Timothy S. Tracy

CYP2C9 Inhibition: Impact of Probe Selection and Pharmacogenetics on in Vitro Inhibition Profiles

Drug-drug interactions may cause serious adverse events in the clinical setting, and the cytochromes P450 are the enzyme system most often implicated in these interactions. Cytochrome P450 2C is the second most abundant subfamily of cytochrome P450 enzymes and is responsible for metabolism of almost 20% of currently marketed drugs. The most abundant isoform of this subfamily is CYP2C9, which is the major clearance pathway for the low therapeutic index drugs warfarin and phenytoin. Considering the importance of CYP2C9 to drug-drug interactions, the in vitro-in vivo extrapolation of drug-drug interactions for CYP2C9 may be confounded by the presence of polymorphic variants and the possibility of multiple binding regions within the CYP2C9 active site, leading to the potential for genotype- and substrate-dependent inhibition. To address the issues of genotype-dependent enzyme inhibition as well as probe substrate correlations, the inhibitory potency (Ki) of 28 effector molecules was assessed with five commonly used probes of CYP2C9 in both the CYP2C9.1 and CYP2C9.3 proteins. The inhibition of CYP2C9.1 and CYP2C9.3 by the battery of inhibitors with five substrate probes demonstrated differential inhibition potency not only between the two genotypes but also across substrate probes. Furthermore, the substrate probes fell into three distinct classes depending on genotype, suggesting that multiple probes may be needed to fully assess inhibition of CYP2C9 in vitro. Thus, both genotype and choice of probe substrate must be considered when attempting to predict potential CYP2C9 drug-drug interactions from in vitro data.

Inhibition of CYP2D6 activity by bupropion

Abstract: The purpose of this study was to assess the effect of bupropion on cytochrome P450 2D6 (CYP2D6) activity. Twenty-one subjects completed this repeated-measures study in which dextromethorphan (30-mg oral dose) was administered to smokers at baseline and after 17 days of treatment with either bupropion sustained-release (150 mg twice daily) or matching placebo. Subjects quit smoking 3 days before the second dextromethorphan administration. To assess CYP2D6 activity, urinary dextromethorphan/dextrorphan metabolic ratios were calculated after an 8-hour urine collection. Thirteen subjects received bupropion, and 8 received placebo. In those receiving active medication, the dextromethorphan/dextrorphan ratio increased significantly at the second assessment relative to the first (0.012 ± 0.012 vs. 0.418 ± 0.302; P < 0.0004). No such change was observed in those randomized to placebo (0.009 ± 0.010 vs. 0.017 ± 0.015; P = NS). At baseline, all subjects were phenotypically extensive CYP2D6 metabolizers (metabolic ratio <0.3); after treatment, 6 of 13 subjects receiving bupropion, but none of those receiving placebo, had metabolic ratios consistent with poor CYP2D6 metabolizers. Bupropion is therefore a potent inhibitor of CYP2D6 activity, and care should be exercised when initiating or discontinuing bupropion use in patients taking drugs metabolized by CYP2D6.

Indices of activity of the nitric oxide system in hemodialysis patients

Arginine deficiency and/or increased levels of circulating nitric oxide (NO) synthesis (NOS) inhibitors can cause reduced NOS, which may contribute to hypertension in patients with end-stage renal disease (ESRD). To test these hypotheses, NO oxidation products (NO(2) + NO(3) = NO(x)) and cyclic guanosine monophosphate (cGMP), the vasodilatory second messenger of NO, were measured in the blood, urine, and dialysate effluent of hemodialysis (HD) patients and compared with the blood and urine of healthy subjects. The subjects ate a controlled low-nitrate diet (approximately 330 micromol/d) for 48 hours before and during blood, dialysis effluent, and 24-hour urine collection. NO(x) output was significantly reduced in HD patients versus controls (552 +/- 51 v 824 +/- 96 micromol/24 h; P < 0.001), whereas cGMP output was not low versus controls. Plasma arginine level was normal and plasma levels of citrulline and the endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA), were markedly elevated in patients with ESRD versus controls. Systolic blood pressure was greater in HD patients compared with controls despite concurrent antihypertensive therapy in most patients with ESRD. These studies suggest NO production is low in patients with ESRD undergoing HD, possibly because of the increased ratio of plasma ADMA to arginine.

