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Six proton pump inhibitors (PPIs), omeprazole, lansoprazole, esomeprazole, dexlansoprazole, pantoprazole, and rabeprazole, were shown to be weak inhibitors of cytochromes P450 (CYP3A4, -2B6, -2D6, -2C9, -2C8, and -1A2) in human liver microsomes. In most cases, IC50values were greater than 40 μM, except for dexlansoprazole and lansoprazole with CYP1A2 (IC50= ∼8 μM) and esomeprazole with CYP2C8 (IC50= 31 μM). With the exception of CYP2C19 inhibition by omeprazole and esomeprazole (IC50ratio, 2.5 to 5.9), there was no evidence for a marked time-dependent shift in IC50(IC50ratio, ≤2) after a 30-min preincubation with NADPH. In the absence of preincubation, lansoprazole (IC50= 0.73 μM) and esomeprazole (IC50= 3.7 μM) were the most potent CYP2C19 inhibitors, followed by dexlansoprazole and omeprazole (IC50= ∼7.0 μM). Rabeprazole and pantoprazole (IC50= ≥25 μM) were the weakest. A similar ranking was obtained with recombinant CYP2C19. Despite the IC50ranking, after consideration of plasma levels (static and dynamic), protein binding, and metabolism-dependent inhibition, it is concluded that omeprazole and esomeprazole are the most potent CYP2C19 inhibitors. This was confirmed after the incubation of the individual PPIs with human primary hepatocytes (in the presence of human serum) and by monitoring their impact on diazepam N-demethylase activity at a low concentration of diazepam (2 μM). Data described herein are consistent with reports that PPIs are mostly weak inhibitors of cytochromes P450 in vivo. However, two members of the PPI class (esomeprazole and omeprazole) are more likely to serve as clinically relevant inhibitors of CYP2C19.

Monkeys have been proposed as an animal model to predict the magnitude of human clinical drug-drug interactions caused by CYP3A4 enzyme induction. To evaluate whether the cynomolgus monkey can be an effective in vivo model, human CYP3A4 inducers were evaluated both in vitro and in vivo. First, a full-length pregnane X receptor (PXR) was cloned from the cynomolgus monkey, and the sequence was compared with those of rhesus monkey and human PXR. Cynomolgus and rhesus monkey PXR differed by only one amino acid (A68V), and both were highly homologous to human PXR (∼96%). When the transactivation profiles of 30 compounds, including known inducers of CYP3A4, were compared between cynomolgus and human PXR, a high degree of correlation with EC50values was observed. These results suggest that cynomolgus and human PXR respond in a similar fashion to these ligands. Second, two known human CYP3A4 inducers, rifampicin and hyperforin, were tested in monkey and human primary hepatocytes for induction of CYP3A enzymes. Both monkey and human hepatocytes responded similarly to the inducers and resulted in increased RNA and enzyme activity changes of CYP3A8 and CYP3A4, respectively. Lastly, in vivo induction of CYP3A8 by rifampicin and hyperforin was shown by significant reductions of midazolam exposure that were comparable with those in humans. These results show that the cynomolgus monkey can be a predictive in vivo animal model of PXR-mediated induction of human CYP3A4 and can provide a useful assessment of the resulting pharmacokinetic changes of affected drugs.

The effect of common organic solvents on the activities of various human cytochromes P450 has been reported. However, very little is known about their influence on CYP2B6 and CYP2C8 enzymes. The purpose of this study was to investigate the effect of solvents on the kinetics of representative CYP2B6 (bupropion hydroxylase) and CYP2C8 (paclitaxel hydroxylase) reactions in human liver microsomes. Methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied at increasing volumes (v/v). Acetonitrile, DMSO, and ethanol were shown to increase the Kmand decrease the intrinsic clearance (CLint) of CYP2B6-mediated bupropion hydroxylation in a concentration-dependent manner. These solvents did not noticeably alter the Vmaxat concentrations of ≤1% (v/v). Unlike the other solvents studied, the effect of methanol (≤0.5%, v/v) on CYP2B6 kinetics was negligible. Both DMSO and ethanol increased the Kmand decreased the CLintof CYP2C8-mediated paclitaxel hydroxylation in a concentration-dependent manner. Acetonitrile had minimal influence on CYP2C8 enzyme kinetics at concentrations of ≤1% (v/v). Methanol decreased the Kmof paclitaxel at low concentrations followed by an increase at concentrations of ≥2% (v/v). This differential influence on Kmresulted in an increased CLintat low concentrations followed by a decrease at high concentrations. The studied solvents had minimal influence on Vmaxof paclitaxel. Collectively, DMSO and ethanol were not suitable for characterizing CYP2B6- and CYP2C8-mediated reactions because they showed concentration-dependent inhibition. Methanol and acetonitrile at concentrations of ≤0.5% and ≤1% (v/v) appeared to be suitable for the measurement of CYP2B6- and CYP2C8-mediated activities, respectively.

