Human Liver Microsomal Cytochrome P450 Research Articles

Antiviral Drug Candidate Repositioning for Streptococcus suis Infection in Non-Tumorigenic Cell Models.

The increasing prevalence of antimicrobial resistance against zoonotic bacteria, including Streptococcus (S.) suis, highlights the need for new therapeutical strategies, including the repositioning of drugs. In this study, susceptibilities of bacterial isolates were tested toward ten different 3-amidinophenyalanine (Phe(3-Am)) derivatives via determination of minimum inhibitory concentration (MIC) values. Some of these protease inhibitors, like compounds MI-432, MI-471, and MI-476, showed excellent antibacterial effects against S. suis. Their drug interaction potential was investigated using human liver microsomal cytochrome P450 (CYP450) measurements. In our work, non-tumorigenic IPEC-J2 cells and primary porcine hepatocytes were infected with S. suis, and the putative beneficial impact of these inhibitors was investigated on cell viability (Neutral red assay), on interleukin (IL)-6 levels (ELISA technique), and on redox balance (Amplex red method). The antibacterial inhibitors prevented S. suis-induced cell death (except MI-432) and decreased proinflammatory IL-6 levels. It was also found that MI-432 and MI-476 had antioxidant effects in an intestinal cell model upon S. suis infection. Concentration-dependent suppression of CYP3A4 function was found via application of all three inhibitors. In conclusion, our study suggests that the potential antiviral Phe(3-Am) derivatives with 2',4' dichloro-biphenyl moieties can be considered as effective drug candidates against S. suis infection due to their antibacterial effects.

Modulation of Major Human Liver Microsomal Cytochromes P450 by Component Alkaloids of Goldenseal: Time-Dependent Inhibition and Allosteric Effects.

Botanical and other natural products (NPs) are often coconsumed with prescription medications, presenting a risk for cytochrome P450 (P450)-mediated NP-drug interactions. The NP goldenseal (Hydrastis canadensis) has exhibited antimicrobial activities in vitro attributed to isoquinoline alkaloids contained in the plant, primarily berberine, (-)-β-hydrastine, and to a lesser extent, hydrastinine. These alkaloids contain methylenedioxyphenyl rings, structural alerts with potential to inactivate P450s through formation of metabolic intermediate complexes. Time-dependent inhibition experiments were conducted to evaluate their ability to inhibit major P450 activities in human liver microsomes by using a cocktail of isozyme-specific substrate probes. Berberine inhibited CYP2D6 (dextromethorphan O-demethylation; K I = 2.7 μM, kinact = 0.065 minute-1) and CYP3A4/5 (midazolam 1'-hydroxylation; K I = 14.8 μM, kinact = 0.019 minute-1); (-)-β-hydrastine inhibited CYP2C9 (diclofenac 4'-hydroxylation; K I = 49 μM, kinact = 0.036 minute-1), CYP2D6 (K I > 250 μM, kinact > 0.06 minute-1), and CYP3A4/5 (K I = 28 μM, kinact = 0.056 minute-1); and hydrastinine inhibited CYP2D6 (K I = 37 μM, kinact = 0.049 minute-1) activity. Berberine additionally exhibited allosteric effects on midazolam hydroxylation, showing both positive and negative heterotropic cooperativity. Experiments with recombinant isozymes showed that berberine activated midazolam 1'-hydroxylation by CYP3A5, lowering K m(app), but showed mixed inhibition and negative cooperativity toward this reaction when catalyzed by CYP3A4. Berberine inactivated CYP3A4 at a much faster rate than CYP3A5 and was a noncompetitive inhibitor of midazolam 4-hydroxylation by CYP3A4 but a strong mixed inhibitor of the CYP3A5 catalyzed reaction. These complex kinetics should be considered when extrapolating the risk for NP-drug interactions involving goldenseal. SIGNIFICANCE STATEMENT: Robust kinetic parameters were determined for the reversible and time-dependent inhibition of CYP2C9, CYP2D6, and CYP3A4/5 activities in human liver microsomes by major component isoquinoline alkaloids contained in the botanical natural product goldenseal. The alkaloid berberine also exhibited opposing, isozyme-specific allosteric effects on midazolam hydroxylation mediated by recombinant CYP3A4 (inhibition) and CYP3A5 (activation). These data will inform the development of a physiologically based pharmacokinetic model that can be used to predict potential clinically relevant goldenseal-drug interactions.

