Impact of Heterotropic Allosteric Modulation on the Time-Dependent Inhibition of Cytochrome P450 3A4.

The current study was designed to investigate the influence of allosteric effectors on the metabolism of the prototypical cytochrome P450 (CYP) 3A4 substrate midazolam (MDZ), and on the determination in vitro time-dependent inhibition (TDI) of CYP3A4 using human liver microsomes (HLM). As the concentration of midazolam increased to 250 µM in HLMs, homotropic cooperativity resulted in a decrease in the 1'-hydroxymidazolam to 4-hydroxymidazolam ratio to a maximum of 1.1. The presence of varying concentrations of testosterone, progesterone (PGS), or carbamazepine (CBZ) in HLMs with MDZ could recapitulate the effect of homotropic cooperativity such that the formation rates of the 1'hydroxymidazolam and 4-hydroxymidazolam were equal even at low concentrations of MDZ. The presence of PGS (10 or 100 µM) and CBZ (100 or 1000 µM) in in vitro TDI determination of four known CYP3A4 time-dependent inactivators (clarithromycin, troleandomycin, mibefradil, raloxifene) simultaneously decreased potency and inactivation rate constant, resulting in fold changes in inactivation efficiency on average of 1.6-fold and 13-fold for the low and high concentrations of allosteric modulator tested, respectively. The formation of a metabolic-intermediate complex (MIC) for clarithromycin and troleandomycin decreased in the presence of the allosteric modulators in a concentration-dependent manner, reaching a new steady state formation that could not be overcome with increased incubation time. Maximum reduction of the MIC formed by clarithromycin was up to ∼91%, while troleandomycin MIC decreased up to ∼31%. These findings suggest that the absence of endogenous allosteric modulators may contribute to the poor translation of HLM-based drug-drug interaction predictions. SIGNIFICANCE STATEMENT: The reported overprediction of in vitro human liver microsome time-dependent inhibition of CYP3A4 and observed drug interactions in vivo remains an issue in drug development. We provide characterization of allosteric modulators on the CYP3A4 metabolism of the prototypical substrate midazolam, demonstrating the ability of the modulators to recapitulate the homotropic cooperativity of midazolam. Furthermore, we demonstrate that allosteric heterotropic cooperativity of CYP3A4 can impact the time-dependent inhibition kinetics of known mechanisms-based inhibitors, providing a potential mechanism to explain the overprediction.

Study on the inhibitory effect of furafylline and troleandomycin in the 7-methoxyresorufin-O-demethylase and nifedipine oxidase activities in hepatic microsomes from four poultry species using high-performance liquid chromatography coupled with fluorescence and ultraviolet detection.

The present study reports the in vitro studies with furafylline and troleandomycin (TAO) as specific inhibitors of activities 7-methoxyresorufin-O-demethylase (MROD) and nifedipine oxidase, catalyzed by cytochrome P450 1 A2 (CYP1 A2) and 3A4 human enzymes, respectively, in hepatic microsomes of quail, duck, turkey and chicken. The results suggest that in chicken and quail the MROD activity is carried out by orthologs CYP1 A4 and 1 A5, meanwhile in duck and turkey by a CYP1 A5 ortholog. The nifedipine oxidase activity is carried out by orthologs of the CYP3A family in the four bird species. The use of furafylline and TAO significantly decreased these activities (P < 0.05) and suggested that the biotransformation of resorufin methyl ether (RME) may be related to more than one avian ortholog.

Evaluation of Strategies for the Assessment of Drug-Drug Interactions Involving Cytochrome P450 Enzymes.

