Troleandomycin Research Articles

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.

Improved Predictions of Drug-Drug Interactions Mediated by Time-Dependent Inhibition of CYP3A.

Time-dependent inactivation (TDI) of cytochrome P450s (CYPs) is a leading cause of clinical drug-drug interactions (DDIs). Current methods tend to overpredict DDIs. In this study, a numerical approach was used to model complex CYP3A TDI in human-liver microsomes. The inhibitors evaluated included troleandomycin (TAO), erythromycin (ERY), verapamil (VER), and diltiazem (DTZ) along with the primary metabolites N-demethyl erythromycin (NDE), norverapamil (NV), and N-desmethyl diltiazem (NDD). The complexities incorporated into the models included multiple-binding kinetics, quasi-irreversible inactivation, sequential metabolism, inhibitor depletion, and membrane partitioning. The resulting inactivation parameters were incorporated into static in vitro-in vivo correlation (IVIVC) models to predict clinical DDIs. For 77 clinically observed DDIs, with a hepatic-CYP3A-synthesis-rate constant of 0.000 146 min-1, the average fold difference between the observed and predicted DDIs was 3.17 for the standard replot method and 1.45 for the numerical method. Similar results were obtained using a synthesis-rate constant of 0.000 32 min-1. These results suggest that numerical methods can successfully model complex in vitro TDI kinetics and that the resulting DDI predictions are more accurate than those obtained with the standard replot approach.

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.

Comparison of the substrate kinetics of pig CYP3A29 with pig liver microsomes and human CYP3A4

CYP (cytochrome P450) 3A29 in pigs could be an important candidate gene responsible for xenobiotic metabolism, similar to CYP3A4 in humans. Accordingly, the tissue expression of CYP3A29 mRNA in domestic pigs has been determined by a real-time PCR. The enzymatic properties of CYP3A29, CYP3A4 and PLM (pig liver microsomes) were compared by kinetic analysis of TST (testosterone) 6β-hydroxylation and NIF (nifedipine) oxidation. CYP3A29 mRNA was highly expressed in the liver and small intestines of domestic pigs. The CYP3A29 enzyme expressed in Sf9 cells had the same TST-metabolizing activity as human CYP3A4 based on their roughly equal in vitro intrinsic clearance values. The affinity of CYP3A29 for NIF was lower than that of CYP3A4 but higher than that of PLM. KET (ketoconazole) was a more potent inhibitor of TST 6β-hydroxylation and NIF oxidation activities of CYP3A29 than TAO (troleandomycin). These findings indicate that pig CYP3A29 is similar to human CYP3A4 in both extent of expression and activity. The results reported in this paper provide a basis for future in vitro toxicity and metabolism studies.

Role of CYP3A and CYP2E1 in Alcohol-Mediated Increases in Acetaminophen Hepatotoxicity: Comparison of Wild-Type andCyp2e1(–/–) Mice

CYP2E1 is widely accepted as the sole form of cytochrome P450 responsible for alcohol-mediated increases in acetaminophen (APAP) hepatotoxicity. However, we previously found that alcohol [ethanol and isopentanol (EIP)] causes increases in APAP hepatotoxicity in Cyp2e1(-/-) mice, indicating that CYP2E1 is not essential. Here, using wild-type and Cyp2e1(-/-) mice, we investigated the relative roles of CYP2E1 and CYP3A in EIP-mediated increases in APAP hepatotoxicity. We found that EIP-mediated increases in APAP hepatotoxicity occurred at lower APAP doses in wild-type mice (300 mg/kg) than in Cyp2e1(-/-) mice (600 mg/kg). Although this result suggests that CYP2E1 has a role in the different susceptibilities of these mouse lines, our findings that EIP-mediated increases in CYP3A activities were greater in wild-type mice compared with Cyp2e1(-/-) mice raises the possibility that differential increases in CYP3A may also contribute to the greater APAP sensitivity in EIP-pretreated wild-type mice. At the time of APAP administration, which followed an 11 h withdrawal from the alcohols, alcohol-induced levels of CYP3A were sustained in both mouse lines, whereas CYP2E1 was decreased to constitutive levels in wild-type mice. The CYP3A inhibitor triacetyloleandomycin (TAO) decreased APAP hepatotoxicity in EIP-pretreated wild-type and Cyp2e1(-/-) mice. TAO treatment in vivo resulted in inhibition of microsomal CYP3A-catalyzed activity, measured in vitro, with no inhibition of CYP1A2 and CYP2E1 activities. In conclusion, these findings suggest that both CYP3A and CYP2E1 contribute to APAP hepatotoxicity in alcohol-treated mice.

