Recombinant Human Cytochrome P450 Enzymes Research Articles

Antifungal activities of novel non-azole molecules against S. cerevisiae and C. albicans

Because of the increasing number of immunocompromised patients and due to problems with antifungal treatment, especially with the most widely used antifungals, azoles, there is an urgent need for new, potent and safe antifungals with fewer cytochrome P450 (CYP)-mediated interactions with other drugs. In the present study, 54 novel non-azole molecules were selected with the help of molecular modelling and virtual molecule database screening to identify new fungistatic or fungicidic compounds with functional groups that would produce reactive intermediates killing the yeast cells. Database screening and selection of tested compounds were based on the construction of two pharmacophores and docking hits to the active site of the CYP51 homology model. Inhibition potency of the compounds was tested against Saccharomyces cerevisiae and/or Candida albicans. Two new structured compounds, 2-({4-[(2-cyanoethyl)(methyl) amino]benzylidene} amino)-5-(3,4-dimethoxyphenyl)-4-methylthiophene-3-carbonitrile and 2-[([1,1′-biphenyl]-4-ylmethylene)amino]-5-(3,4-dimethoxyphenyl)-4-methylthiophene-3-carbonitrile were discovered to have promising antifungal properties based on bioassays. Inhibition screen of human hepatic CYP enzymes revealed that these two compounds did not inhibit potently five human recombinant CYP enzymes. The results of this study indicate that the functional groups of the two compounds may produce reactive intermediates when located at the active site of CYP51.

Disposition and Drug-Drug Interaction Potential of Veliparib (ABT-888), a Novel and Potent Inhibitor of Poly(ADP-ribose) Polymerase

The disposition of veliparib [(R)-2-(2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxamide, ABT-888], a novel and potent inhibitor of poly(ADP-ribose) polymerase for the treatment of cancers, was investigated in rats and dogs after intravenous and oral administration of [(3)H]veliparib and compared with that of humans. Veliparib absorption was high. Dosed radioactivity was widely distributed in rat tissues. The majority of drug-related material was excreted in urine as unchanged drug (approximately 54, 41, and 70% of the dose in rats, dogs, and humans, respectively). A lactam M8 and an amino acid M3 were two major excretory metabolites in animals. In the circulation of animals and humans, veliparib was the major drug-related component, and M8 was one of the major metabolites. Monooxygenated metabolite M2 was significant in the rat and dog, and M3 was also significant in the dog. Veliparib biotransformation occurred on the pyrrolidine moiety via formation of a lactam, an amino acid, and an N-carbamoyl glucuronide, in addition to oxidation on benzoimidazole carboxamide and sequential glucuronidation. In vitro experiments using recombinant human cytochrome P450 (P450) enzymes identified CYP2D6 as the major enzyme metabolizing veliparib with minor contributions from CYP1A2, 2C19, and 3A4. Veliparib did not inhibit or induce the activities of major human P450s. Veliparib was a weak P-glycoprotein (P-gp) substrate, showing no P-gp inhibition. Taken together, these studies indicate a low potential for veliparib to cause clinically significant P-gp or P450-mediated drug-drug interactions (DDIs). Overall, the favorable dispositional and DDI profiles of veliparib should be beneficial to its safety and efficacy.

Characterization of metabolites and human P450 isoforms involved in the microsomal metabolism of mesaconitine

Mesaconitine (MA), a major Aconitum alkaloid, provides effects against rheumatosis with high toxicity. To supply information for clinical safety, this study aims to investigate the metabolism of MA in male human liver microsomes (MHLMs) and the CYP isoforms involved in its metabolism.Metabolism studies were performed in vitro using MHLMs. Selective chemical inhibitors and recombinant human cytochrome P450 enzymes were used to confirm that the CYP isoforms contributed to MA metabolism.A total of nine metabolites were found and characterized in the MHLM incubations. The metabolic pathways were demethylation, dehydrogenation, hydroxylation, and demethylation–dehydrogenation. Results showed that the inhibitor of CYP3A had a strong inhibitory effect; the inhibitors of CYP2C8, CYP2C9, CYP2C19, and CYP2D6 had modest inhibitory effects, whereas inhibitors of CYP1A2 and CYP2E1 had no obvious inhibitory effects on MA metabolism. Recombinant human cytochrome P450 isoforms CYP3A4 and CYP3A5 contributed greatly to the formation of MA metabolites, and CYP2C8, CYP2C9, and CYP2D6 played a minor role in the formation of MA metabolites.MA could be transformed into at least nine metabolites in MHLMs. MA might be metabolized by CYP3A4, CYP3A5, CYP2C8, CYP2C9, and CYP2D6 in MHLMs.