BACKGROUND AND OBJECTIVES: Drug-drug interactions (DDIs) can occur when one drug alters the metabolism of another drug. Drug metabolism mediated by cytochrome P450 enzymes (CYPs) is responsible for the majority of metabolism of known drugs and inhibition of CYP enzymes is a well-known cause of DDIs. In the current study, the use of various human liver microsomes (HLM)-based methods to determine occurrence of CYP-mediated metabolism-dependent inhibition (MDI) and possible follow-up studies were evaluated. METHODS: Human CYP inhibition was studied using the following methodologies: direct inhibition and (non-diluted) IC50-shift assays, a ferricyanide-based reversibility assay, a spectrophotometric metabolic intermediate complex (MIC) assay, and recording of reduced carbon monoxide (CO)-difference spectra. HLM incubations in the presence and absence of NADPH and glutathione (GSH) were performed to study the possible formation of CYP-dependent GSH adducts. HLM incubations with the radiolabeled inhibitors mifepristone and paroxetine were performed to study CYP-mediated covalent binding. RESULTS: Dihydralazine and furafylline displayed irreversible MDI of CYP1A2. Paroxetine displayed both quasi-irreversible and irreversible MDI of CYP2D6, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Mifepristone displayed irreversible MDI of CYP3A4, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Troleandomycin and verapamil displayed quasi-irreversible MDI of CYP3A4; MIC formation was observed, while no formation of CYP-dependent GSH adducts occurred. CONCLUSIONS: This study gives a representative overview of current methodologies that can be used to study CYP inhibition. The here presented strategy can be applied as a tool during risk evaluation of CYP-mediated DDIs.

Metabolism of megestrol acetate in vitro and the role of oxidative metabolites.

1. There is limited knowledge regarding the metabolism of megestrol acetate (MA), as it was approved by FDA in 1971, prior to the availability of modern tools for identifying specific drug-metabolizing enzymes. We determined the cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) that metabolize MA, identified oxidative metabolites and determined pharmacologic activity at the progesterone, androgen and glucocorticoid receptors (PR, AR and GR, respectively). 2. Oxidative metabolites were produced using human liver microsomes (HLMs), and isolated for mass spectral (MS) and nuclear magnetic resonance (NMR) analyses. We screened recombinant P450s using MA at 62 µM (HLM Km for metabolite 1; M1) and 28 µM (HLM Km for metabolite 2; M2). UGT isoforms were simultaneously incubated with UDPGA, nicotinamide adenine dinucleotide phosphate (NADPH), CYP3A4 and MA. Metabolites were evaluated for pharmacologic activity on the PR, AR and GR. CYP3A4 and CYP3A5 are responsible for oxidative metabolism of 62 µM MA. 3. At 28 µM substrate concentration, CYP3A4 was the only contributing enzyme. Mass spectral and NMR data suggest metabolism of MA to two alcohols. After oxidation, MA is converted into two secondary glucuronides by UGT2B17 among other UGTs. MA, M1 and M2 had significant pharmacologic activity on the PR while only MA showed activity on the AR and GR.

Carbamazepine-Induced Liver Injury Requires CYP3A-Mediated Metabolism and Glutathione Depletion in Rats.

Carbamazepine (CBZ) is widely used as an antiepileptic agent and causes rare but severe liver injury in humans. It has been generally recognized that reactive metabolites formed via the metabolic activation reaction contribute to the onset of liver injuries by several drugs. However, the role of CBZ metabolism in the development of liver injury is not fully understood. In this study, we developed a novel rat model of CBZ-induced liver injury and attempted to elucidate the associated mechanisms by focusing on the metabolism of CBZ. The repeated administration of CBZ for 5 days in combination with l-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor, resulted in increases in the plasma alanine aminotransferase (ALT) levels and centrilobular necrosis in the liver that were observed in various degrees. The CBZ and 2-hydroxy-CBZ concentrations in the plasma after the last CBZ administration were lower in the rats with high plasma ALT levels compared with those with normal plasma ALT levels, showing the possibility that the further metabolism of CBZ and/or 2-hydroxy-CBZ is associated with the liver injury. Although a single administration of CBZ did not affect the plasma ALT levels, even when cotreated with BSO, pretreatment with dexamethasone, a CYP3A inducer, increased the plasma ALT levels. In addition, the rats cotreated with troleandomycin or ketoconazole, CYP3A inhibitors, suppressed the increased plasma ALT levels. In conclusion, reactive metabolite(s) of CBZ produced by CYP3A under the GSH-depleted condition might be involved in the development of liver injury in rats.

Demethylation of neferine in human liver microsomes and formation of quinone methide metabolites mediated by CYP3A4 accentuates its cytotoxicity.

