Studies on the metabolic fate of M17055, a novel diuretic (6). Assessment for drug-drug interactions of M17055 in metabolism,distribution and excretion

1. The potential of M17055, a novel diuretic candidate, to affect the activities of human CYP enzymes, alter the plasma unbound fraction and compete with concomitant drugs in renal secretion as part of an assessment for drug-drug interactions in metabolism, distribution and excretion was investigated. 2. The effects of M17055 on the activities of human CYP1A2, CYP2E1, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were considered negligible at clinically relevant concentrations. 3. The majority of M17055 (99%) was bound to human plasma proteins, but it is unlikely to alter the binding of other clinically relevant drugs. 4. The renal clearance of M17055 (corrected for the plasma unbound fraction in male rats) substantially exceeded the glomerular filtration rate and was markedly reduced by treatment with probenecid, suggesting that the renal excretion of M17055 is controlled predominantly by an active secretion mechanism. 5. The results show that M17055 is unlikely to cause or undergo significant pharmacokinetic interactions with concomitant drugs in metabolism and distribution. However, when it is administered simultaneously with certain organic anions, drug-drug interactions during kidney excretion may be possible.     

Studies on the metabolic fate of M17055, a novel diuretic (6). Assessment for drug-drug interactions of M17055 in metabolism,distribution and excretion

1. The potential of M17055, a novel diuretic candidate, to affect the activities of human CYP enzymes, alter the plasma unbound fraction and compete with concomitant drugs in renal secretion as part of an assessment for drug-drug interactions in metabolism, distribution and excretion was investigated. 2. The effects of M17055 on the activities of human CYP1A2, CYP2E1, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were considered negligible at clinically relevant concentrations. 3. The majority of M17055 (99%) was bound to human plasma proteins, but it is unlikely to alter the binding of other clinically relevant drugs. 4. The renal clearance of M17055 (corrected for the plasma unbound fraction in male rats) substantially exceeded the glomerular filtration rate and was markedly reduced by treatment with probenecid, suggesting that the renal excretion of M17055 is controlled predominantly by an active secretion mechanism. 5. The results show that M17055 is unlikely to cause or undergo significant pharmacokinetic interactions with concomitant drugs in metabolism and distribution. However, when it is administered simultaneously with certain organic anions, drug-drug interactions during kidney excretion may be possible.     

Cytochrome P450-dependent metabolism of gefitinib

The in vitro metabolism of [(14)C]-gefitinib (1-3 microM) was investigated using human liver microsomes and a range of expressed human cytochrome P450 enzymes, with particular focus on the formation of O-desmethyl-gefitinib (M523595), the major metabolite observed in human plasma. High-performance liquid chromatography with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of metabolites. On incubation with pooled human liver microsomes, [(14)C]-gefitinib underwent rapid and extensive metabolism to a number of metabolites, although M523595 was only a minor microsomal product. Formation of most metabolites was markedly decreased by ketoconazole, but M523595 production was inhibited only by quinidine. Gefitinib was metabolized extensively by expressed CYP3A4, producing a similar range of metabolites to liver microsomes, but M523595 was not formed. CYP1A2, 2C9 and 2C19 produced no measurable metabolism of gefitinib, while CYP3A5 produced a range of metabolites similar to CYP3A4, but to a much lower degree. In contrast, CYP2D6 catalysed rapid and extensive metabolism of gefitinib to M523595. While formation of M523595 was CYP2D6 mediated, the overall metabolism of gefitinib was dependent primarily on CYP3A4, and this was not obviously diminished in liver microsomes from CYP2D6 poor metabolizers.     

Cytochrome P450-dependent metabolism of gefitinib

The in vitro metabolism of [¹⁴C]-gefitinib (1–3 µM) was investigated using human liver microsomes and a range of expressed human cytochrome P450 enzymes, with particular focus on the formation of O-desmethyl-gefitinib (M523595), the major metabolite observed in human plasma. High-performance liquid chromatography with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of metabolites. On incubation with pooled human liver microsomes, [¹⁴C]-gefitinib underwent rapid and extensive metabolism to a number of metabolites, although M523595 was only a minor microsomal product. Formation of most metabolites was markedly decreased by ketoconazole, but M523595 production was inhibited only by quinidine. Gefitinib was metabolized extensively by expressed CYP3A4, producing a similar range of metabolites to liver microsomes, but M523595 was not formed. CYP1A2, 2C9 and 2C19 produced no measurable metabolism of gefitinib, while CYP3A5 produced a range of metabolites similar to CYP3A4, but to a much lower degree. In contrast, CYP2D6 catalysed rapid and extensive metabolism of gefitinib to M523595. While formation of M523595 was CYP2D6 mediated, the overall metabolism of gefitinib was dependent primarily on CYP3A4, and this was not obviously diminished in liver microsomes from CYP2D6 poor metabolizers.     