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n=36) consist of compounds isolated from the Papaveraceae family (n=20), synthetic analogs (n=15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.

Metabolism of Ramelteon in Human Liver Microsomes and Correlation with the Effect of Fluvoxamine on Ramelteon Pharmacokinetics

Ramelteon is a melatonin receptor agonist used as a treatment for insomnia. It is subject to a remarkably large drug-drug interaction (DDI) caused by fluvoxamine coadministration, resulting in a more than 100-fold increase in exposure. The objective of this study was to determine whether the DDI could be estimated using in vitro metabolism data. Ramelteon was shown to undergo hydroxylation in human liver microsomes to eight metabolites via six pathways. The main routes of metabolism included hydroxylation on the ethyl side chain and the benzylic position of the cyclopentyl ring, as assessed through enzyme kinetic measurements. Hydroxylation at the other benzylic position was observed in human intestinal microsomes. Ramelteon metabolism was catalyzed by CYP1A2, CYP2C19, and CYP3A4 as shown through the use of recombinant human cytochrome P450 enzymes and specific inhibitors. In liver, CYP1A2, CYP2C19, and CYP3A4 were estimated to contribute 49, 42, and 8.6%, respectively, whereas in intestine only CYP3A4 contributes. The in vitro data were used to estimate the magnitudes of DDI caused by ketoconazole, fluconazole, and fluvoxamine. The DDIs caused by the former were reliably estimated (1.82-fold estimated versus 1.82-fold actual for ketoconazole; 2.99-fold estimated versus 2.36-fold actual for fluconazole), whereas for fluvoxamine it was underestimated (11.4-fold estimated versus 128-fold actual). This suggests that there may be a limit on the magnitude of DDI that can be estimated from in vitro data. Nevertheless, the example of the fluvoxamine-ramelteon DDI offers a unique example wherein one drug can simultaneously inhibit multiple enzymatic pathways of a second drug.

Comparison between recombinant P450s and human liver microsomes in the determination of cytochrome P450 Michaelis–Menten constants

Non-linear dose–exposure (supra-proportionality) occurs when plasma drug concentrations increase in a non-linear fashion with increasing dose. To predict the likelihood of this, an understanding is required of the KM, which reflects a drug ability to saturate a specific enzyme involved in its metabolism.This study assessed the accuracy of KM and Vmax determinations for compounds using a substrate-depletion approach with those determined using the product-formation approach, using both recombinant human cytochrome P450 (CYP) enzymes and human liver microsomes.For the vast majority of the compounds studied, the KM’s using recombinant CYPs and human liver microsomes in the two approaches predicted within two-fold. Further comparisons between the KM and Vmax-values were made between those measured using the product-formation approach and those estimated following simultaneous fitting of the Michaelis–Menten equation to all substrate depletion plots. In each case values were comparable.In conclusion, the current study showed the substrate-depletion approach can be used to estimate KM and Vmax using both human liver microsomes and recombinant P450s. Estimation of these parameters during early discovery will aid in the understanding of dosages at which non-linearity may occur, but potentially aid predictions of likely clinical drug–drug interactions.

Pharmacogenetics of a PARP inhibitor ABT-888 metabolic pathway

e14556 Background: Poly(ADP-ribose) polymerase (PARP) is essential for single-stranded DNA break repair and repair of DNA damage can lead to radio- and chemo-resistance. Thus, inhibition of PARP activity can sensitize cells to cytotoxic therapies. ABT-888 is a potent, orally bioavailable PARP inhibitor. Preclinical studies suggest that ABT-888 potentiates multiple cytotoxic agents and its efficacy is correlated with plasma/tumor drug concentrations. The objective of this study was to determine the pharmacogenetic effect of genetic variants in the ABT-888 metabolic pathway, with the aim to better understand molecular basis of the variation in ABT-888 pharmacokinetics (PK) and therapeutic outcome. Methods: The major enzymes responsible for ABT-888 metabolism were identified by in vitro metabolism studies with specific recombinant human cytochrome P450 (CYP) enzymes. The functional significance of genetic variants of the identified enzymes was assessed by examining ABT-888 metabolic kinetics by candidate variant enzymes and microsomes. The association of the functional significant genetic variants with the PK and clinical outcome is being evaluated in the context of an ongoing phase I trial in which ABT-888 is administered in combination with irinotecan in patients with advanced solid tumors. Results: ABT-888 was metabolized predominantly by human CYP2D6, to a less extent by CYP1A1, and to a negligible extent by CYP1A2, 2C9, 2C19, 3A4, and 3A5. CYP2D6*10 exhibited markedly reduced catalytic capability in ABT-888 overall metabolism and the metabolite (A-925088) formation, with in vitro maximum clearance being 31% and 5.3%, respectively, of that estimated from the wild-type CYP2D6. In human liver microsomes carrying homozygous CYP2D6*4, the rates of parent drug disappearance and metabolite formation were significantly lower than those observed in the microsomes carrying wild-type CYP2D6, P &lt; 0.05. Conclusions: CYP2D6 is the predominant enzyme responsible for the hepatic metabolism of ABT-888. Common allelic variants CYP2D6*10 and *4 are associated with significantly reduced metabolic activity towards ABT-888. CYP2D6 polymorphisms may influence the PK and therapeutic outcome of ABT-888. Its clinical relevance remains to be determined. No significant financial relationships to disclose.