Neferine is a bisbenzylisoquinoline alkaloid isolated from the seed embryos of Nelumbonucifera Gaertn (Lotus) with various potent pharmacological effects. Recently, neferine has attracted attention for its anti-tumor activities. Our study explored its metabolism and cytotoxicity mechanism. Approaches using chemical inhibitors and recombinant human enzymes to characterize the involved enzymes and kinetic studies indicated that the demethylation of neferine by cytochrome P450 (CYP) 2D6 and CYP3A4 fitted a biphasic kinetic profile. Glutathione (GSH) was used as a trapping agent to identify reactive metabolites of neferine, and four novel GSH conjugates were detected with [M+H](+) ions at m/z 902.4, 916.2, 916.1, and 930.4. Based on its structure containing para-methylene phenol and results from a product ion scan, GSH tends to conjugate with C9' after undergoing oxidative metabolism to form the binding site predominated by CYP3A4. Furthermore, the addition of recombinant human GSTA1, GSTT1, and GSTP1 had little effect on the production of the GSH conjugates. In a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay, combined with the GSH modulators l-buthionine sulfoximine or N-acetyl-l-cysteine, neferine treatment of MDCK-hCYP3A4 and HepG2 cells revealed that CYP3A4 expression and cellular GSH content could cause an EC50 shift. Metabolic activation mediated by CYP3A4 and GSH depletion significantly enhanced neferine-induced cytotoxicity.

Drug-drug interactions and metabolism in cytochrome P450 2C knockout mice: application to troleandomycin and midazolam.

Drug-drug interactions (DDIs) may cause serious drug toxicity and delay development of candidate drugs. Screening using human liver microsomes and hepatocytes can help predict DDIs but do not always provide the degree of certainty required for confident progression of a candidate drug. Thus a suitable in vivo test system could be of great value. Here a Cyp2c knockout (KO) mouse was investigated for studying DDIs using midazolam (MDZ) a standard human CYP3A4 substrate and troleandomycin (TAO) a potent human CYP3A4 inhibitor. Pharmacokinetics (PK) and biotransformation of MDZ were investigated following dosing to Cyp2c KO and wild type mice before and after TAO treatment. The noteworthy differences in the metabolism of MDZ in Cyp2c KO compared to wild type mice confirms the important role that Cyp2c enzymes play in the murine metabolism of MDZ in vivo. The impact of Cyp3a inhibition produced a further increase in circulating MDZ concentrations in all individuals from both strains of mice though the impact of the elimination of the Cyp2c pathway in the KO mice on the AUC was less than perhaps expected. We have shown that TAO produces an increase in the MDZ concentration and a reduction in the 1'hydroxymidazolam/midazolam formation ratio but the expected difference in the magnitude of this effect between the wild type and the Cyp2c KO mice was not seen. The magnitude of the TAO effect was also smaller than is reported in humans. Hence further work is required before this animal model could be used to predict clinical interactions.

Green tea extract affects the cytochrome P450 3A activity and pharmacokinetics of simvastatin in rats.

Effects of green tea extract (GTE) on the activity of cytochrome P450 (CYP) enzymes and pharmacokinetics of simvastatin (SIM) were investigated in rats. Inhibitory effects of GTE on CYP3A activity were investigated in rat hepatic microsomes (RHM) using midazolam (MDZ) 1'-hydroxylation as a probe reaction. SD female rats received a single oral dose of GTE (400 mg/kg) or troleandomycin (TAO, a CYP3A selective inhibitor, 500 mg/kg), followed 30 min later by SIM (20 mg/kg). Plasma concentrations of SIM and its active metabolite, simvastatin acid, were determined up to 6 h after the SIM administration using LC/MS/MS. In RHM, GTE inhibited MDZ 1'-hydroxylation with IC50 and K(i)(app) values of 12.5 and 18.8 µg/mL, respectively, in a noncompetitive manner. Area under plasma concentration-time curves for SIM in the GTE and TAO groups were increased by 3.4- and 10.2-fold, respectively, compared with the control. The maximum concentrations of SIM were higher in the GTE (3.3-fold) and TAO (9.5-fold) groups. GTE alters the pharmacokinetics of SIM, probably by inhibiting intestinal CYP3A.

Time-dependent inhibition and estimation of CYP3A clinical pharmacokinetic drug-drug interactions using plated human cell systems.