Nitric oxide production is low in end-stage renal disease patients on peritoneal dialysis

To test the hypothesis that nitric oxide (NO) deficiency occurs in end-stage renal disease (ESRD), NO oxidation products (NO2 + NO3 = NOx) and cGMP were measured in blood, urine, and dialysate effluent of peritoneal dialysis (PD) patients and compared with blood and urine of healthy subjects. All subjects were on a controlled low-nitrate diet (approximately 330 micromol/day). NOx and cGMP outputs were significantly reduced in PD patients (334 +/- 50 micromol/24 h and 55 +/- 13 nmol/24 h, respectively) vs. controls (823 +/- 101 micromol/24 h and 149 +/- 46 nmol/24 h). Plasma arginine was borderline low, plasma citrulline was elevated and plasma levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine were approximately five time higher in PD patients (2.2 +/- 0.3 microM) vs. controls (0.4 +/- 0.1 microM). Although blood pressure (BP) was not different between groups at the time of study, 10 of 11 PD patients were on medication for hypertension. These studies demonstrate that total NO production is low in ESRD, and with appropriate caution, we conclude that this NO deficiency may contribute to the increased BP that occurs in ESRD.To test the hypothesis that nitric oxide (NO) deficiency occurs in end-stage renal disease (ESRD), NO oxidation products (NO2 + NO3 = NOx) and cGMP were measured in blood, urine, and dialysate effluent of peritoneal dialysis (PD) patients and compared with blood and urine of healthy subjects. All subjects were on a controlled low-nitrate diet (∼330 μmol/day). NOx and cGMP outputs were significantly reduced in PD patients (334 ± 50 μmol/24 h and 55 ± 13 nmol/24 h, respectively) vs. controls (823 ± 101 μmol/24 h and 149 ± 46 nmol/24 h). Plasma arginine was borderline low, plasma citrulline was elevated and plasma levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine were approximately five time higher in PD patients (2.2 ± 0.3 μM) vs. controls (0.4 ± 0.1 μM). Although blood pressure (BP) was not different between groups at the time of study, 10 of 11 PD patients were on medication for hypertension. These studies demonstrate that total NO production is low in ESRD, and with appropriate caution, we conclude that this NO deficiency may contribute to the increased BP that occurs in ESRD.

Alterations in drug disposition during pregnancy:: implications for drug therapy

The disposition of many medications is altered during pregnancy. Due to changes in many physiological parameters as well as variability in the activity of maternal drug-metabolizing enzymes, the efficacy and toxicity of drugs used by pregnant women can be difficult to predict. Enzymatic activity exhibited by the placenta and fetus may affect maternal drug distribution and clearance also. In addition, efflux transporters have been detected in high amounts within placental tissue, potentially limiting fetal exposure to xenobiotics. Dosage adjustments of antiepileptic drugs, antidepressants and anti-infectives administered during pregnancy have been required due to these changes in drug metabolism and disposition. As such, pregnant women may require different dosing regimens than both men and non-pregnant women.

Alterations in drug disposition during pregnancy

The disposition of many medications is altered during pregnancy. Due to changes in many physiological parameters as well as variability in the activity of maternal drug-metabolizing enzymes, the efficacy and toxicity of drugs used by pregnant women can be difficult to predict. Enzymatic activity exhibited by the placenta and fetus may affect maternal drug distribution and clearance also. In addition, efflux transporters have been detected in high amounts within placental tissue, potentially limiting fetal exposure to xenobiotics. Dosage adjustments of antiepileptic drugs, antidepressants and anti-infectives administered during pregnancy have been required due to these changes in drug metabolism and disposition. As such, pregnant women may require different dosing regimens than both men and non-pregnant women.

Modeling Kinetic Data from In Vitro Drug Metabolism Enzyme Experiments

Modeling of in vitro enzyme kinetic data derived from drug metabolism experiments can greatly facilitate the drug development process because estimation of kinetic parameters can facilitate decision making regarding whether to continue development of a compound. From this information, predictions can be made regarding the “metabolic stability” of a compound and even the in vivo intrinsic clearance of the drug. Many drugs exhibit typical Michaelis–Menten‐type kinetics in vitro that result in a hyperbolic kinetic profile from which Km and Vm can be readily estimated. However, it is increasingly being recognized that many drug compounds exhibit “atypical” enzyme kinetics in vitro, requiring use of more complex kinetic models for data fitting and parameter estimation. These atypical kinetic profiles may include sigmoidal kinetics (autoactivation), biphasic kinetics, substrate inhibition kinetics, and heterotropic cooperativity (activation). This article briefly summarizes the types of equations necessary to adequately model both typical and atypical kinetic profiles in order to facilitate correct estimation of the relevant kinetic parameters.

Effects of genetic variants of human P450 oxidoreductase on catalysis by CYP2D6 in vitro.