Ginkgolide A contributes to the potentiation of acetaminophen toxicity by Ginkgo biloba extract in primary cultures of rat hepatocytes

The present cell culture study investigated the effect of Ginkgo biloba extract pretreatment on acetaminophen toxicity and assessed the role of ginkgolide A and cytochrome P450 3A (CYP3A) in hepatocytes isolated from adult male Long-Evans rats provided ad libitum with a standard diet. Acetaminophen (7.5-25 mM for 24 h) conferred hepatocyte toxicity, as determined by the lactate dehydrogenase (LDH) assay. G. biloba extract alone increased LDH leakage in hepatocytes at concentrations > or =75 mug/ml and > or =750 mug/ml after a 72 h and 24 h treatment period, respectively. G. biloba extract (25 or 50 mug/ml once every 24 h for 72 h) potentiated LDH leakage by acetaminophen (10 mM for 24 h; added at 48 h after initiation of extract pretreatment). The effect was confirmed by a decrease in [(14)C]-leucine incorporation. At the level present in a modulating concentration (50 mug/ml) of the extract, ginkgolide A (0.55 mug/ml), which increased CYP3A23 mRNA levels and CYP3A-mediated enzyme activity, accounted for part but not all of the potentiating effect of the extract on acetaminophen toxicity. This occurred as a result of CYP3A induction by ginkgolide A because triacetyloleandomycin (TAO), a specific inhibitor of CYP3A catalytic activity, completely blocked the effect of ginkgolide A. Ginkgolide B, ginkgolide C, ginkgolide J, quercetin, kaempferol, isorhamnetin, and isorhamnetin-3-O-rutinoside did not alter the extent of LDH leakage by acetaminophen. In summary, G. biloba pretreatment potentiated acetaminophen toxicity in cultured rat hepatocytes and ginkgolide A contributed to this novel effect of the extract by inducing CYP3A.

A reporter gene cellular model for evaluating induction of CYP3A4 by new chemical entities

AbstractA cell‐based reporter gene assay to study CYP3A4 induction was developed in the present study. The pregnane X receptor (PXR) gene or variant (PXR2) was cloned into the pSG5 vector and cotransfected into HepG2 cells, with a construct, pGL3‐3A4‐Luc, containing the promoter/enhancer region of the CYP3A4 gene. The two systems were validated using rifampicin (RIF). The variant, PXR2, did not mediate induction of CYP3A4 by rifampicin (10 µM) whereas PXR showed dose‐dependent induction of CYP3A4, with a fold change of 40–60, compared to the vehicle control, 0.1% dimethylsulfoxide (DMSO). Further validation of the PXR/3A4 system was performed using other inducers of CYP3A4, after which CYP3A4 inducibility by a Wyeth compound, Tanaproget (TNPR), and the synthetic steroid, 3‐ketodesogestrel (3‐KDG), were assessed. At the highest concentrations tested, troleandomycin (TROL) and phenobarbital (PB) induced CYP3A4 by 15–25‐fold, while carbamazepine (CMZ), dexamethasone (DEX), and erythromycin (ERY) produced fold induction of &lt;3. Pregnenolone 16‐α carbonitrile (PCN), a rat‐specific CYP3A inducer, did not induce CYP3A4. TNPR did not induce CYP3A4 at concentrations up to 10 µM while 3‐KDG produced a concentration‐dependent induction, with a fold change of 14 at 20 µM. These data show that the variant PXR2 is not activated by rifampicin. The PXR/3A4 system described can be used to study CYP3A4 induction and provides a robust, specific, and reproducible in vitro system that can be used to assess CYP3A4 inducibility by compounds in the drug development process. Drug Dev. Res. 67:470–475, 2006. © 2006 Wiley‐Liss, Inc.