Acetaminophen bioactivation by human cytochrome P450 enzymes and animal microsomes

Acetaminophen is a widely used analgesic antipyretic agent. When used at low doses, it is a safe drug, but at higher doses it can cause acute hepatic necrosis in humans and experimental animals. The key mechanism in the hepatotoxicity is cytochrome P450 (CYP)-catalysed formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) that is capable of binding to cellular macromolecules and in that way an LC/MS liquid chromatography/mass spectrometry (LC/MS) method was developed to measure NAPQI formation by trapping it to reduced glutathione. This method was used to determine the bioactivation of acetaminophen at two concentrations: 50 μM therapeutic and 1 mM toxic by using nine human recombinant CYP enzymes: CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4; and with different microsomes from experimental animals. At the toxic concentration the formation of NAPQI–glutathione was highest with CYP3A4 followed by CYP2E1, CYP1A2, and CYP2D6. At the therapeutic concentration, CYP3A4 had also the highest bioactivation capacity. In a comparison of the enzyme kinetics, CYP3A4 was the most efficient CYP with the lowest Km value 130 μM (95% confidence interval = 63–210 μM). Dexamethasone-induced rat liver microsomes had the most effective bioactivation capacity at therapeutic and toxic acetaminophen concentrations. This study suggests that CYP3A4 is the major CYP enzyme form catalysing acetaminophen oxidation to NAPQI in human liver.

Oxidation of carcinogenic 2-nitroanisole by rat cytochromes P450 - similarity between human and rat enzymes

2-Nitroanisole (2-NA) is an important industrial pollutant and a potent carcinogen for rodents. Understanding which cytochrome P450 (CYP) enzymes are involved in its metabolism are important to assess an individual's susceptibility to this environmental carcinogen. The aim of this study was to evaluate the efficiency of rat hepatic CYPs to oxidize 2-NA, to examine the metabolites formed during such an oxidation, and to compare such efficiencies of rat CYPs with those of human. 2-NA is oxidized by rat hepatic microsomes to 2-nitrophenol (2-NP) as the major metabolite, and to 2,6-dihydroxynitrobenzene (2,6-DNB) and 2,5-dihydroxynitrobenzene (2,5-DNB) as the minor products. All these metabolites are suggested as detoxication products. Using hepatic microsomes of rats pre-treated with specific CYP inducers and microsomes from Baculovirus transfected insect cells expressing recombinant rat and human CYP enzymes we found that rat recombinant CYP2E1, 2D2, 2B2, 2C6 and 1A1, as well as orthologous human CYP enzymes are the most efficient enzymes metabolizing 2-NA. However, human CYP1A1 oxidize 2-NA with a higher efficiency than the enzyme of rats. The results show the participation of orthologous CYPs in 2-NA oxidation by both species and underline the suitability of rat species as a model to evaluate human susceptibility to 2-NA.

Development of immobilized enzyme reactors based on human recombinant cytochrome P450 enzymes for phase I drug metabolism studies

Two cytochrome P450 (CYP)-based immobilized enzyme reactors (IMERs) were developed to perform automated on-line phase I drug metabolism studies. For this purpose, biotinylated recombinant CYP2D6 or CYP3A4 reconstituted systems were anchored to the surface of two monolithic mini-columns (2 mm × 6 mm I.D.), which had been covalently grafted with NeutrAvidin. After optimization of immobilization conditions, the obtained IMERs were integrated on-line into a LC hyphenated to an electrospray ionization MS/MS system. Studies with probe substrates and a known competitive inhibitor were performed, showing the potential of CYP-based IMERs in drug metabolism. In the optimized conditions, ca. 15 experiments were carried out with each bioreactor.