Mass spectrometric identification of in vitro-generated metabolites of two emerging organophosphate flame retardants: V6 and BDP

The aim of the present study was to investigate the in vitro metabolism of two emerging organophosphate flame retardants, namely tetrekis(2-chlorethyl)dichloroisopentyldiphosphate (V6) and bisphenol-A bis-diphenyl phosphate (BDP) in human liver microsomes (HLMs), HLM S9 fractions and in human serum. In particular, the role of cytochrome P450 (CYPs) enzymes and/or paraoxonases (PONs) in the formation of V6 and BDP phase I metabolites was studied. Mono-, di-hydroxylated and hydrolytic phase I metabolites of V6 were mainly formed by CYPs in HLMs, while hydrolytic and O-dealkylated phase I metabolites of BDP were generated by PONs mainly in serum experiments. Limited number of glucuronidated and sulfated phase II metabolites were also identified for the two chemicals. The activity of seven recombinant CYPs (rCYPs) including rCYP1A2, rCYP2B6, rCYP2C9, rCYP2C19, rCYP2D6, rCYP2E1 and rCYP3A4 in the in vitro phase I metabolism of V6 and BDP was investigated. The formation of V6 metabolites was catalyzed by several enzymes, especially rCYP1A2 that was responsible for the exclusive formation of two metabolites, one primary (M1) and its secondary metabolite (M9). For BDP, only one phase I metabolite (MM1) was catalyzed by the seven rCYPs. Collectively, these results indicate that CYPs have a predominant role in the metabolism of V6, while PONs have a predominant role in BDP in vitro metabolism. These results are a starting point for future studies involving the study of the toxicity, bioaccumulation and in vivo biomonitoring of V6 and BDP.

Suitable albumin concentrations for enhanced drug oxidation activities mediated by human liver microsomal cytochrome P450 2C9 and other forms predicted with unbound fractions and partition/distribution coefficients of model substrates

Albumin has reportedly enhanced cytochrome P450 (P450)-mediated drug oxidation rates in human liver microsomes. Consequently, measurements of clearances and fractions metabolized could vary depending on the experimental albumin concentrations used.In this study, the oxidation rates of diclofenac and warfarin by human liver microsomes were significantly enhanced in the presence of 0.10% (w/v) bovine serum albumin, whereas those of tolbutamide and phenytoin required 1.0% and 2.0% of albumin for significant enhancement. Values of the fractions metabolized by P450 2C9 for four substrates did not markedly change in the presence of albumin at the above-mentioned concentrations.The oxidation rates of bupropion, omeprazole, chlorzoxazone and phenacetin in human liver microsomes were reportedly enhanced by 0.5%, 1%, 2% and 2% of albumin, respectively. Analysis of reported intrinsic clearance values and suitable albumin concentrations for the currently analyzed substrates and the reported substrates revealed an inverse correlation, with warfarin as an outlier.Suitable albumin concentrations were multivariately correlated with physicochemical properties, that is, the plasma unbound fractions, octanol–water partition coefficient and acid dissociation constant (r = 0.98, p<.0001, n = 10). Therefore, multiple physicochemical properties may be determinants of suitable albumin concentrations for substrate oxidations in human liver microsomes.

Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration

The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. et al. (2017) J. Biol. Chem. 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug-metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme-thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.).

Influence of Derivatives of Resveratrol on Enzyme Activities in Human Liver Microsomal Cytochromes P450

Influence of Derivatives of Resveratrol on Enzyme Activities in Human Liver Microsomal Cytochromes P450

Inhibitory Effects of 12 Ginsenosides on the Activities of Seven Cytochromes P450 in Human Liver Microsomes

Ginseng, a traditional herbal drug, has been used in Eastern Asia for more than 2000 years. Various ginsenosides, which are the major bioactive components of ginseng products, have been shown to exert numerous beneficial effects on the human body when co-administered with drugs. However, this may give rise to ginsenoside-drug interactions, which is an important research consideration. In this study, acassette assay was performed the inhibitory effects of 12 ginsenosides on seven cytochrome P450 (CYP) isoforms in human liver microsomes (HLMs) using LC-MS/MS to predict the herb-drug interaction. After incubation of the 12 ginsenosides with seven cocktail CYP probes, the generated specific metabolites were quantified by LC-MS/MS to determine their activities. Ginsenoside Rb1 and F2 showed strong selective inhibitory effect on CYP2C9-catalyzed diclofenac 4′-hydroxylation and CYP2B6-catalyzed bupropion hydroxylation, respectively. Ginsenosides Rd showed weak inhibitory effect on the activities of CYP2B6, 2C9, 2C19, 2D6, 3A4, and compound K, while ginsenoside Rg3 showed weak inhibitory effects on CYP2B6. Other ginsenosides, Rc, Rf, Rg1, Rh1, Rf, and Re did not show significant inhibitory effects on the activities of the seven CYPs in HLM. Owing to the poor absorption of ginsenosides after oral administration in vivo, ginsenosides may not have significant side effects caused by interaction with other drugs.