Metabolic activation of a pentafluorophenylethylamine derivative: Formation of glutathione conjugates in vitro in the rat

The aim was to investigate the metabolic activation potential of a pentafluorophenylethylamine derivative (compound I) in vitro in the rat and to identify the cytochrome P450 (CYP) enzymes that catalyse these metabolic activation processes. Reduced glutathione (GSH) was fortified in rat hepatocytes and liver microsomes to trap possible reactive intermediates. Four glutathione conjugates (M1–4) were identified by LC-MSn following incubation of compound I in GSH-enriched rat hepatocytes and liver microsomes. Three of these conjugates (M2–4) have not been reported previously for pentafluorophenyl derivatives. Elemental composition analysis of these conjugates was obtained using high-resolution quadrupole time-of-flight mass spectrometry. The formation of GSH conjugate M1 was rationalized as a direct nucleophilic replacement of fluoride by glutathione, whereas the formation of the GSH conjugates M2–4 was proposed to occur by NADPH-dependent metabolic activation of the pentafluorophenyl ring via arene oxide, quinone and/or quinoneimine reactive intermediates. Formation of these conjugates was enhanced three- to five-fold in liver microsomes obtained from phenobarbital- and dexamethasone-treated rats. In incubations with pooled rat liver microsomes and recombinant rat CYP3A1 and CYP3A2, troleandomycin (TAO) reduced the formation of GSH conjugates M2–4 by 80–90%, but it had no effect on the formation of M1. Incubation of compound I with rat supersomes indicated that only CYP3A1 and CYP3A2 were capable of mediating these metabolic activation processes.

Macrolides in the treatment of asthma and cystic fibrosis

Asthma and cystic fibrosis are two respiratory diseases characterized by chronic inflammation, leading to remodelling of the airways. Macrolides are widely used antibiotics, with a peculiar anti-inflammatory effect. On the basis of the methodologies used by the Cochrane collaboration, this review discusses the evidence for their long-term use as anti-inflammatory agents in these two diseases. Three randomized-controlled trials (RCTs) were identified for both asthma and cystic fibrosis. A positive effect of macrolides on reducing eosinophil numbers and markers of eosinophilic inflammation was demonstrated in patients with asthma. Data on cystic fibrosis demonstrated an effect on lung function with an increase of 5.4% in forced vital capacity (FVC) in patients treated with macrolide vs. placebo, but without a significant effect on FEV1. Side-effects were rare, mild and reversible on withdrawal of treatment. Although preliminary data from small studies are promising, the role of macrolides in the treatment of these chronic disorders needs to be more firmly established with larger, well-designed trials, targeted to investigate major clinical outcomes.

Intravenous and oral alfentanil as in vivo probes for hepatic and first-pass cytochrome P450 3A activity: Noninvasive assessment by use of pupillary miosis