The current studies assessed the utility of freshly plated hepatocytes, cryopreserved plated hepatocytes, and cryopreserved plated HepaRG cells for the estimation of inactivation parameters k(inact) and K(I) for CYP3A. This was achieved using a subset of CYP3A time-dependent inhibitors (fluoxetine, verapamil, clarithromycin, troleandomycin, and mibefradil) representing a range of potencies. The estimated k(inact) and K(I) values for each time-dependent inhibitor were compared with those obtained using human liver microsomes and used to estimate the magnitude of clinical pharmacokinetic drug-drug interaction (DDI). The inactivation kinetic parameter, k(inact), was most consistent across systems tested for clarithromycin, verapamil, and troleandomycin, with a high k(inact) of 0.91 min(-1) observed for mibefradil in HepaRG cells. The apparent K(I) estimates derived from the various systems displayed a range of variability from 3-fold for clarithromycin (5.4-17.7 µM) to 6-fold for verapamil (1.9-12.6 µM). In general, the inactivation kinetic parameters derived from the cell systems tested fairly replicated what was observed in time-dependent inhibition studies using human liver microsomes. Despite some of the observed differences in inactivation kinetic parameters, the estimated DDIs derived from each of the tested systems generally agreed with the clinically reported DDI within approximately 2-fold. In addition, a plated cell approach offered the ability to conduct longer primary incubations (greater than 30 min), which afforded improved ability to identify the weak time-dependent inhibitor fluoxetine. Overall, results from these studies suggest that in vitro inactivation parameters generated from plated cell systems may be a practical approach for identifying time-dependent inhibitors and for estimating the magnitude of clinical DDIs.

Pharmacokinetic and pharmacodynamic interaction between nifedipine and metformin in rats: competitive inhibition for metabolism of nifedipine and metformin by each other via CYP isozymes.

It has been reported that hypertension exponentially increases in the patients with type 2 diabetes mellitus. Thus, this study was performed to investigate the pharmacokinetic and pharmacodynamic interactions between nifedipine and metformin, since both drugs were commonly metabolized via hepatic CYP2C and 3A subfamilies in rats. Nifedipine (3 mg/kg) and metformin (100 mg/kg) were simultaneously administered intravenously or orally to rats. Concentrations (I) of each drug in the liver and intestine, maximum velocity (V(max)), Michaelis-Menten constant (K(m)), and intrinsic clearance (CL(int)) for the disappearance of each drug, apparent inhibition constant (K(i)) and [I]/K(i) ratios of each drug in liver and intestine were determined. Also the metabolism of each drug in rat and human CYPs and blood pressure were also measured. After the simultaneous single intravenous administration of both drugs together, the AUCs of each drug were significantly greater than that in each drug alone due to the competitive inhibition for the metabolism of nifedipine by metformin via hepatic CYP3A1/2 and of metformin by nifedipine via hepatic CYP2C6 and 3A1/2. After the simultaneous single oral administration of both drugs, the significantly greater AUCs of each drug than that in each drug alone could have mainly been due to the competitive inhibition for the metabolism of nifedipine and metformin by each other via intestinal CYP3A1/2 in addition to competitive inhibition for the hepatic metabolism of each drug as same as the intravenous study.

Effects of cytochrome P450 inhibitors on peroxidase activity.

