upon several lines of in vitro evidence, including the following. 1) Incubations with clindamycin in hepatic microsomes from a panel of human donors showed that clindamycin sulfoxide formation correlated with CYP3A-catalyzed testosterone 6beta-hydroxylase activity; 2) coincubation with ketaconazole, a CYP3A4-specific inhibitor, markedly inhibited clindamycin S-oxidase activity; and 3) when clindamycin was incubated across a battery of recombinant heterologously expressed human cytochrome P450 (P450) enzymes, CYP3A4 possessed the highest clindamycin S-oxidase activity. A potential role for flavin-containing monooxygenases (FMOs) in clindamycin S-oxidation in human liver was also evaluated. Formation of clindamycin sulfoxide in human liver microsomes was unaffected either by heat pretreatment or by chemical inhibition (e.g., methimazole). Furthermore, incubations with recombinant FMO isoforms revealed no detectable activity toward the formation of clindamycin sulfoxide. Beyond identifying the drug-metabolizing enzyme responsible for clindamycin S-oxidation, the ability of clindamycin to inhibit six human P450 enzymes was also evaluated. Of the P450 enzymes examined, only the activity of CYP3A4 was inhibited (approximately 26%) by coincubation with clindamycin (100 microM). Thus, it is concluded that CYP3A4 appears to account for the largest proportion of the observed P450 catalytic clindamycin S-oxidase activity in vitro, and this activity may be extrapolated to the in vivo condition.     

In vitro metabolism of gefitinib in human liver microsomes

1.?The in vitro metabolism of gefitinib was investigated by incubating [¹⁴C]-gefitinib, as well as M537194, M387783 and M523595 (the main metabolites of gefitinib observed in man), at a concentration of 100?μM with human liver microsomes (4?mg?ml^?1) for 120?min. These relatively high substrate and microsomal protein concentrations were used in an effort to generate sufficient quantities of metabolites for identification.

2.?HPLC with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of a large number of metabolites. Although 16 metabolites were identified, metabolism was restricted to three regions of the molecule.

3.?The major pathway involved morpholine ring-opening and step-wise removal of the morpholine ring and propoxy side chain. O-demethylation of the quinazoline methoxy group was a quantitatively less important pathway, in contrast to the clinical situation, where O-desmethyl gefitinib (M523595) is the predominant plasma metabolite. The third metabolic route, oxidative defluorination, was only a minor route of metabolism. Some metabolites were formed by a combination of these processes, but no metabolism was observed in other parts of the molecule.

4.?Incubation of gefitinib produced ten identified metabolites, but the use of the three main in vivo metabolites as additional substrates enabled a more comprehensive metabolic pathway to be constructed and this has been valuable in supporting the more limited data available from the human in vivo study.     

In vitro metabolism of gefitinib in human liver microsomes

The in vitro metabolism of gefitinib was investigated by incubating [14C]-gefitinib, as well as M537194, M387783 and M523595 (the main metabolites of gefitinib observed in man), at a concentration of 100 microM with human liver microsomes (4 mg ml(-1)) for 120 min. These relatively high substrate and microsomal protein concentrations were used in an effort to generate sufficient quantities of metabolites for identification. HPLC with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of a large number of metabolites. Although 16 metabolites were identified, metabolism was restricted to three regions of the molecule. The major pathway involved morpholine ring-opening and step-wise removal of the morpholine ring and propoxy side chain. O-demethylation of the quinazoline methoxy group was a quantitatively less important pathway, in contrast to the clinical situation, where O-desmethyl gefitinib (M523595) is the predominant plasma metabolite. The third metabolic route, oxidative defluorination, was only a minor route of metabolism. Some metabolites were formed by a combination of these processes, but no metabolism was observed in other parts of the molecule. Incubation of gefitinib produced ten identified metabolites, but the use of the three main in vivo metabolites as additional substrates enabled a more comprehensive metabolic pathway to be constructed and this has been valuable in supporting the more limited data available from the human in vivo study.     

In vitro phase I metabolism of vinclozolin by human liver microsomes

1.?Vinclozolin (Vin) is a fungicide used in agricultural settings and is classified as an endocrine disruptor. Vin is non-enzymatically hydrolyzed to 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) metabolites. There is no information about Vin biotransformation in humans, therefore, the aim of this study was to characterize its in vitro metabolism using human liver microsomes.

2.?Vin was metabolized to the [3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione] (M4) and N-(2,3,4-trihydroxy-2-methyl-1-oxo)-3,5-dichlorophenyl-1-carbamic acid (M7) metabolites, which are unstable and gradually converted to 3′,5′-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (DTMBA, formerly denoted as M5). M4 and DTMBA metabolites co-eluted in the same HPLC peak; this co-elute peak exhibited a Michaelis-Menten kinetic, whereas M7 showed a substrate inhibition kinetics. The K_{M app} for co-eluted M4/DTMBA and M7 was 24.2?±?5.6 and 116.0?±?52.6?μM, the V_{Max app} was 0.280?±?0.015 and 0.180?±?0.060 nmoles/min/mg protein, and the CL_{int app} was 11.5 and 1.5?mL/min/g protein, respectively. The K_i for M7 was 133.2?±?63.9?μM. Cytochrome P450 (CYP) chemical inhibitors furafylline (CYP1A2), ketoconazole (CYP3A4), pilocarpine (CYP2A6) and sulfaphenazole (CYP2C9) inhibited M4/DTMBA and M7 formation, suggesting that Vin is metabolized in humans by CYP.

3.?DTMBA is a stable metabolite and specific of Vin, therefore, it could be used as a biomarker of Vin exposure in humans to perform epidemiological studies.