Bioconversion of Mono- and Sesquiterpenoids by Recombinant Human Cytochrome P450 Monooxygenases

Cytochrome P450 monooxygenases play an important role in the biosynthesis and metabolism of terpenoids. We explored the potential of recombinant human liver cytochrome P450 monooxygenases CYP1A2, CYP2C9, and CYP3A4, heterologously expressed in Escherichia coli, to convert mono- and sesquiterpenoids to human metabolites. This natural product group is a diverse class of secondary metabolites and includes several industrially and pharmaceutically interesting compounds. Incubation of cedrol with CYP3A4 resulted in a bioconversion of 74% (±8.9%) after 1 h of the unknown metabolites 2-hydroxycedrol and 4-hydroxycedrol, which have been structurally elucidated by 1H and 13C NMR and GC-MS. We conclude that recombinant human cytochrome P450 enzymes can be useful tools in a combinatorial biosynthesis strategy for the production of new natural products and for in vitro metabolization studies.

Metabolism of CJ-036878, N-(3-phenethoxybenzyl)-4-hydroxybenzamide, in liver microsomes and recombinant cytochrome P450 enzymes: metabolite identification by LC-UV/MSn and 1H-NMR

The identification of metabolites in the early stages of drug discovery is important not only for guiding structure–activity relationships (SAR) and structure–metabolism relationships (SMR) strategies, but also for predicting the potential for adverse events. The present study investigated the phase I metabolism of CJ-036878 (N-(3-phenethoxybenzyl)-4-hydroxybenzamide), a potent antagonist of the N-methyl-D-asparatate (NMDA) receptor, using liver microsomes and representative recombinant cytochrome P450 enzymes. The structures of the oxidative metabolites M1–M11 were confirmed by LC-UV/MSn and/or 1H-nuclear magnetic resonance (NMR). It was found that CJ-036878 is metabolized through three routes: (1) aliphatic hydroxylation that generates M1 and M2; (2) aromatic hydroxylation that produces M3–M5, M7 and M8; and (3) dimerization through an oxidative phenol coupling reaction that yields M10 and M11. The use of recombinant human cytochrome P450 enzymes suggested that CYP3A4 is the major enzyme involved in the oxidative metabolism of CJ-036878, with minor contributions from CYP1A2, CYP2C19, and CYP2D6.

Study on the cytochrome P450-mediated oxidative metabolism of the terpene alcohol linalool: Indication of biological epoxidation

The cytochrome P450-mediated oxidative metabolism of the terpene alcohol linalool was studied in vitro by enzymatic assays using recombinant human cytochrome P450 enzymes. Three different enzymatic products of allylic hydroxylation and epoxidation were identified by gas chromatography-mass spectrometry. Identified enzymatic products were 8-hydroxylinalool ((R/S)-3,7-dimethyl-1,6-octadiene-3,8-diol) and the cyclic ethers pyranoid-linalool oxide ((R/S)-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-ol) and furanoid-linalool oxide (R/S)-2-(1,1-dimethylethyl)-5-methyl-5-vinyltetrahydrofuran. The cyclic ethers result most likely from the epoxidation of the 6,7-carbon double carbon bond of (R/S)-linalool, followed by the intramolecular rearrangement of the 6,7-epoxy-linalool. Allylic-hydroxylation of the 8-methyl group of linalool was catalyzed by CYP2C19 and CYP2D6 while the enzymatic epoxidation of linalool was only observed with CYP2D6. The results indicate that the electrophilic oxidation products of linalool such as 6,7-epoxy-linalool which may cause sensitization and irritational skin reactions are not only produced by auto-oxidation reactions in the presence of air-oxygen as published in the past, but also by P450-mediated oxidative biological transformation.