OBJECTIVES: Of several enzymes metabolizing xenobiotics, cytochrome P450 (CYP) and peroxidase enzymes seem to be most important. One of the major challenges in studies investigating metabolism of xenobiotics is to resolve which of these two groups of enzymes is predominant to metabolize individual xenobiotic compounds. Utilization of selective inhibitors of CYP and peroxidase enzymes might be a useful tool to identify the contribution of these enzymes to metabolism of xenobiotics in samples, where both types of enzymes are present. The aim of this study was to investigate specificities of several known CYP inhibitors to these enzymes; whether they inhibit only the CYP enzymes and do not inhibit peroxidases. METHODS: Since the oxidation of o-anisidine catalyzed by a model peroxidase used, horseradish peroxidase (HRP), is a two-substrate reaction, the inhibition potential of tested chemicals was studied with respect to both peroxidase substrates, o-anisidine and hydrogen peroxide. Initial velocities of o-anisidine oxidation by HRP under various conditions were determined spectrophotometrically. RESULTS: The CYP inhibitors metyrapone, troleandomycine, disulfiram, sulfaphenazole, quinidine and 1-aminobenzotriazole do not inhibit o-anisidine oxidation catalyzed by HRP. In contrast, ketoconazole, diethyldithiocarbamate, ellipticine, α-naphtoflavone, proadifen SKF525A, piperonylbutoxide, were found to inhibit not only the CYPs, but also the HRP-mediated oxidation of o-anisidine. Interestingly, α-naphtoflavone inhibits oxidation of o-anisidine by HRP with respect to H2O2, but not with respect to o-anisidine. Diethyldithiocarbamate is the most potent peroxidase inhibitor of o-anisidine oxidation with Ki with respect to o-anisidine of 10 µM and Ki with respect to H2O2 of 60 µM, being even the better peroxidase inhibitor than the classical "peroxidase inhibitor" - propyl gallate (Ki with respect to o-anisidine of 60 µM and Ki with respect to H2O2 of 750 µM). CONCLUSIONS: The results of the present study demonstrate that 1-aminobenzotriazole, a potent inhibitor of various CYP enzymes, seems to be the best candidate suitable for utilization in studies evaluating participation of CYP enzymes in metabolism of xenobiotics in various complex biological materials containing both CYP and peroxidase enzymes. Moreover, precaution to prevent misinterpretation of results is necessary in cases when proadifen SKF525A, piperonylbutoxide, diethyldithiocarbamate, ketoconazole, α-naphtoflavone and ellipticine are used in similar studies (as CYP inhibitors in various complex biological materials containing both CYP and peroxidase enzymes), since these chemicals can except of CYP enzymes inhibit also peroxidase-mediated reactions.

Determination of time-dependent inactivation of CYP3A4 in cryopreserved human hepatocytes and assessment of human drug-drug interactions.

Assessment of time-dependent inhibition (TDI), especially CYP3A4, is an important parameter for preclinical and clinical development. The use of human liver microsomes (HLM) is the most common in vitro matrix to assess TDI, but this often leads to an overprediction of an actual effect observed clinically. Recently, the use of human hepatocytes has been hypothesized as a more relevant and possibly predictive matrix for the assessment of CYP3A4 TDI. Our work evaluates and optimizes three different human hepatocyte assays for the assessment of CYP3A4 TDI using pooled cryopreserved human hepatocytes. Using two of the optimized methods, the time-dependent inhibition kinetic parameters (K(I) and k(inact)) for four known CYP3A4 TDI (diltiazem, erythromycin, verapamil, and troleandomycin) were determined. When comparing TDI in HLM, the K(I) values from hepatocytes were in general 4- to 13-fold higher than that in HLM, whereas the k(inact) values in human hepatocytes were similar or slightly higher or lower depending on the inhibitor. The inactivation potency (k(inact)/K(I)) for four tested CYP3A4 inactivators in human hepatocytes was generally lower than that in HLM due to either lower affinity (K(I)) or lower inactivation rate (k(inact)) or both. When drug interactions were simulated with Simcyp using either HLM or human hepatocyte data, the predictions using the kinetic parameters from human hepatocytes resulted in a much better simulated change in pharmacokinetics compared with observed clinical data.

Cytochromes P450 catalyze the reduction of α,ß-unsaturated aldehydes.

The metabolism of α,ß-unsaturated aldehydes, e.g., 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently, we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O(2), and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 and rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice a diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of the reduction of α,ß-unsaturated aldehydes in the liver.

Cholesterol 25-hydroxylation activity of CYP3A.

To date, many studies have been conducted using 25-hydroxycholesterol, which is a potent regulator of lipid metabolism. However, the origins of this oxysterol have not been entirely elucidated. Cholesterol 25-hydroxylase is one of the enzymes responsible for the metabolism of 25-hydroxycholesterol, but the expression of this enzyme is very low in humans. This oxysterol is also synthesized by sterol 27-hydroxylase (CYP27A1) and cholesterol 24-hydroxylase(CYP46A1), but it is only a minor product of these enzymes. We now report that CYP3A synthesizes a significant amount of 25-hydroxycholesterol and may participate in the regulation of lipid metabolism. Induction of CYP3A by pregnenolone-16α-carbonitrile caused the accumulation of 25-hydroxycholesterol in a cell line derived from mouse liver. Furthermore, treatment of the cells with troleandomycin, a specific inhibitor of CYP3A, significantly reduced cellular 25-hydroxycholesterol concentrations. In cells that overexpressed human recombinant CYP3A4, the activity of cholesterol 25-hydroxylation was found to be higher than that of cholesterol 4ß-hydroxylation, a known marker activity of CYP3A4. In addition, 25-hydroxycholesterol concentrations in normal human sera correlated positively with the levels of 4ß-hydroxycholesterol (r = 0.650, P < 0.0001, n = 78), but did not significantly correlate with the levels of 27-hydroxycholesterol or 24S-hydroxycholesterol. These results demonstrate the significance of CYP3A on the production of 25-hydroxycholesterol.