Influence of Sulforaphane Metabolites on Activities of Human Drug-Metabolizing Cytochrome P450 and Determination of Sulforaphane in Human Liver Cells.

The influence of metabolites of sulforaphane, natural compounds present in broccoli (Brassica oleracea var. botrytis italica) and in other cruciferous vegetables, on drug-metabolizing cytochrome P450 (CYP) enzymes in human liver microsomes and possible entry of sulforaphane into human hepatic cells were investigated. Metabolites studied are compounds derived from sulforaphane by the mercapturic acid pathway (conjugation with glutathione and by following reactions), namely sulforaphane glutathione and sulforaphane cysteine conjugates and sulforaphane-N-acetylcysteine. Their possible effect on four drug-metabolizing CYP enzymes, CYP3A4 (midazolam 1'-hydroxylation), CYP2D6 (bufuralol 1'-hydroxylation), CYP1A2 (7-ethoxyresorufin O-deethylation), and CYP2B6 (7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation), was tested. Inhibition of four prototypical CYP activities by sulforaphane metabolites was studied in pooled human liver microsomes. Sulforaphane metabolites did not considerably affect biological function of drug-metabolizing CYPs in human liver microsomes except for CYP2D6, which was found to be inhibited down to 73-78% of the original activity. Analysis of the entry of sulforaphane into human hepatocytes was done by cell disruption by sonication, methylene chloride extraction, and modified high-performance liquid chromatography method. The results have shown penetration of sulforaphane into the human hepatic cells.

Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities

1. The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically.2. The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with Ki of 35.95 ± 6.96 and 60.56 ± 3.53 μmol/l and CYP3A4 (inhibited by genistein with Ki of 23.25 ± 5.85 μmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (Ki of 57.69 ± 2.36 μmol/l) and equol (Ki of 38.47 ± 2.32 μmol/l).3. Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.

Evaluation of possible inhibition of human liver drug metabolizing cytochromes P450 by two new acetylcholinesterase oxime-type reactivators

Two non-symmetric bispyridine oxime – based reactivators of acetylcholinesterase enzyme (AChE), labeled as K027 (1-(4-carbamoylpyridinium)-3-(4-hydroxyiminomethylpyridinium)-propane dibromide) and K203 ((E)-1-(4- carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide) were tested for their potential to inhibit activities of human liver microsomal cytochromes P450 (CYP). Both oximes are very potent reactivators of organophosphate-inhibited AChE.An interaction of both compounds with CYP in human liver microsomal preparation was detected using difference spectroscopy. The compounds were shown to bind to CYP enzymes with spectral binding constants of 5.04 ± 1.79 nM (K027) and 5.2 ± 2.6 nM (K203). Enzymology studies were subsequently performed aimed at determining which of the nine most important CYP involved in drug is affected by this interaction. The results have shown no prominent inhibition of individual CYP activities with either compounds except in the case of CYP2E1 and K203. Diagnostic Dixon plot revealed that K203 acted as an uncompetitive inhibitor of CYP2E1. Inhibition of this activity however is not as prominent as to make a potent drug interaction likely. Hence, the interaction of K027 and K203 oxime-type AChE reactivators with human liver microsomal CYP enzymes does not seem to be of prominent clinical importance and both compounds could be safely used in this respect as antidotes with low risk of drug interactions.