Systemic clearance of intravenous (IV) alfentanil (ALF) is an in vivo probe for hepatic cytochrome P450 (CYP) 3A activity, miosis is a surrogate for plasma ALF concentrations, and IV ALF miosis is a noninvasive probe for hepatic CYP3A. This investigation characterized the bioavailability and first-pass metabolism of oral ALF and tested the hypotheses that (1) first-pass ALF clearance reflects first-pass CYP3A activity, (2) miosis after oral ALF will reflect intestinal and hepatic CYP3A activity, and (3) miosis can approximate plasma concentration-based pharmacokinetic measures for IV and oral ALF as a noninvasive in vivo probe for hepatic and first-pass CYP3A activity and drug interactions. Results were compared with those for midazolam (MDZ), an alternative CYP3A probe. Ten volunteers were studied by use of a randomized, 9-way, crossover design after administration of rifampin (INN, rifampicin) (hepatic and intestinal CYP3A induction), troleandomycin (TAO) (hepatic and intestinal CYP3A inhibition), grapefruit juice (selective intestine CYP3A inhibition), or nothing (control). For each condition, they received 1 mg IV MDZ and then 15 microg/kg IV ALF, as well as 3 mg oral MDZ and then oral ALF (23 or 60 microg/kg) on another day. Plasma concentrations were determined by liquid chromatography-mass spectrometry. Dark-adapted pupil diameters were measured coincident with blood sampling. ALF effect was analyzed similarly to concentration to yield an effect "clearance" (Dose/Area under the pupil diameter change versus time curve). Bioavailability (Foral), hepatic extraction (EH), and intestinal availability (FG) were 0.26 +/- 0.08, 0.52 +/- 0.09, and 0.56 +/- 0.20, respectively, for MDZ and 0.42 +/- 0.15, 0.28 +/- 0.09, and 0.56 +/- 0.18, respectively, for ALF. Oral clearance (CL/F) was 34.7 +/- 12.8 and 10.9 +/- 3.5 mL.kg -1.min -1 , respectively, for MDZ and ALF. After rifampin, TAO, and grapefruit juice, ALF F oral was 0.04 +/- 0.02 (P <.05, versus control), 0.99 +/- 0.18 (P <.05, versus control), and 0.62 +/- 0.18 (P <.05, versus control), respectively; E H was 0.69 +/- 0.14 (P < .05, versus control), 0.04 +/- 0.01 (P <.05, versus control), and 0.26 +/- 0.08, respectively; F G was 0.16 +/- 0.10 (P <.05, versus control), 1.0 +/- 0.2 (P <.05, versus control), and 0.85 +/- 0.30 (P <.05, versus control), respectively; CL/F was 339 +/- 233 (P <.05, versus control), 0.62 +/- 0.26 (P <.05, versus control), and 6.7 +/- 2.5 (P <.05, versus control), respectively, and effect clearance was 2.1 +/- 1.1 (P <.05, versus control), 0.087 +/- 0.056 (P <.05, versus control), and 0.54 +/- 0.30 (0.73 +/- 0.43 mg.mm -1.h -1 in controls), respectively. There were significant correlations between ALF and MDZ systemic clearances (r2= 0.92), EH (r2=0.93), and CL/F (r2= 0.97), as well as between oral ALF effect (miosis) clearance and oral clearance (r2=0.59). ALF and MDZ have similar intestinal extraction but low and intermediate hepatic extraction, respectively. Systemic and oral clearances of ALF are excellent in vivo probes for hepatic and first-pass CYP3A activities and drug interactions. Miosis was an acceptable surrogate for plasma ALF. ALF miosis may be a suitable noninvasive in vivo probe for both hepatic and first-pass CYP3A.

Identification of cytochromes P450 2C9 and 3A4 as the major catalysts of phenprocoumon hydroxylation in vitro.

This in-vitro study aimed at an identification of cytochrome P(450) (CYP) enzymes catalysing the (S)- and (R)-hydroxylation of the widely used anticoagulant phenprocoumon (PPC) to its major, inactive metabolites. Relevant catalysts were identified by kinetic, correlation and inhibition experiments using human liver microsomes and recombinant enzymes. Kinetics revealed (S)-7-hydroxylation as quantitatively most important. Biphasic Eadie-Hofstee plots indicated more than one catalyst for the 4'-, 6- and 7-hydroxylation of both enantiomers with mean K(m1) and K(m2) of 144.5+/-34.9 and 10.0+/-6.49 microM, respectively. PPC hydroxylation rates were significantly correlated with CYP2C9 and CYP3A4 activity and expression analysing 11 different CYP-specific probes. Complete inhibition of PPC hydroxylation was achieved by combined addition of the CYP3A4-specific inhibitor triacetyloleandomycin (TAO) and a monoclonal, inhibitory antibody (mAb) directed against CYP2C8, 9, 18 and 19, except for the (R)-4'-hydroxylation that was, however, inhibited by ~80% using TAO alone. (S)-PPC hydroxylation was reduced by approximately 2/3 and approximately 1/3 using mAb2C8-9-18-19 and TAO, respectively, but (R)-6- and 7-hydroxylation by approximately 50% each. Experiments with mAbs directed against single CYP2C enzymes clearly indicated CYP2C9 as a major catalyst of the 6- and 7-hydroxylation for both enantiomers. However, CYP2C8 was equally important regarding the (S)-4'-hydroxylation. Recombinant CYP2C8 and CYP2C9 were high-affinity catalysts (K(m) <5 microM), whereas CYP3A4 operated with low affinity (K(m) >100 microM). CYP2C9 and CYP3A4 are major catalysts of (S)- and (R)-PPC hydroxylation, while CYP2C8 partly catalysed the (S)-4'-hydroxylation. Increased vigilance is warranted when PPC treatment is combined with substrates, inhibitors, or inducers of these enzymes.