The effects of route of administration on the stereoselective pharmacokinetics of tramadol (T) and its active metabolite (M1) were studied in rats. A single 20 mg/kg dose of racemic T was administered through intravenous, intraperitoneal, or oral route to different groups of rats, and blood and urine samples were collected. Samples were analyzed using chiral chromatography, and pharmacokinetic parameters (mean ± SD) were estimated by noncompartmental methods. Following intravenous injection, there was no stereoselectivity in the pharmacokinetics of T. Both enantiomers showed clearance values (62.5 ± 27.2 and 64.4 ± 39.0 ml/min/kg for (+)‐ and (−)‐T, respectively) that were equal or higher than the reported liver blood flow in rats. Similar to T, the area under the plasma concentration–time curves (AUCs) of M1 did not exhibit stereoselectivity after intravenous administration of the parent drug. However, the systemic availability of (+)‐T was significantly (P< 0.05) higher than that of its antipode following intraperitoneal (0.527 ± 0.240 vs. 0.373 ± 0.189) and oral (0.307 ± 0.136 vs. 0.159 ± 0.115) administrations. The AUC of the M1 enantiomers, on the other hand, remained mostly nonstereoselective regardless of the route of administration. Pharmacokinetic analysis indicated that the stereoselectivity in the pharmacokinetics of oral T is due to stereoselective first pass metabolism in the liver and, possibly, in the gastrointestinal tract. The direction and extent of stereoselectivity in the pharmacokinetics of T and M1 in rats were in agreement with those previously reported in humans, suggesting that the rat may be a suitable model for enantioselective studies of T pharmacokinetics. Chirality, 2007. © 2006 Wiley‐Liss, Inc.

The rapid and direct effects of nitric oxide (NO) donors sodium nitroprusside (SNP) and isosorbide dinitrate (ISDN) on the hepatic and biliary disposition of chlorzoxazone (CZX), a marker of CYP2E1, and dextromethorphan (DEM), a marker of CYP2D1, were studied in a single-pass isolated perfused rat liver model. Livers (n = 30) were perfused with constant concentrations of NO donors (0–120 min) in addition to infusion of CZX or DEM (60–120 min), and periodical outlet and bile samples were collected. Both ISDN and SNP significantly reduced (30 and 60%, respectively) the hepatic extraction ratio of CZX and decreased (50 and 70%, respectively) the recovery of the CYP2E1-mediated metabolite, 6-hydroxychlorzoxazone, in the outlet perfusate and bile. As for DEM, both NO donors increased (up to 3.5-fold) the recovery of the CYP2D1-mediated metabolite dextrorphan (DOR) in the outlet perfusate. However, this was associated with a simultaneous decrease (50–75%) in the excretion of the metabolite into the bile, thus resulting in no change in the overall recovery of DOR as a result of NO donor treatment. The decrease in the biliary excretion of DOR was caused by NO-induced simultaneous reductions in both the conjugation of DOR and biliary clearance of DOR conjugate. Additionally, both SNP and ISDN significantly reduced the metabolism of DEM to 3-hydroxymorphinan, which is mostly regulated by CYP3A2. These studies in an intact liver model confirm the selectivity of the inhibitory effects of NO donors on cytochrome P450 enzymes, which was recently reported in microsomal studies, and expand these inhibitory effects to conjugation pathways.

Nitric oxide (NO) has recently been shown to decrease cytochrome P450 (P450) enzyme activity rapidly (≤30 min), concentration dependently, and enzyme-selectively in the rat liver. Interestingly, among all the studied P450 enzymes, only CYP2D1 was not affected by NO donors. However, these studies were conducted using only a single concentration of the substrates, thus lacking information about the possible simultaneous changes in both maximum velocity (Vmax) and affinity (Km) of the enzymes. In the present study, we systematically evaluated the effects of NO on the enzyme kinetic parameters of marker substrates for a range of P450 enzymes, including 2D1. Livers were perfused (1 h) in the absence (control) or presence of two NO donors with different mechanisms of NO release. At the end of the perfusion, microsomes were prepared and used for kinetic analysis. Except for 2D1, NO reduced the Vmax of all the model reactions studied, although to a varying degree. However, the effects of NO donors on Km were more diverse. Whereas the Km values for testosterone 6{szligbeta}-hydroxylation (3A2) and 16α-hydroxylation (2C11) significantly decreased, the values for chlorzoxazone 6-hydroxylation (2E1), dextromethorphan N-demethylation (3A2), and high affinity ethoxyresorufin O-dealkylation (1A1/2) significantly increased in the presence of NO donors. Furthermore, the Km values for the high-affinity component of dextromethorphan O-demethylation and benzyloxyresorufin O-dealkylation remained unchanged. These results indicate that NO can potentially change both the Vmax and Km of various substrates selectively and confirm our previous findings that the activity of CYP2D1 is not affected by NO donors.