Characterization of human liver cytochrome P450 enzymes involved in the metabolism of rutaecarpine

Rutaecarpine has recently been characterized to have an anti-inflammatory activity through cyclooxygenase-2 inhibition. The incubation of rutaecarpine with human liver microsomes in the presence of NADPH generated six isobaric mono-hydroxylated metabolites. The specific cytochrome P450 (CYP) isozymes responsible for rutaecarpine metabolites were identified using the combination of chemical inhibition, immuno-inhibition and metabolism by cDNA expressed CYP enzymes. The results suggested that CYP3A4 might play major roles in the metabolism of rutaecarpine in human liver microsomes. The production of M1, M2, M3, M4 and M6 formed in human liver microsomes was inhibited by ketoconazole, a selective CYP3A4 inhibitor, and anti-CYP3A4 antibody. CYP1A2 and CYP2C9 played minor roles in the metabolism of rutaecarpine. These results were confirmed in microsomes derived from cDNA expressed lymphoblastoid cells. CYP3A4 microsome clearly formed M1, M2, M3 and M6. CYP1A2 and CYP2C9 microsomes comparably formed M5.     

In vitro metabolism of MK-0767 [(+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl) phenyl]methyl]benzamide], a peroxisome proliferator-activated receptor alpha/gamma agonist. I. Role of cytochrome P450, methyltransferases, flavin monooxygenases, and esterases.

The metabolism of MK-0767, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl) phenyl]methyl]benzamide, a thiazolidinedione (TZD)-containing peroxisome proliferator-activated receptor alpha/gamma agonist, was studied in liver microsomes and hepatocytes from humans and rat, dog, and rhesus monkey, to characterize the enzyme(s) involved in its metabolism. The major site of metabolism is the TZD ring, which underwent opening catalyzed by CYP3A4 to give the mercapto derivative, M22. Other metabolites formed in NADPH-fortified liver microsomes included the TZD-5-OH derivative (M24), also catalyzed by CYP3A4, and the O-desmethyl derivative (M28), whose formation was catalyzed by CYP2C9 and CYP2C19. Metabolite profiles from hepatocyte incubations were different from those generated with NADPH-fortified microsomal incubations. In addition to M22, M24, and M28, hepatocytes generated several S-methylated metabolites, including the methyl mercapto (M25), the methyl sulfoxide amide (M16), and the methyl sulfone amide (M20) metabolites. Addition of the methyl donor, S-adenosyl methionine, in addition to NADPH, to microsomal incubations enhanced the turnover and resulted in metabolite profiles similar to those in hepatocyte incubations. Collectively, these results indicated that methyltransferases played a major role in the metabolism of MK-0767. Using enzyme-specific inhibitors, it was concluded that microsomal thiol methyltransferases play a more important role than the cytosolic thiopurine methyltransferase. Baculovirus-expressed human flavin-containing monooxygenase 3, as well as CYP3A4, oxidized M25 to M16, whereas further oxidation of M16 to M20 was catalyzed mainly by CYP3A4. Esterases were involved in the formation of the methyl sulfone carboxylic acids, minor metabolites detected in hepatocytes.     