A method for selecting cis-acting regulatory sequences that respond to small molecule effectors.

BACKGROUND: Several cis-acting regulatory sequences functioning at the level of mRNA or nascent peptide and specifically influencing transcription or translation have been described. These regulatory elements often respond to specific chemicals. RESULTS: We have developed a method that allows us to select cis-acting regulatory sequences that respond to diverse chemicals. The method is based on the beta-lactamase gene containing a random sequence inserted into the beginning of the ORF. Several rounds of selection are used to isolate sequences that suppress beta-lactamase expression in response to the compound under study. We have isolated sequences that respond to erythromycin, troleandomycin, chloramphenicol, meta-toluate and homoserine lactone. By introducing synonymous and non-synonymous mutations we have shown that at least in the case of erythromycin the sequences act at the peptide level. We have also tested the cross-activities of the constructs and found that in most cases the sequences respond most strongly to the compound on which they were isolated. CONCLUSIONS: Several selected peptides showed ligand-specific changes in amino acid frequencies, but no consensus motif could be identified. This is consistent with previous observations on natural cis-acting peptides, showing that it is often impossible to demonstrate a consensus. Applying the currently developed method on a larger scale, by selecting and comparing an extended set of sequences, might allow the sequence rules underlying the activity of cis-acting regulatory peptides to be identified.

[Effect of CYP3A and P-glycoprotein on the absorption of buagafuran in rat intestinal lumen].

The rat single-pass intestinal perfusion model was applied to study the effect of CYP3A and P-glycoprotein on the absorption of buagafuran in lumen of rats. Buagafuran concentrations in intestinal perfusate and blood in vena mesenterica collected at different time points after perfusion were determined by GC-MS. Permeability coefficient of buagafuran was calculated by the equation [P(lumen) = -(Q/2pirl)Ln(C(out)/C(in)) and P(blood) = (deltaM(B)/deltat)/(2pirl)]. The effects of troleandomycin (TAO, CYP3A inhibitor), cyclosporin A (CYP3A/p-glycoprotein inhibitor) on the absorption of buagafuran in lumen were observed. After rat single-pass intestinal perfusion, the cumulative amount of buagafuran in mesenteric vein of rat was 73.4, 82.9, and 98.3 pmol x cm(-2) and were increased 3.9, 4.6, and 5.6 fold by addition of inhibitor of P-gp (LSN335984), CYP3A (TAO) or P-gp and CYP3A (CsA), respectively. Moreover, the metabolized fraction of buagafuran was decreased by 12%, 11% and 21% with inhibitors. The results suggested that the poor bioavailability of buagafuran was mostly due to the interplay of P-gp and CYP3A on the absorption, transport and metabolism of buagafuran in intestine of rats.

[Metabolism of 3-cyanomethyl-4-methyl-DCK, a new anti-HIV candidate, in human intestinal microsomes].