Mechanism-based inhibition of CYP3A4 and CYP3A5 by seven inhibitors

We evaluated the potential for inhibition of CYP3A4 and CYP3A5 using seven mechanism-based inhibitors: troleandomycin (TAO), erythromycin (ERY), 6′, 7′-dihydroxybergamottin (DHB), diltiazem (DIL), verapamil (VER), ethynyl estradiol (EE) and ritonavir. These inhibitors were preincubated with NADPH and recombinant enzymes (CYP3A4 and CYP3A5) for 20 minutes. Enzyme activity was determined by 1-hydroxy triazolam (1-OH TRZ) formation from triazolam (250μM). Using recombinant enzymes, IC50 values were reduced by preincubation with inhibitors. IC50 values with preincubation with CYP3A4 using TAO, ERY, DHB, DIL, VER, EE and ritonavir were: 1.7, 2.9, 0.9, 7, 3.3, 4.4 and 0.06 μM respectively. With CYP3A5, IC50 values with preincubation were: 44.5, 77.5, 1.2, 21.8, 8, 17 and 0.1 μM respectively. The pattern of 1-OH TRZ IC50 values suggested two classes of mechanism-based inhibitors, with nonmacrolides showing less than a 4-fold difference between CYP3A4 and CYP3A5, and macrolides showing more than a 25-fold difference. Individuals polymorphically expressing CYP3A5 in significant amounts may have greater susceptibility to interactions involving nonmacrolide mechanism-based inhibitors than that seen with reversible inhibitors. However, macrolide mechanism-based inhibitors may be less susceptible to interactions in these subjects. Clinical Pharmacology & Therapeutics (2004) 75, P83–P83; doi: 10.1016/j.clpt.2003.11.316

Inhibitory effect of troleandomycin on the metabolism of omeprazole is CYP2C19 genotype-dependent

1. Eighteen healthy CYP2C19 genotyped male subjects were administered a 20-mg oral dose of omeprazole (OP) alone or received troleandomycin (TAO) 500 mg daily for 2 days before the dose of OP was administered. Blood samples were obtained and OP 5-hydroxyomeprazole (5-OH-OP) and OP sulfone in plasma were determined by reversed-phase HPLC. 2. The mean C max, AUC and CL for OP in poor metabolizers (PMs) were greater with TAO than without TAO. The C max and AUC of 5-OH-OP in PMs were significantly (p < 0.05) less with TAO than without TAO. The differences in 5-OH-OP between heterozygous extensive metabolizers (EMs) with TAO versus without TAO were similar to those observed in PMs, except for the AUC. However, in homozygous EMs, there were no statistical differences for the effect of TAO. 3. The effect of TAO on the metabolism of OP and its two principal metabolites differs in different genotype groups of CYP2C19. CYP3A4 not only plays a dominant role in the formation of OP sulfone, but also it contributes to the 5-hydroxylation of OP. Both CYP2C19 and CYP3A contribute to the further elimination of 5-OH-OP and OP sulfone.