Enantiospecific effects of chiral drugs on cytochrome P450 inhibition in vitro

1. The aim of this work was to examine the differences in the inhibitory potency of individual enantiomers and racemic mixtures of selected chiral drugs on human liver microsomal cytochromes P450.2. The interaction of enantiomeric forms of six drugs (tamsulosin, tolterodine, citalopram, modafinil, zopiclone, ketoconazole) with nine cytochromes P450 (CYP3A4, CYP2E1, CYP2D6, CYP2C19, CYP2C9, CYP2C8, CYP2B6, CYP2A6, CYP1A2) was examined. HPLC methods were used to estimate the extent of the inhibition of specific activity in vitro.3. Tamsulosin (TAM) and tolterodine (TOL) inhibited CYP3A4 activity with an enantiospecific pattern. The inhibition of CYP3A4 activity differed for R-TAM (Ki 2.88 ± 0.12 µM) and S-TAM (Ki 14.22 ± 0.53 µM) as well as for S-TOL (Ki 1.71 ± 0.03 µM) and R-TOL (Ki 4.78 ± 0.17 µM). Also, the inhibition of CYP2C19 by ketoconazole (KET) cis-enantiomers exhibited enantioselective behavior: the (+)-KET (IC50 23.64 ± 6.25 µM) was more potent than (−)-KET (IC50 66.12 ± 12.6 µM). The inhibition of CYP2C19 by modafinil (MOD) enantiomers (R-MOD IC50 = 51.79 ± 8.58 µM, S-MOD IC50 = 48.62 ± 9.74 µM) and the inhibition of CYP2D6 by citalopram (CIT) enantiomers (R-CIT IC50 = 68.17 ± 5.70 µM, S-CIT IC50 = 62.63 ± 7.89 µM) was not enantiospecific.4. Although enantiospecific interactions were found (TAM, TOL, KET), they are probably not clinically relevant as the plasma levels are generally lower than the drug concentration needed for prominent inhibition (at least 50% of CYP activity).

Interaction of rocuronium with human liver cytochromes P450

Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied.Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75–80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium.To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively.The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed.

Inhibitory effects of dioscin on cytochrome P450 enzymes

Dioscin, a natural product, shows various pharmacological activities. However, the drug–drug interaction (DDI) potential of dioscin via the inhibition of cytochrome P450 (CYP) enzymes is still unknown. In this paper, the inhibitory effects of dioscin on six major CYP isoforms (1A2, 2A6, 2C9, 2D6, 2E1 and 3A4) were investigated. Using human liver microsomes (HLM), we found that dioscin inhibited the activities of CYP2C9, CYP2E1 and CYP3A4, with IC50 values of 22.60, 17.40 and 12.59 μM, respectively. Further research indicated that dioscin was a typical competitive inhibitor for CYP2C9, CYP2E1 and CYP3A4 (Ki = 5.0, 10.4 and 15.5 μM, respectively), as demonstrated by Lineweaver–Burk plots. In addition, dioscin exhibited time- and NADPH-dependent inhibitions of CYP3A4, and weak inhibitions of CYP1A2, CYP2A6 and CYP2D6 were also observed. In cell and animal experiments, dioscin markedly down-regulated the protein expressions of CYP2C9, CYP2E1 and CYP3A4 in primary rat hepatocytes and livers in a dose-dependent manner. This is the first paper on the inhibitory effects of dioscin on CYP in HLMs to predict the potential for dioscin–drug interaction and toxicity. Nevertheless, the clinical significance of the data presented herein should be further evaluated with in vivo research.

Interaction of selected platinum(II) complexes containing roscovitine-based CDK inhibitors as ligands with human liver microsomal cytochrome P450

We studied the interaction of oxaliplatin derivatives involving cytotoxic adenine-based cyclin-dependent kinase inhibitors, with human liver microsomal cytochrome P450. The activities of 9 human liver microsomal CYP forms (CYPs 1A2, 7-ethoxyresorufin O-deethylation; 2A6, coumarin 7-hydroxylation; 2B6, 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation; 2C8, luciferin-6´ methyl ether demethylation; 2C9, diclofenac 4´-hydroxylation, 6´-deoxyluciferin hydroxylation; 2C19, (S)-mephenytoin 4´-hydroxylation; 2D6, bufuralol 1´-hydroxylation, 2E1, chlorzoxazone 6-hydroxylation; 3A4, testosterone 6β-hydroxylation, luciferin-6´ benzyl ether debenzylation) were tested using HPLC, fluorescence and luminescence product detection. At 100 µM platinum(II) oxalato complex concentration, CYP inhibition was in general 25%-50%, except for the CYP3A4 form which showed roughly twice the inhibition (72%-95%). At low complex concentration (10 µM), the difference in inhibition of CYP3A4 and other forms was even more pronounced. Dixon and Lineweaver-Burk plots indicated a partially noncompetitive mechanism of CYP3A4 inhibition. The tested complexes significantly inhibit human liver microsomal CYP3A4 activity even at clinically relevant concentrations. This could be a serious drawback for the use of these compounds in clinical practice.