Identification of human cytochrome P450 enzymes involved in the metabolism of IN-1130, a novel activin receptor-like kinase-5 (ALK5) inhibitor

1. The in vitro metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1,130), a selective activin receptor-like kinase-5 (ALK5) inhibitor and a candidate drug for fibrotic disease, was studied. 2. The cytochrome P450s (CYPs) responsible for metabolism of IN-1,130 in liver microsomes of rat, mouse, dog, monkey and human, and in human CYP supersomestrade mark, were identified using specific CYP inhibitors. The order of disappearance of IN-1,130 in various liver microsomal systems studied was as follows: monkey, mouse, rat, human, and dog. 3. Five distinct metabolites (M1-M5) were identified in all the above microsomes and their production was substantially inhibited by CYP inhibitors such as SKF-525A and ketoconazole. Among nine human CYP supersomestrade mark examined, CYP3A4, CYP2C8, CYP2D6 1, and CYP2C19 were involved in the metabolism of IN-1,130, and the production of metabolites were significantly inhibited by specific CYP inhibitors. IN-1,130 disappeared fastest in CYP2C8 supersomes. CYP3A4 produced four metabolites of IN-1,130 (M1-M4), whereas supersomes expressing human FMO cDNAs, such as FMO1, FMO3, and FMO5, produced no metabolites. 4. Hence, it is concluded that metabolism of IN-1,130 is mediated by CYP3A4, CYP2C8, CYP2D6 1, and CYP2C19.     

In vitro metabolism of genistein and tangeretin by human and murine cytochrome P450s

Recombinant cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6 enzymes obtained from Escherichia coli and human liver microsomes samples were used to investigate the ability of human CYP enzymes to metabolize the two dietary flavonoids, genistein and tangeretin. Analysis of the metabolic profile from incubations with genistein and human liver microsomes revealed the production of five different metabolites, of which three were obtained in sufficient amounts to allow a more detailed elucidation of the structure. One of these metabolites was identified as orobol, the 3'-hydroxylated metabolite of genistein. The remaining two metabolites were also hydroxylated metabolites as evidenced by LC/MS. Orobol was the only metabolite formed after incubation with CYP1A2. The two major product peaks after incubation of tangeretin with human microsomes were identical with 4'-hydroxy-5,6,7,8-tetramethoxyflavone and 5,6-dihydroxy-4',7,8-trimethoxyflavone, previously identified in rat urine in our laboratory. By comparison with UV spectra and LC/MS fragmentation patterns of previously obtained standards, the remaining metabolites eluting after 14, 17 and 20 min. were found to be demethylated at the 4',7-, 4',6-positions or hydroxylated at the 3'- and demethylated at the 4'-positions, respectively. Metabolism of tangeretin by recombinant CYP1A2, 3A4, 2D6 and 2C9 resulted in metabolic profiles that qualitatively were identical to those observed in the human microsomes. Inclusion of the CYP1A2 inhibitor fluvoxamine in the incubation mixture with human liver microsomes resulted in potent inhibition of tangeretin and genistein metabolism. Other isozymes-selective CYP inhibitors had only minor effects on tangeretin or genistein metabolism. Overall the presented observations suggest major involvement of CYP1A2 in the hepatic metabolism of these two flavonoids.     

Identification of human cytochrome P450 enzymes involved in the metabolism of IN-1130, a novel activin receptor-like kinase-5 (ALK5) inhibitor

1.?The in vitro metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130), a selective activin receptor-like kinase-5 (ALK5) inhibitor and a candidate drug for fibrotic disease, was studied.

2.?The cytochrome P450s (CYPs) responsible for metabolism of IN-1130 in liver microsomes of rat, mouse, dog, monkey and human, and in human CYP supersomes?, were identified using specific CYP inhibitors. The order of disappearance of IN-1130 in various liver microsomal systems studied was as follows: monkey, mouse, rat, human, and dog.

3.?Five distinct metabolites (M1–M5) were identified in all the above microsomes and their production was substantially inhibited by CYP inhibitors such as SKF-525A and ketoconazole. Among nine human CYP supersomes? examined, CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19 were involved in the metabolism of IN-1130, and the production of metabolites were significantly inhibited by specific CYP inhibitors. IN-1130 disappeared fastest in CYP2C8 supersomes. CYP3A4 produced four metabolites of IN-1130 (M1–M4), whereas supersomes expressing human FMO cDNAs, such as FMO1, FMO3, and FMO5, produced no metabolites.

4.?Hence, it is concluded that metabolism of IN-1130 is mediated by CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19.     