Study on the cytochrome P450-mediated oxidative metabolism of the terpene alcohol linalool: Indication of biological epoxidation

The cytochrome P450-mediated oxidative metabolism of the terpene alcohol linalool was studied in vitro by enzymatic assays using recombinant human cytochrome P450 enzymes. Three different enzymatic products of allylic hydroxylation and epoxidation were identified by gas chromatography-mass spectrometry. Identified enzymatic products were 8-hydroxylinalool ((R/S)-3,7-dimethyl-1,6-octadiene-3,8-diol) and the cyclic ethers pyranoid-linalool oxide ((R/S)-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-ol) and furanoid-linalool oxide (R/S)-2-(1,1-dimethylethyl)-5-methyl-5-vinyltetrahydrofuran. The cyclic ethers result most likely from the epoxidation of the 6,7-carbon double carbon bond of (R/S)-linalool, followed by the intramolecular rearrangement of the 6,7-epoxy-linalool. Allylic-hydroxylation of the 8-methyl group of linalool was catalyzed by CYP2C19 and CYP2D6 while the enzymatic epoxidation of linalool was only observed with CYP2D6. The results indicate that the electrophilic oxidation products of linalool such as 6,7-epoxy-linalool which may cause sensitization and irritational skin reactions are not only produced by auto-oxidation reactions in the presence of air-oxygen as published in the past, but also by P450-mediated oxidative biological transformation.

Cytochrome P450-Mediated Metabolism of Haloperidol and Reduced Haloperidol to Pyridinium Metabolites

Haloperidol (HP) has been reported to undergo cytochrome P450 (P450)-mediated metabolism to potentially neurotoxic pyridinium metabolites; however, the chemical pathways and specific enzymes involved in these reactions remain to be identified. The aims of the current study were to (i) fully identify the cytochrome P450 enzymes capable of metabolizing HP to the pyridinium metabolite, 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-oxobutylpyridinium (HPP(+)), and reduced HP (RHP) to 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-hydroxybutylpyridinium (RHPP(+)); and (ii) determine whether 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-oxobutyl-1,2,3,6-tetrahydropyridine (HPTP) and 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-hydroxybutyl-1,2,3,6-tetrahydropyridine (RHPTP) were metabolic intermediates in these pathways. In vitro studies were conducted using human liver microsomal preparations and recombinant human cytochrome P450 enzymes (P450s 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19 2D6, 2E1, 3A4, 3A5, and 3A7) expressed in bicistronic format with human NADPH cytochrome P450 reductase in Escherichia coli membranes. Pyridinium formation from HP and RHP was highly correlated across liver preparations, suggesting the same enzyme or enzymes were responsible for both reactions. Cytochrome P450s 3A4, 3A5, and 3A7 were the only recombinant enzymes which demonstrated significant catalytic activity under optimized conditions, although trace levels of activity could be catalyzed by NADPH-P450 reductase alone. NADPH-P450 reductase-mediated activity was inhibited by reduced glutathione but not catalase or superoxide dismutase, suggesting O(2)-dependent oxidation. No evidence was obtained to support the contention that HPTP and RHPTP are intermediates in these pathways. K(m) values for HPP(+) (34 +/- 5 microM) and RHPP(+) (64 +/- 4 microM) formation by recombinant P450 3A4 agreed well with those obtained with human liver microsomes, consistent with P450 3A4 being the major catalyst of pyridinium metabolite formation in human liver.

Role of Polymorphic Human Cytochrome P450 Enzymes in Estrone Oxidation

Estrogen and its metabolites are believed to play important roles in breast cancer. The influence of genetic polymorphisms in the enzymes responsible for formation and disposition of estrogen on breast cancer risk may shed light on the importance of estrogen metabolites in this disease. However, for such studies to be valid, it is important to correctly identify the enzymes involved in estrogen bioactivation. Therefore, we assessed the human cytochrome P450-dependent oxidation of estrone using substrate concentrations that more closely approximate the maximum expected concentrations in breast tissue. The in vitro metabolism of estrone by recombinant human cytochrome P450 enzymes and human liver microsomes was studied. The formation of estrone metabolites (2-hydroxyestrone, 4-hydroxyestrone, and 16alpha-hydroxyestrone) was monitored by high-performance liquid chromatography. 2-Hydroxyestrone formation was catalyzed predominantly by CYP1A2, CYP1A1, and CYP1B1 enzymes; 4-hydroxyestrone formation was catalyzed predominantly by CYP1B1, CYP1A2, and CYP1A1 enzymes; and 16alpha-hydroxyestrone formation was catalyzed predominantly by CYP2C19, CYP1A1, and CYP3A5. This study confirms the important role of members of the CYP1 family in the 2-hydroxylation and 4-hydroxylation of estrone, but the enzymes identified as responsible for the 16alpha-hydroxylation of estrone are different from those previously identified. The relative importance of these enzymes in vivo would depend on the specific tissue expression of the enzymes. These enzymes are all known to be genetically variant in the human population, and additional studies to assess the role CYP1A2, CYP2C19, and CYP3A5 in breast cancer risk are indicated.