Drug interactions of diclofenac and its oxidative metabolite with human liver microsomal cytochrome P450 1A2-dependent drug oxidation

1. The purpose of this study was to investigate the inhibitory effects of diclofenac on human cytochrome P450 1A2-, 2C19- and 3A4-mediated drug oxidations and to evaluate the drug interaction potential of diclofenac and 4′-hydroxydiclofenac.2. Diclofenac was converted to 4′-hydroxydiclofenac by recombinantly expressed human P450 1A2 with Km and Vmax values of 33 µM and 0.20 min−1, respectively. Diclofenac and 4′-hydroxydiclofenac suppressed flurbiprofen 4′-hydroxylation by P450 2C9 strongly and moderately, respectively; however, they did not affect P450 2C19-dependent S-mephenytoin hydroxylation or P450 3A4-dependent midazolam hydroxylation.3. Although the caffeine 3-N-demethylation activity of liver microsomal P450 1A2 was inhibited by simultaneous incubation with diclofenac, the riluzole N-hydroxylation activities of recombinant P450 1A2 and human liver microsomes were inhibited after preincubation with diclofenac or 4′-hydroxydiclofenac for 20 min in the presence of NADPH. Using the inhibition constant (37 µM) of diclofenac on caffeine 3-N-demethylation and the reported 95th percentiles of maximum plasma concentration (10.5 µM) after an oral dose of diclofenac, the in vivo estimated increase in area under the plasma concentration–time curve was 29%.4. These results suggest that diclofenac could inhibit drug clearance to a clinically important degree that depends on P450 1A2. Clinically relevant drug interactions in vivo with diclofenac are likely to be invoked via human P450 1A2 function in addition to those caused by the effect of diclofenac on P450 2C9.

Characterization of SfmD as a Heme Peroxidase That Catalyzes the Regioselective Hydroxylation of 3-Methyltyrosine to 3-Hydroxy-5-methyltyrosine in Saframycin A Biosynthesis

Saframycin A (SFM-A) is a potent antitumor antibiotic that belongs to the tetrahydroisoquinoline family. Biosynthetic studies have revealed that its unique pentacyclic core structure is derived from alanine, glycine, and non-proteinogenic amino acid 3-hydroxy-5-methyl-O-methyltyrosine (3-OH-5-Me-OMe-Tyr). SfmD, a hypothetical protein in the biosynthetic pathway of SFM-A, was hypothesized to be responsible for the generation of the 3-hydroxy group of 3-OH-5-Me-OMe-Tyr based on previously heterologous expression results. We now report the in vitro characterization of SfmD as a novel heme-containing peroxidase that catalyzes the hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine using hydrogen peroxide as the oxidant. In addition, we elucidated the biosynthetic pathway of 3-OH-5-Me-OMe-Tyr by kinetic studies of SfmD in combination with biochemical assays of SfmM2, a methyltransferase within the same pathway. Furthermore, SacD, a counterpart of SfmD involved in safracin B biosynthesis, was also characterized as a heme-containing peroxidase, suggesting that SfmD-like heme-containing peroxidases may be commonly involved in the biosynthesis of SFM-A and its analogs. Finally, we found that the conserved motif HXXXC is crucial for heme binding using comparative UV-Vis and Magnetic Circular Dichroism (MCD) spectra studies of SfmD wild-type and mutants. Together, these findings expand the category of heme-containing peroxidases and set the stage for further mechanistic studies. In addition, this study has critical implications for delineating the biosynthetic pathway of other related tetrahydroisoquinoline family members.