Selective induction of abamectin metabolism by dexamethasone, 3-methylcholanthrene, and phenobarbital in Colorado potato beetle, Leptinotarsa decemlineata (Say)

Oxidative metabolism of abamectin as a principal resistance mechanism in the Colorado potato beetle, Leptinotarsa decemlineata (Say), was validated using dexamethasone (DEX)-, 3-methylcholanthrene (3-MC)-, and phenobarbital (PB)-induced beetles of susceptible (SS) and abamectin-resistant (AB-F) strains compared to non-induced strains, respectively. Significantly decreased mortality to abamectin in DEX- and 3-MC-induced versus non-induced SS beetles indicated that induction by DEX and 3-MC resulted in increased abamectin tolerance. The toxicity of abamectin in PB-induced SS and AB-F beetles compared to non-induced SS and AB-F beetles, respectively, was not significantly different, indicating induction by PB in either strain did not affect abamectin tolerance. Significant synergism by piperonyl butoxide (PBO) in DEX- and 3-MC-induced SS beetles was suggestive of increased oxidative metabolism as a mechanism responsible for the increased tolerance to abamectin. Mortality to abamectin was not significantly synergized by PBO in PB-induced SS beetles, indicating that oxidative metabolism of abamectin is not likely increased by PB induction. Three major metabolites of [ 3H]avermectin B 1a (24-OH, 26-OH (tentative identification), and 3″DM) were produced under both in vivo and in vitro conditions. DEX-induced SS beetles and microsomes prepared from DEX-induced SS beetles produced significantly more 3″DM versus non-induced beetles and microsomes, respectively, indicating that DEX was involved in a specific induction pathway but less involved in production of the two hydroxylated metabolites (24-OH and 26-OH). 3-MC-induced SS beetles produced significantly greater amounts of all three metabolites than non-induced beetles, which suggests that 3-MC was involved in multiple induction pathways. The enhanced production of 3″DM by DEX-induced microsomes was significantly inhibited by PBO and troleandomycin (TAO) in vitro. Because DEX is a specific inducer and TAO is a specific inhibitor of CYP3A family of cytochrome P450 monooxygenases, it appears that this family or a related family of P450s is involved in oxidative metabolism of abamectin to 3″DM and may be responsible for this aspect of resistance. The specific and significant increase in the production of 3″DM by DEX induction and its selective inhibition by TAO may provide an efficient means to obtain sequence of this monooxygenase by differential display or isotope-coded affinity tagging/mass spectrometry techniques.

Species Difference in Nisoldipine Oxidation Activity in the Small Intestine

Species difference in nisoldipine oxidation activities was investigated using small intestinal microsomes of rats, guinea pigs, dogs, monkeys and humans. The oxidation activities were estimated by measuring metabolites formation (BAY o 3199 and BAY r 9425) of nisoldipine. For the preparation of small intestinal microsomes of various animal species, the effect of protease inhibitors was preliminarily investigated. The formation of BAY o 3199 significantly increased in the rat small intestinal microsomes prepared with trypsin inhibitor. Using the trypsin inhibitor-treated small intestinal microsomes of various animals, metabolic intrinsic clearances (CL(int, in vitro)) for BAY o 3199 and BAY r 9425 formations were estimated based on an Eadie-Hofstee plot. The total CL(int,in vitro) estimated by the sum of CL(int, in vitro) for both formations in the small intestines of all species was much lower than that in the liver. There was a marked species difference in the nisoldipine oxidation activities in the small intestines, with the rank order being humans=monkeys>dogs>rats>guinea pigs, versus the following order in the liver: rats>monkeys=guinea pigs>humans>dogs. The formations of both BAY o 3199 and BAY r 9425 in the human intestinal microsomes were inhibited by pretreatment with troleandomycin (TAO) and antiserum against CYP3A4. Similar inhibition profile by TAO was obtained from the monkey intestinal microsomes. These results implied that monkeys would be a good predictor of human small intestinal metabolism for CYP3A4 substrates.