In vitro metabolism of a new oxazolidinedione hypoglycemic agent utilizing liver microsomes and recombinant human cytochrome P450 enzymes

The compound, 5-{4-[3-(4-cyclohexyl-2-propylphenoxy)propoxy]phenyl}-1,3-oxazolidine-2,4-dione (compound A) is a peroxisome proliferator-activated receptor-γ (PPARγ) agonist. PPARγ agonists have proven useful in the treatment of type 2 diabetes, which is characterized by hyperglycemia, insulin resistance and/or abnormal insulin secretion. The metabolism of this oxazolidinedione (OZD) was investigated in male rat, dog, monkey and human liver microsomes, and recombinant human cytochrome P450 enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in the presence of NADPH. Routes of metabolism included monohydroxylation of the cyclohexane ring at multiple positions, monohydroxylation of the n-propyl side chain or the tether linkage, and OZD ring opening, giving rise to the keto amide and alcohol amide entities. Liver microsomes showed subtle qualitative and quantitative metabolic differences among rat, dog, monkey and human preparations. Further, CYP2C8 and CYP2C19 did not display different regioselectivity for hydroxylation on the cyclohexane ring with both of them giving rise to C-3 and C-4 hydroxy metabolites, but they did display different stereoselectivity with CYP2C8 preferring cyclohexane hydroxylation in equatorial positions and CYP2C19 in axial positions.

Identification of the cytochrome P450 enzymes involved in the metabolism of domperidone

1. The objective was to identify the major cytochrome P450 enzyme(s) involved in the metabolism of domperidone.2. Experiments were performed using human liver microsomes (HLMs), recombinant human cytochrome P450 enzymes, cytochrome P450 chemical inhibitors and monoclonal antibodies directed against cytochrome P450 enzymes.3. Four metabolites were identified from incubations performed with HLMs and excellent correlations were observed between the formation of domperidone hydroxylated metabolites (M1, M3 and M4), N-desalkylated domperidone metabolite (M2) and enzymatic markers of CYP3A4/5 (r2 = 0.9427, 0.951, 0.9497 and 0.8304, respectively).4. Ketoconazole (1 μM) decreased the formation rate of M1, M2, M3 and M4 by 83, 78, 75 and 88%, respectively, whereas the effect of other inhibitors (quinidine, furafylline and sulfaphenazole) was minimal. Important decreases in the formation rate of M1 (68%), M2 (64%) and M3 (54%) were observed with anti-CYP3A4 antibodies.5. Formation of M1, M2 and M3 in HLMs exhibited Michaelis–Menten kinetics (Km: 166, 248 and 36 μM, respectively). Similar Km values were observed for M1, M2 and M3 when incubations were performed with recombinant human CYP3A4 (Km: 107, 273 and 34 μM, respectively).6. The data suggest that CYP3As are the major enzymes involved in the metabolism of domperidone.

Identification of CYP3A4 and CYP2C8 as the major cytochrome P450 s responsible for morphine N -demethylation in human liver microsomes

1. The aim was to identify the cytochrome P450 (CYP) enzymes responsible for the N -demethylation of morphine in vitro. 2. In human liver microsomes, normorphine formation followed Michaelis-Menten kinetics with mean K m and V max of 12.4 ± 2.2 mM and 1546 ± 121 pmol min − 1 mg − 1, respectively. In microsomes from a panel of 14 human livers phenotyped for 10 CYP enzymes, morphine N -demethylation correlated with testosterone 6 β -hydroxylation (r = 0.91, p <0.001) and paclitaxel 6- α hydroxylation (r = 0.72, p <0.001), two specific markers of CYP3A4 and CYP2C8, respectively. Normorphine formation decreased when incubated in the presence of troleandomycin or quercetin (by 46 and 33-36%, respectively), which further corroborates the contribution of CYP3A4 and CYP2C8. 3. Among eight recombinant human CYP enzymes tested, CYP3A4 and CYP2C8 exhibited the highest intrinsic clearance. More than 90% of morphine N -demethylation could be accounted for via the action of both CYP3A4 and CYP2C8. 4. The in vitro findings suggest that hepatic CYP3A4, and to a lesser extent CYP2C8, play an important role in the metabolism of morphine into normorphine.

Metabolism of Cytotoxic Drugs by Recombinant Human Cytochrome P450 Enzymes and Identification of Putative Metabolites by LC-MS/MS

Metabolism of Cytotoxic Drugs by Recombinant Human Cytochrome P450 Enzymes and Identification of Putative Metabolites by LC-MS/MS