In vitro interaction of a novel neutrophil growth factor with human liver microsomal cytochromes P450 and the contribution of UDP-glucuronosyltransferases to its metabolism

Neutrophil growth factors (NGFs) stimulate neutrophil growth and survival. The first synthetic cytokinin-derived NGF was recently discovered and is a prospective drug owing to its potential use in anti-inflammatory therapy. The metabolism of some cytokinin-derived drugs (e.g. R-roscovitine, olomoucine II) has already been studied and it has been shown that they may give rise to drug-drug interactions.In this in vitro study, the interactions of the novel neutrophil growth factor NGF1568 with two of the main classes of human drug-metabolizing enzymes, cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs), were tested. Of the CYPs evaluated, NGF1568 was found to inhibit only CYP2C9, by an uncompetitive mechanism and with a Ki value of 349 μM.Formation of a glucuronide of NGF1568 was detected by LC/MS/MS analysis after it was incubated with human liver microsomes and UDP-glucuronic acid. The human recombinant UGT1A9 enzyme (major liver expression) and UGT1A7, UGT1A8, UGT1A10 enzymes (expressed in gastrointestinal tract instead of liver) were found to be responsible for NGF1568 glucuronidation.These results show that interaction of NGF1568 with CYPs is not as important as it is in the case of the cytokinin CDK inhibitors R-roscovitine and olomoucine II, but the conjugation enzymes (UGTs) play a major role in its metabolism. Thus, possible interference of NGF1568 with metabolism of other coadministered drugs at least on level of liver, kidney or intestinal UGTs should be thoroughly considered.

Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter‐system extrapolation factors

The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended.

Human Liver Microsomal Cytochrome P450 3A Enzymes Involved in Thalidomide 5-Hydroxylation and Formation of a Glutathione Conjugate

(R)-Thalidomide was oxidized to 5-hydroxythalidomide and 5'-hydroxythalidomide by NADPH-fortified liver microsomes from humans and monkeys. (R)-Thalidomide was hydroxylated more efficiently than (S)-thalidomide. Recombinant human P450s 3A4, 3A5, and 3A7 and monkey P450s 3A8 and 3A5 (coexpressed with NADPH-P450 reductase in bacterial membranes) also catalyzed (R)-thalidomide 5-hydroxylation. Purified human P450s 2C19, 3A4, and 3A5 mediated (R)-thalidomide 5-hydroxylation at similar rates in reconstituted systems. P450 2C19 showed a rather nonsaturable substrate-velocity curve; however, P450s 3A4 and 3A5 showed sigmoidal curves. P450 also oxidized 5-hydroxythalidomide to an epoxide or dihydroxy compound. Liquid chromatography-mass spectrometry analysis revealed the formation of a glutathione conjugate from (R)- and (S)-5-hydroxythalidomide, catalyzed by liver microsomal P450s 3A4 and 3A5 in the presence of glutathione (assigned as a conjugate of 5-hydroxythalidomide formed on the phenyl ring). These results indicate that human P450s 3A4 and 3A5 mediate thalidomide 5-hydroxylation and further oxidation leading to a glutathione conjugate, which may be of relevance in the pharmacological and toxicological actions of thalidomide.

Inhibition of human liver microsomal CYP by nateglinide

Nateglinide is metabolized by CYP2C9 and CYP3A4, therefore drug-drug interactions through cytochrome P450 (CYP) inhibition may occur. In this study, we examined the inhibitory effects of nateglinide and its major metabolite N-[trans-4-(1-hydroxy-1-methylethyl)-cyclohexanecarbonyl]-D-phenylalanine (M1) on various CYP isoforms in human liver microsomes. We used typical substrates (7-ethoxyresorufin for CYP1A1/2, tolbutamide for CYP2C9, S-mephenytoin for CYP2C19, bufuralol for CYP2D6, chlorzoxazone for CYP2E1 and midazolam for CYP3A4) in the evaluation of the inhibitory effects, and examined the possibility of mechanism-based inhibition (MBI) by evaluating the influence of pre-incubation in the inhibition. The results showed that nateglinide inhibited CYP2C9 and CYP2C19 with an IC50(app) (apparent value of the 50% inhibitory concentration) of 125 micromol/l and 946 micromol/l, respectively, while M1 did not inhibit any of the CYP isoforms. The inhibition constant (K(i)) value of the inhibitory effect of nateglinide on CYP2C9 and the 1 + I(in,max,u)/K(i) value were estimated (where I(in,max,u)= the maximum unbound concentration of nateglinide). The 1 + I(in,max,u)/K(i) value was 1.02 (close to 1), suggesting a low risk of drug-drug interactions. The influence of pre-incubation on the inhibition by nateglinide of CYP3A4, CYP2C9 and CYP2C19 was examined. The results revealed that the inhibition of CYP by nateglinide was not influenced by pre-incubation, and that the possibility of MBI is very low. The possibility of drug-drug interactions involving nateglinide that might be attributable to CYP inhibition is low.