In vitro metabolism of a triclyclic alkaloid (M445526) in human liver microsomes and hepatocytes

The in vitro metabolism of M445,526 (ZD6,126 phenol) was investigated by incubating [(14)C]-M445,526 at a concentration of 10 microg ml(-1) with human hepatic microsomes (4 mg ml(-1)) or human hepatocytes (2 x 10(6) cells ml(-1)) for up to 180 min. Following incubation with microsomes and hepatocytes, up to 78% and 40% of [(14)C]-M445,526 was metabolized after 180 and 120 min, respectively. High-performance liquid chromatography (HPLC) with radiochemical detection confirmed extensive metabolism of [(14)C]-M445,526 by microsomes and hepatocytes. Mass spectrometry and (1)H-NMR spectroscopy enabled structural identification of up to eight metabolites. Human liver microsomes formed one major (O-desmethyl) and three minor (a further O-desmethyl and two different hydroxylated) phase I metabolites. Human hepatocytes produced one major metabolite, a sulphate conjugate of the major O-desmethyl metabolite formed by microsomes. Four minor metabolites were also formed, primarily by O-demethylation with subsequent glucuronidation. Taken collectively, [(14)C]-M445,526 underwent extensive in vitro metabolism by human liver fractions. These data were confirmed by subsequent human in vivo studies.     

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.     

In vitro metabolism of the calmodulin antagonist DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) by human liver microsomes: involvement of cytochromes p450 in atypical kinetics and potential drug interactions.

Human cytochrome P450 (P450) isozyme(s) responsible for metabolism of the calmodulin antagonist 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e) and kinetic profiles for formation of its six primary metabolites [M3, M5, M6, M7, M8, and DY-9836 (3-[2-[4-(3-chloro-2-methylphenyl)piperazinyl]ethyl]-5,6-dimethoxyindazole)] were identified using human liver microsomes and recombinant P450 enzymes. In vitro experiments, including an immunoinhibition study, correlation analysis, and reactions with recombinant P450 enzymes, revealed that CYP3A4 is the primary P450 isozyme responsible for the formation of the DY-9760e metabolites, except for M5, which is metabolized by CYP2C9. Additionally, at clinically relevant concentrations, CYP2C8 and 2C19 make some contribution to the formation of M3 and M5, respectively. The formation rates of DY-9760e metabolites except for M8 by human liver microsomes are not consistent with a Michaelis-Menten kinetics model, but are better described by a substrate inhibition model. In contrast, the enzyme kinetics for all metabolites formed by recombinant CYP3A4 can be described by an autoactivation model or a mixed model of autoactivation and biphasic kinetics. Inhibition of human P450 enzymes by DY-9760e in human liver microsomes was also investigated. DY-9760e is a very potent competitive inhibitor of CYP2C8, 2C9 and 2D6 (Ki 0.25-1.7 microM), a mixed competitive and noncompetitive inhibitor of CYP2C19 (Ki 2.4 microM) and a moderate inhibitor of CYP1A2 and 3A4 (Ki 11.4-20.1 microM), suggesting a high possibility for human drug-drug interaction.     

In vitro metabolism of BIIB021, an inhibitor of heat shock protein 90, in liver microsomes and hepatocytes of rats, dogs, and humans and recombinant human cytochrome P450 isoforms

Inhibition of heat shock protein 90 (HSP90) results in the degradation of oncoproteins that drive malignant progression and induce cell death, thus making HSP90 a potential target of cancer therapy. 6-Chloro-9-(4-methoxy-3, 5-dimethyl-pyridin-2-ylmethyl)-9H-purin-2-ylamine (BIIB021), a synthetic HSP90 inhibitor, exhibited promising antitumor activity in preclinical models. It is currently in phase II clinical trials for the oral treatment of breast cancer. The objective of this study was to obtain both quantitative and qualitative metabolic profiles of [(14)C]BIIB021 in rat, dog, and human liver microsomes and hepatocytes to provide support for in vivo safety and clinical studies. The metabolites of [(14)C]BIIB021 were identified using liquid chromatography-tandem mass spectrometry coupled with radiometric detection. BIIB021 was extensively metabolized in both liver microsomes and hepatocytes. The major oxidative metabolic pathways identified for all species were due to hydroxylation (M7) and O-demethylation (M2) of the methoxy-dimethylpyridine moiety. The majority of M7 in dog hepatocytes was further conjugated to form the glucuronide (M4). Oxidative dechlorination (M6), monooxygenation (M10), and oxidative N-dealkylation of the methoxy-dimethylpyridine moiety (M11 and M12) were observed as the minor metabolic pathways in hepatocytes of all three species. A glutathione conjugate (M18) was also identified in all species. Its formation was catalyzed, in part, by soluble glutathione transferase via direct displacement of the chlorine on the amino-chloropurine moiety. Subsequent minor secondary metabolites M13, M14, M15, and M17 were observed in human, dog, and rat hepatocytes. Results from incubations of BIIB021 with human recombinant cytochrome P450 (P450) isoforms and a P450 antibody inhibition study in human liver microsomes suggested that the formation of M7 is mainly catalyzed by CYP2C19 and CYP3A4, whereas the formation of minor metabolite M2 in human liver microsomes probably could be attributed to CYP3A4.     