Species difference in stereoselective involvement of CYP3A in the mono-N-dealkylation of disopyramide

1. To determine which CYP isoenzyme is involved in the N-dealkylation of disopyramide (DP) metabolism in human and dog, and to determine the stereoselectivity of DP metabolism with human CYP and dog CYP isoenzymes, the following in vitro metabolism studies of DP were conducted: correlation between human CYP isoenzyme activities and DP metabolism with human liver microsomes; inhibition of DP metabolism in human and dog liver microsomes with chemical inhibitors of CYP isoenzymes; inhibition of DP metabolism inhuman microsomes withhuman CYPantibodies; inhibition of DP metabolism in dog liver microsomes with human and dog CYP antibodies; metabolism of DP with human (CYP3A4) and dog (CYP3A12) cDNA-expressed isoenzymes; determination of Km and Vmax of DP enantiomers by using cDNA-expressed CYP3A4 and CYP3A12. 2. In human liver microsomes, the formation of the mono-N-dealkylated disopyramide (MNDP) metabolite was best correlated with CYP3A4 activities. DP metabolism was substantially inhibited by ketoconazole, troleandomycin (TA) and human CYP3A4 antibody. DP was metabolized by cDNA-expressed CYP3A isoenzymes. In dog liver microsomes, DP metabolism was inhibited by ketoconazole, TA and dog anti-CYP3A12. DP was also metabolized by cDNA-expressed CYP3A12. 3. CYP3A4 and CYP3A12 are the principal isoenzymes involved in DP metabolism in human and dog respectively. There was no stereoselectivity in N-dealkylation of DP by human CYP3A4. However, there was notable stereoselectivity in the N-dealkylation by dog CYP3A12.

In-vitro and in-vivo evaluation of the drug-drug interaction between fluvoxamine and clozapine.

The drug-drug interaction between fluvoxamine (FLV) and clozapine (CLZ) was evaluated by in-vitro and in-vivo methods. In-vitro studies were conducted using human hepatic microsomal preparations with standard chemical inhibitors of the cytochrome P450 (CYP 450) isozyme system. Furafyline, FLV, troleandomycin (TAO) and erythromycin were used as the chemical inhibitors. For the in-vivo study, nine male schizophrenic patients were administered a single dose of CLZ 50 mg on two separate occasions with a 2-week FLV treatment of 50 mg twice a day in between each CLZ dose. Blood samples were obtained over 48 h following CLZ administration. CLZ and its two principle metabolites, clozapine N-oxide (CNO) and desmethylclozapine (DCLZ), were measured by high performance liquid chromatography with ultraviolet detection for both in-vitro and in-vivo studies. The in-vitro formation of DCLZ was inhibited by furafyline and FLV by 42.0% and 48.5% (P<0.01), respectively. TAO and erythromycin had only modest inhibition effects on DCLZ formation of 18.3% and 21.0% (P = NS), respectively. CNO in-vitro formation was significantly reduced by TAO and erythromycin by 44.5% and 45.0% (P<0.01), respectively. Furafyline and FLV had only modest effects of 19.2% and 8.5% (P = NS), respectively. In schizophrenic patients, FLV resulted in a pronounced increased in CLZ plasma concentrations with the total mean CLZ AUC increased by a factor of 2.58 from 780.8 ng/ml per hour to 2218.0 ng/ml per hour (P<0.001). All patients were sedated during combined FLV and CLZ use. During FLV treatment, CNO and DCLZ AUC both decreased by 18.8% (P = 0.07) and 9.0% (P = NS), respectively. These results indicate that in-vitro evaluations may not always accurately reflect changes in drug-drug interaction observed in-vivo. Careful patient monitoring is recommended during FLV/CLZ co-administration.

Role of CYP3A in haloperidol N-dealkylation and pharmacokinetics in rats.