Nitric oxide (NO) is implicated as a mediator in the decreased catalytic activities of cytochrome P450 (P450) enzymes during inflammation or infection. Here, we examined the time course and the reversibility of the NO effect on P450s using isolated perfused rat livers. Livers were perfused at a constant rate with the NO donor sodium nitroprusside (SNP) for 0.5 or 1 h, followed by washout periods of 0 to 2.5 h. At the end of perfusion, microsomes were prepared and analyzed for P450 activities and other metabolic markers. Whereas 0.5 h of NO exposure caused an irreversible decline (approximately 30%) in total P450 content, a greater decline after 1 h of NO (approximately 55%) was mostly (approximately 30%) reversible, a pattern identical to that observed for the microsomal heme content. NO exposure also caused an enzyme-selective and time-dependent decline in P450 activities. Whereas the pattern of decline and reversibility of activities were qualitatively similar for CYP3A2, 2C11, 2E1, and 1A1/2, they differed for 2B1/2 and 2D1 in that the decline in the activity was delayed (1 h) for 2B1/2 and not observed for 2D1. This may be attributed to the accessibility of heme or cysteine thiolate and/or the presence/reactivity of critical cysteinyl amino acid residues in various P450 enzymes. Additionally, for most enzymes, the activity showed a biphasic decline, one within 1 h of SNP perfusion and another after 2 h of washout. This was associated with an identical biphasic decline in the microsomal free thiols, presumably due to the rapid and slow reaction of NO and peroxynitrite, respectively, with critical P450 thiols. The short-term effects of NO on P450 are time-dependent and enzyme-selective, with both reversible and irreversible mechanisms.

Purpose.We recently showed that the perfusion of isolated rat livers with perfusates containing bovine serum albumin (BSA) would significantly stimulate the release of tumor necrosis factor (TNF)-α. Here, we hypothesize that BSA-induced increase in the release of TNF-α, and possibly other cytokines, would affect cytochrome P450 (CYP)-mediated drug metabolism. Methods.Rat livers were perfused ex vivofor 1, 2, or 3 h with a physiologic buffer containing or lacking 1% BSA (n= 4-5/group). At the end of perfusion, liver microsomes were prepared and analyzed for their total CYP, CYP2E1, CYP3A2, and CYP2C11 protein contents and the activities of cytochrome c reductase, CYP2E1, CYP3A2, CYP2C11, CYP2E1, CYP2D1, CYP1A1, and CYP2B1/2. In addition, the concentrations of various cytokines and nitric oxide were quantified in the outlet perfusate. Results.In the absence of BSA, the perfusate levels of all measured cytokines and nitric oxide were low. However, when the perfusate contained BSA, the levels of TNF-α, interleukin-6, and nitric oxide increased significantly (p < 0.005). Perfusion of the livers for 3 h with the BSA-containing perfusate resulted in significant (p < 0.05) decreases in the total CYP (41%), CYP2E1 (59%), CYP3A2 (68%), and CYP2C11 (50%) protein contents and activities of cytochrome c reductase (31%), CYP2E1 (66%), CYP3A2 (54%), and CYP2C11 (51%). In contrast, perfusion of livers for 1 or 2 h with the BSA perfusate did not have any significant effect on CYP-mediated metabolism. The CYP1A2, CYP2D1, and CYP2B1/2 activities were not affected by BSA, regardless of perfusion time. Conclusion.Addition of BSA to perfusates, which is a routine practice in isolated rat liver studies, can reduce CYP-mediated drug metabolism by a mechanism independent of protein-binding effect.

Mehta: Kyowa Kirin: Current Employment. Patel: Kyowa Kirin: Current Employment. Vuppugalla: Kyowa Kirin: Current Employment. Tudor: Kyowa Kirin: Current Employment. Dwyer: Kyowa Kirin: Current Employment; Bristol Myers Squibb: Current Employment. Hruska: Viatris: Current equity holder in publicly-traded company; Viking: Current equity holder in publicly-traded company; Durect: Current equity holder in publicly-traded company; CymaBay: Current equity holder in publicly-traded company; Takeda: Current equity holder in publicly-traded company; Pfizer: Current equity holder in publicly-traded company; Merck: Current equity holder in publicly-traded company; Johnson & Johnson: Current equity holder in publicly-traded company; GlaxoSmithKline: Current equity holder in publicly-traded company; Genmab: Current equity holder in publicly-traded company; Gilead: Current equity holder in publicly-traded company; Biohaven: Current equity holder in publicly-traded company; AbbVie: Current equity holder in publicly-traded company; Bristol Myers Squibb: Current equity holder in publicly-traded company; Kyowa Kirin: Current Employment. Marsteller: Kyowa Kirin: Current Employment.Yes. Mogamulizumab is a CCR4-directed monoclonal antibody indicated for the treatment of adult patients with relapsed or refractory mycosis fungoides or Sézary syndrome after at least one prior systemic therapy - new dosing regimen under examination.

Purandare: Bristol-Myers Squibb: Employment. McDevitt:Bristol-Myers Squibb: Employment. Gottardis:Bristol-Myers Squibb: Employment. You:Bristol-Myers Squibb: Employment. Lombardo:Bristol_Myers Squibb: Employment. Penhallow:Bristol-Myers Squibb: Employment. Vuppugalla:Bristol-Myers Squibb: Employment. Trainor:Bristol-Myers Squibb: Employment. Lorenzi:Bristol-Myers Squibb: Employment.

Abstract 4112