The biotransformation, CYP reaction phenotyping, the impact of CYP inhibitors and enzyme kinetics of 3-cyanomethyl-4-methyl-DCK (CMDCK), a new anti-HIV preclinical candidate belonging to DCK analogs, were investigated in human intestinal microsomes and recombinant cytochrome P450 (CYP) enzymes. CMDCK (4 micromol L(-1)) was incubated with a panel of rCYP enzymes (CYP1A2, 2C9, 2C19, 2D6 and 3A4) in vitro. The remaining parent drug in incubates was quantitatively analyzed by a LC-MS method. CYP3A4 was identified as the principal CYP isoenzyme responsible for its metabolism in intestinal microsomes. The major metabolic pathway of CMDCK was oxidation and a number of oxidative metabolites were screened with LC-MS. The Km, Vmax, CLint and T1/2 of CMDCK obtained from human intestinal microsome were 45.6 micromol L(-1), 0.33 micromol L(-1) min(-1), 12.1 mL min(-1) kg(-1) and 25.7 min, respectively. Intestinal clearance of CMDCK was estimated from in vitro data to be 3.3 mL min(-1) kg(-1), and was almost equal to the intestinal blood flow rate (4.6 mL min(-1) kg(-1)). The selective CYP3A4 inhibitors, ketoconazole, troleandomycin and ritonavir demonstrated significant inhibitory effects on CMDCK intestinal metabolism, which suggested that co-administration of CMDCK with potent CYP3A inhibitors, such as ritonavir, might decrease its intestinal metabolic clearance and subsequently improve its bioavailability in body.

CYP3A catalyses schizandrin biotransformation in human, minipig and rat liver microsomes.

Schizandrin is recognized as the major absorbed effective constituent of Fructus schisandrae, which is extensively applied in Chinese medicinal formula. The present study aimed to profile the phase I metabolites of schizandrin and identify the cytochrome P450 (CYP) isoforms involved. After schizandrin was incubated with human liver microsomes, three metabolites were isolated by high-performance liquid chromatography (HPLC) and their structures were identified to be 8(R)-hydroxyl-schizandrin, 2-demethyl-8(R)-hydroxyl-schizandrin, 3-demethyl-8(R)-hydroxyl-schizandrin, by liquid chromatography-mass spectrometry (LC-MS), (1)H-nuclear magnetic resonance (NMR), and (13)C-NMR, respectively. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs, and enzyme kinetics indicated that CYP3A4 was the main hepatic isoform that cleared schizandrin. Rat and minipig liver microsomes were included when evaluating species differences, and the results showed little difference among the species. In conclusion, CYP3A4 plays a major role in the biotransformation of schizandrin in human liver microsomes. Minipig and rat could be surrogate models for man in schizandrin pharmacokinetic studies. Better knowledge of schizandrin's metabolic pathway could provide the vital information for understanding the pharmacokinetic behaviours of schizandrin contained in Chinese medicinal formula.

Effect of hepatic CYP inhibitors on the metabolism of sildenafil and formation of its metabolite, N-desmethylsildenafil, in rats in vitro and in vivo.

OBJECTIVES: It has been reported that hepatic cytochrome P450 (CYP)2C9 and CYP3A4 are responsible for the metabolism of sildenafil and formation of its metabolite, N-desmethylsildenafil, in humans. However, in-vivo studies in rats have not been reported. METHODS: Sildenafil (20 mg/kg) was administered intravenously to rats pretreated with sulfaphenazole, cimetidine, quinine hydrochloride or troleandomycin, inhibitors of CYP2C6, CYP2C11, CYP2D subfamily and CYP3A1/2, respectively. In-vitro studies using rat liver microsomes were also performed. KEY FINDINGS: The area under the plasma-concentration time curve (AUC) was increased and clearance of sildenafil decreased in rats pretreated with cimetidine or troleandomycin. The AUC ratio for N-desmethylsildenafil (0-4 h) : sildenafil (0-infinity) was significantly decreased only in rats pretreated with cimetidine. Similar results were obtained in the in-vitro study using rat liver microsomes. CONCLUSIONS: Sildenafil is metabolised via hepatic CYP2C11 and 3A1/2, and N-desmethylsildenafil is mainly formed via hepatic CYP2C11 in rats. Thus, rats could be a good model for pharmacokinetic studies of sildenafil and N-desmethylsildenafil in humans.

Structures of triacetyloleandomycin and mycalamide A bind to the large ribosomal subunit of Haloarcula marismortui.

Structures have been obtained for the complexes that triacetyloleandomycin and mycalamide A form with the large ribosomal subunit of Haloarcula marismortui. Triacetyloleandomycin binds in the nascent peptide tunnel and inhibits the activity of ribosomes by blocking the growth of the nascent peptide chain. Mycalamide A binds to the E site and inhibits protein synthesis by occupying the space normally occupied by the CCA end of E-site-bound tRNAs.