In vitro metabolic study of temsirolimus: preparation, isolation, and identification of the metabolites.

The in vitro metabolism of temsirolimus, (rapamycin-42-[2,2-bis-(hydroxymethyl)]-propionate), an antineoplastic agent, was studied using human liver microsomes as well as recombinant human cytochrome P450s, namely CYP3A4, 1A2, 2A6, 2C8, 2C9, 2C19, and 2E1. Fifteen metabolites were detected by liquid chromatography (LC)-tandem mass spectrometry (MS/MS or MS/MS/MS). CYP3A4 was identified as the main enzyme responsible for the metabolism of the compound. Incubation of temsirolimus with recombinant CYP3A4 produced most of the metabolites detected from incubation with human liver microsomes, which was used for large-scale preparation of the metabolites. By silica gel chromatography followed by semipreparative reverse-phase high-performance liquid chromatography, individual metabolites were separated and purified for structural elucidation and bioactivity studies. The minor metabolites (peaks 1-7) were identified as hydroxylated or desmethylated macrolide ring-opened temsirolimus derivatives by both positive and negative mass spectrometry (MS) and MS/MS spectroscopic methods. Because these compounds were unstable and only present in trace amounts, no further investigations were conducted. Six major metabolites were identified as 36-hydroxyl temsirolimus (M8), 35-hydroxyl temsirolimus (M9), 11-hydroxyl temsirolimus with an opened hemiketal ring (M10 and M11), N- oxide temsirolimus (M12), and 32-O-desmethyl temsirolimus (M13) using combined LC-MS, MS/MS, MS/MS/MS, and NMR techniques. Compared with the parent compound, these metabolites showed dramatically decreased activity against LNCaP cellular proliferation.     

Preclinical pharmacokinetics and metabolism of BMS-214778, a novel melatonin receptor agonist

BMS-214778 is a novel melatonin receptor agonist that may be a useful treatment for sleep disorders that result from disruption of circadian rhythms. Pharmacokinetic studies following intravenous and oral administration and 1 month oral steady-state studies were carried out in rats and monkeys. Rat brain was analyzed for BMS-214778 to determine the extent of its penetration from plasma. Equilibrium dialysis was employed to determine the extent of binding of [(14)C]-BMS-214778 to rat, monkey, and human sera proteins. In vitro metabolism studies with BMS-214778 in rat, monkey, and human liver homogenate preparations (S-9), with monkey and human liver slice preparations, and with pooled human liver microsomes were performed and the incubates analyzed for potential metabolites. Recombinant microsomes expressing specific human cytochrome P(450) (CYP) enzymes were employed to identify possible human metabolic pathways. BMS-214778 showed a high hepatic extraction and high degree of tissue distribution. BMS-214778 also displayed non-linear oral pharmacokinetics. Systemic exposures following oral doses in rats and monkeys increased more than proportionally to the increment in dose. Loss of systemic exposure to BMS-214778 upon chronic oral dosing was observed in male rats where exposure was one-half to two-thirds compared to a single dose, while modest decreases in exposure were observed upon chronic dosing in both sexes of monkey. This was suggestive of induction of BMS-214778 clearance and/or excretion mechanisms. BMS-214778 distributed from the plasma to brain in the rat (mean +/- SD brain:plasma ratio of 0.9 +/- 0.1, N = 3). [(14)C]-BMS-214778 was moderately bound to serum proteins (<91% bound) in all species examined. In vitro metabolism of BMS-214778 was mostly by hydroxylation and dehydrogenation, with CYP1A1, 1A2, 2D6, and 2C9 being the most likely isoforms to be involved in its metabolism in humans.     