Haloperidol (HP), an antipsychotic drug, is N-dealkylated by cytochrome P450 (CYP) to 4-fluorobenzoylpropionic acid (FBPA). The purpose of this study was to identify whether CYP3A metabolizes HP to FBPA in hepatic microsomes of rats and to investigate whether an inhibitor or an inducer of CYP3A affects HP pharmacokinetics in rats. The rate of FBPA formation was determined in hepatic microsomes from 8-week-old male Sprague-Dawley rats. Among several specific CYP isozyme inhibitors including troleandomycin (TAO), diethyldithiocarbamate, furafylline and quinine, only TAO showed marked inhibition of FBPA formation. Anti-rat CYP3A serum inhibited FBPA formation by 76.4%, while other anti-rat CYP sera (1A1, 1A2, 2B1, 2C11, 2E1) only slightly did. In a pharmacokinetic study, 8-week-old male Sprague-Dawley rats were given 0.5 mg/kg HP intravenously after treatment with 100 mg/kg erythromycin, a CYP3A inhibitor, or 80 mg/kg dexamethasone, a CYP3A inducer, intraperitoneally once a day for 7 days or 2 days, respectively or untreated. HP half-life was prolongated to 171% of the average control value by erythromycin and shortened to 49% of control by dexamethasone. HP clearance was reduced to 63% of control by erythromycin and was increased to 167% of control by dexamethasone. These results suggested that CYP3A mainly catalyzed HP to FBPA in rats, and the modification of this enzyme activity would affect the pharmacokinetics of HP.

Cytochrome P450 isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of NS-21

1. Cytochrome P450 (P450) isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of (±)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2- hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21) have been identified in rat and man. 2. Anti-CYP2C11 antibody inhibited the N-deethylation of S- and R-NS-21 in rat hepatic microsomes by 84 and 66% respectively, indicating that CYP2C11 is mainly responsible for these activities in male rats. 3. Of several human recombinant P450 isoforms, CYP3A4 had the activities for the Ndeethylation of S- and R-NS-21. In addition, triacetyloleandomycin (TAO), an inhibitor of the CYP3A subfamily, significantly inhibited the N-deethylation of S- and R-NS-21 in human hepatic microsomes by 67 and 69%, respectively. CYP3A4 therefore contributes to it in man. 4. Quinine, an inhibitor of the rat CYP2D subfamily, significantly inhibited the cyclohexane-4-cis-hydroxylation of S-NS-21 by 48% in rat hepatic microsomes. In contrast, this inhibitor had little effect on the cyclohexane-4-trans-hydroxylation of S-NS21, and the cyclohexane-4-cis-and trans-hydroxylation of R-NS-21. 5. Human recombinant CYP3A4 catalysed the cyclohexane-4-trans-hydroxylation of S-NS-21, and CYP2D6 supported the cyclohexane-4-cis- and trans-hydroxylation of S-NS-21. Quinidine, an inhibitor of human CYP2D6, had little effect on these latter activities in human hepatic microsomes. TAO significantly inhibited the cyclohexane-4-trans-hydroxylation of S-NS-21 by 75%, indicating that CYP3A4 catalyses this reaction.

Identification of cytochromes P450 involved in human liver microsomal metabolism of ecabapide, a prokinetic agent

1. In vitro studies identified the hepatic cytochrome P450 (CYP) enzyme(s) involved in the major metabolism of ecabapide in human. 2. Ecabapide mainly underwent N-dealkylation to form M1 and 6-hydroxylation of the benzamide moiety to form M6. 3. The rates of formation of the major metabolites M1 and M6 were significantly correlated with CYP3A-selective testosterone 6beta-hydroxylase activities in 14 different human liver microsomes. The formation of both metabolites was markedly decreased by ketoconazole, miconazole or troleandomycin (TAO), CYP3A-selective inhibitors, and also was inhibited by anti-CYP3A antibodies. 4. These results strongly indicate that CYP3A is the predominant isozyme responsible for the major metabolism of ecabapide in human liver microsomes. 5. Marginal inhibition of the formation of M1 and M6 by nifedipine, a substrate of CYP3A with a Ki>100 muM, suggested that nifedipne has a limited potential to inhibit the major metabolic pathways of ecabapide.