Identification of enzymes involved in phase I metabolism of ciclesonide by human liver microsomes.

Ciclesonide, a novel inhaled corticosteroid, is currently being developed for the treatment of asthma. Here, the enzymes catalysing the human hepatic metabolism of ciclesonide were investigated. When incubated with human liver microsomes (HLM), [14C]ciclesonide was first metabolised to the active metabolite M1 (des-isobutyryl-ciclesonide, des-CIC) and to at least two additional metabolites, M2 and M3. M3 comprises a 'family' of structurally similar metabolites that are inactive. 16-Hydroxyprednisolone was also formed in microsomal incubations of [14C]des-CIC, but at approximately one-tenth the amount of both M2 and M3. bis-p-Nitrophenylphosphate and SKF 525-A respectively inhibited des-CIC formation from [14C]ciclesonide by 82% and 49% and M2/M3 formation by 82-84% and 87-89%. Regression analysis showed significant negative correlations (r = -0.96, -0.79 and -0.71, respectively) of M2 formation with CYP3A4/5, CYP2B6 and CYP2C8 activities; M3 formation significantly correlated with CYP4A9/11 (r = 0.47). Troleandomycin and diethyldithiocarbamate inhibited M2 and M3 formation by 85% and 45%, respectively. Sulphaphenazole and quinidine had no inhibitory effects. CYP3A4 Supersomes catalysed notable formation of both M2 and M3 from [14C]des-CIC; CYP2C8 and CYP2D6, but not CYP4A11 formed smaller amounts. It is concluded that the human hepatic metabolism of ciclesonide is primarily catalysed by one or more esterases and, subsequently, by CYP3A4.     

Characterization of human liver cytochrome P450 enzymes involved in the metabolism of a new H+/K+-ATPase inhibitor KR-60436

KR-60436 ([1-(4-methoxy-2-methylphenyl)-4-[(2-hydroxyethyl)amino]-6-trifluoromethoxy-2,3-dihydropyrrolo [3,2-c]quinoline]) is a new reversible H+/K+-ATPase inhibitor. The isoforms of human liver cytochrome P450 (CYP) responsible for the hepatic transformation of KR-60436 is identified. Dihydropyrrole oxidation and O-demethylation are major pathways for the metabolism of KR-60436 in human liver microsomes, whereas N-dehydroxyethylation and hydroxylation are minor pathways. The specific CYP isozymes responsible for KR-60436 oxidation to four major metabolites, pyrrole-KR-60436, O-demethylpyrrole-KR-60436, N-dehydroxyethyl-KR-60436 and an active metabolite, O-demethyl-KR-60436 were identified using the combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by expressed recombinant CYP enzymes. The inhibitory potency of KR-60436 on clinically major CYPs was investigated in human liver microsomes. The results show that CYP3A4 contributes to the oxidation of KR-60436 to pyrrole-KR-60436, O-demethylpyrrole-KR-60436 and N-dehydroxyethyl-KR-60436, and CYP2C9 and CYP2D6 play roles in demethylation of KR-60436 to form the active metabolite, O-demethyl-KR-60436. KR-60436 was found to inhibit potently the metabolism of CYP1A2 substrates.     

Characterization of cytochrome P450s mediating ipriflavone metabolism in human liver microsomes

Ipriflavone, a synthetic flavonoid for the prevention and treatment of osteoporosis, has been reported to be extensively metabolized in man to seven metabolites (M1-M7). This study was performed to characterize the human liver cytochrome P450s (CYP) responsible for the metabolism of ipriflavone. Hydroxylation at the beta-ring to M3, O-dealkylation to M1 and oxidation at isopropyl group to M4 and M5 are major pathways for ipriflavone metabolism in three different human liver microsome preparations. The specific CYPs responsible for ipriflavone oxidation to the active metabolites, M1, M3, M4 and M5 were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by expressed recombinant CYP enzymes. The inhibitory potencies of ipriflavone and its five metabolites, M1-M5 on seven clinically important CYPs were investigated in human liver microsomes. Our results demonstrate that CYP3A4 plays the major role in O-dealkylation of ipriflavone to M1 and CYP1A2 plays a dominant role in the formation of M3, M4 and M5. Ipriflavone and/or its five metabolites were found to inhibit potently the metabolism of CYPs 1A2, 2C8, 2C9 and 2C19 substrates.     

Metabolism of MK-0524, a prostaglandin D2 receptor 1 antagonist, in microsomes and hepatocytes from preclinical species and humans.

(3R)-4-(4-Chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl acetic acid (MK-0524) is a potent orally active human prostaglandin D(2) receptor 1 antagonist that is currently under development for the prevention of niacin-induced flushing. The major in vitro and in vivo metabolite of MK-0524 is the acyl glucuronic acid conjugate of the parent compound, M2. To compare metabolism of MK-0524 across preclinical species and humans, studies were undertaken to determine the in vitro kinetic parameters (K(m) and V(max)) for the glucuronidation of MK-0524 in Sprague-Dawley rat, beagle dog, cynomolgus monkey, and human liver microsomes, human intestinal microsomes, and in recombinant human UDP glucuronosyltransferases (UGT). A comparison of K(m) values indicated that UGT1A9 has the potential to catalyze the glucuronidation of MK-0524 in the liver, whereas UGT1A3 and UGT2B7 have the potential to catalyze the glucuronidation in the intestine. MK-0524 also was subject to phase I oxidative metabolism; however, the rate was significantly lower than that of glucuronidation. The rate of phase I metabolism was ranked as follows: rat approximately monkey > human intestine > dog > human liver with qualitatively similar metabolite profiles across species. In all the cases, the major metabolites were the monohydroxylated epimers (M1 and M4) and the keto-metabolite, M3. Use of inhibitory monoclonal antibodies and recombinant human cytochromes P450 suggested that CYP3A4 was the major isozyme involved in the oxidative metabolism of MK-0524, with a minor contribution from CYP2C9. The major metabolite in hepatocyte preparations was the acyl glucuronide, M2, with minor amounts of M1, M3, M4, and their corresponding glucuronides. Overall, the in vivo metabolism of MK-0524 is expected to proceed via glucuronidation, with minor contributions from oxidative pathways.     

In vitro identification of metabolic pathways and cytochrome P450 enzymes involved in the metabolism of etoperidone

1. In vitro studies have been carried out to investigate the metabolic pathways and identify the hepatic cytochrome P450 (CYP) enzymes involved in etoperidone (Et) metabolism. 2. Ten in vitro metabolites were profiled, quantified and tentatively identified after incubation with human hepatic S9 fractions. Et was metabolized via three metabolic pathways: (A) alkyl hydroxylation to form OH-ethyl-Et (M1); (B) phenyl hydroxylation to form OH-phenyl-Et (M2); and (C) N-dealkylation to form 1-m-chlorophenylpiperazine (mCPP, M8) and triazole propyl aldehyde (M6). Six additional metabolites were formed by further metabolism of M1, M2, M6 and M8. 3. Kinetic studies revealed that all metabolic pathways were monophasic, and the pathway leading to the formation of OH-ethyl-Et was the most efficient at eliminating the drug. On incubation with microsomes expressing individual recombinant CYPs, formation rates of M1-3 and M8 were 10-100-fold greater for CYP3A4 than that for other CYP forms. The formation of these metabolites was markedly inhibited by the CYP3A4-specific inhibitor ketoconazole, whereas other CYP-specific inhibitors did not show significant effects. In addition, the production of M1-3 and M8 was strongly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activities in 13 different human liver microsome samples. 4. Dealkylation of the major metabolite M1 to form mCPP (M8) was also investigated using microsomes containing recombinant CYP enzymes. The rate of conversion of M1 to mCPP by CYP3A4 was 503.0 +/- 3.1 pmole nmole(-1) min(-1). Metabolism of M1 to M8 by other CYP enzymes was insignificant. In addition, this metabolism in human liver microsomes was extensively inhibited by the CYP3A4 inhibitor ketoconazole, but not by other CYP-specific inhibitors. In addition, conversion of M1 to M8 was highly correlated with CYP3A4-mediated testosterone 6beta-hydroxylase activity. 5. The results strongly suggest that CYP3A4 is the predominant enzyme-metabolizing Et in humans.