Objectives Cytochrome P450 (P450) oxidoreductase (POR) donates electrons to all microsomal cytochrome P450s, including drug-metabolizing and steroidogenic enzymes. Severe POR mutations cause skeletal malformations and disordered steroidogenesis. The POR polymorphism A503V is found on approximately 28% of human alleles and decreases activities of CYP3A4 and steroidogenic CYP17, but not the activities of steroidogenic CYP21 or drug-metabolizing CYP1A2 and CYP2C19. CYP2D6 metabolizes about 25% of clinically used drugs; we assessed the capacity of POR variants to support the activities of human CYP2D6. Methods N-27 forms of wildtype (WT), Q153R, A287P, R457H and A503V POR, and WT CYP2D6 were expressed in Escherichia coli. POR proteins in bacterial membranes were reconstituted with purified CYP2D6. Support of CYP2D6 was measured by metabolism of EOMCC (2H-1-benzopyran-3-carbonitrile,7-(ethoxy-methoxy)-2-oxo-(9Cl)), dextromethorphan and bufuralol. Michaelis constant (Km) and maximum velocity (Vmax) were determined in three triplicate experiments for each reaction; catalytic efficiency is expressed as Vmax/Km. Results Compared with WT POR, disease-causing POR mutants A287P and R457H supported no detectable CYP2D6 activity with EOMCC, but A287P supported approximately 25% activity with dextromethorphan and bufuralol. Q153R had increased function with CYP2D6 (128% with EOMCC, 198% with dextromethorphan, 153% with bufuralol). A503V supported decreased CYP2D6 activity: 85% with EOMCC, 62% with dextromethorphan and 53% with bufuralol. Conclusion POR variants have different effects depending on the substrate metabolized. Disease-causing POR mutations R457H and A287P had poor activities, suggesting that diminished drug metabolism should be considered in affected patients. The common A503V polymorphism impaired CYP2D6 activities with two commonly used drugs by 40–50%, potentially explaining some genetic variation in drug metabolism.

Glucuronidation of Dihydrotestosterone and trans-Androsterone by Recombinant UDP-Glucuronosyltransferase (UGT) 1A4: Evidence for Multiple UGT1A4 Aglycone Binding Sites

UDP-glucuronosyltransferase (UGT) 1A4-catalyzed glucuronidation is an important drug elimination pathway. Although atypical kinetic profiles (nonhyperbolic, non-Michaelis-Menten) of UGT1A4-catalyzed glucuronidation have been reported occasionally, systematic kinetic studies to explore the existence of multiple aglycone binding sites in UGT1A4 have not been conducted. To this end, two positional isomers, dihydrotestosterone (DHT) and trans-androsterone (t-AND), were used as probe substrates, and their glucuronidation kinetics with HEK293-expressed UGT1A4 were evaluated both alone and in the presence of a UGT1A4 substrate [tamoxifen (TAM) or lamotrigine (LTG)]. Coincubation with TAM, a high-affinity UGT1A4 substrate, resulted in a concentration-dependent activation/inhibition effect on DHT and t-AND glucuronidation, whereas LTG, a low-affinity UGT1A4 substrate, noncompetitively inhibited both processes. The glucuronidation kinetics of TAM were then evaluated both alone and in the presence of different concentrations of DHT or t-AND. TAM displayed substrate inhibition kinetics, suggesting that TAM may have two binding sites in UGT1A4. However, the substrate inhibition kinetic profile of TAM became more hyperbolic as the DHT or t-AND concentration was increased. Various two-site kinetic models adequately explained the interactions between TAM and DHT or TAM and t-AND. In addition, the effect of TAM on LTG glucuronidation was evaluated. In contrast to the mixed effect of TAM on DHT and t-AND glucuronidation, TAM inhibited LTG glucuronidation. Our results suggest that multiple aglycone binding sites exist within UGT1A4, which may result in atypical kinetics (both homotropic and heterotropic) in a substrate-dependent fashion.

Involvement of multiple cytochrome P450 isoforms in naproxen O-demethylation.

AbstractObjective: A series of studies was undertaken to determine the cytochrome P450 isoform(s) involved in naproxen demethylation and whether this included the same isoforms reported to be involved in the metabolism of other NSAIDs. Methods: (S)-Naproxen was incubated with human liver microsomes in the presence of a NADPH-generating system and the formation of desmethylnaproxen was measured by high-performance liquid chromatography (HPLC). To further clarify the specific isoforms involved, experiments were conducted with preparations expressing only a single P450 isoform (vaccinia virus-expressed cells and microsomes derived from a lymphoblastoid cell line, each transfected with specific P450 cDNAs) as well as inhibition studies using human liver microsomes and putative specific P450 inhibitors. Results: In human liver microsomes (n=7), desmethylnaproxen formation was observed with a mean kM of 92 (21) μmol · l−1, Vmax of 538 pmol · min−1 · mg−1 protein and Cint2 (reflective of a second binding site) of 0.36 μl · min−1 · mg−1 protein. This Cint2 term was added since Eadie-Scatchard analysis suggested the involvement of more than one enzyme. Studies using putative specific P450 inhibitors demonstrated inhibition of this␣reaction by sulfaphenazole, (apparent Ki= 1.6 μmol · l−1), warfarin (apparent Ki=27 μmol · l−1), piroxicam (apparent Ki=23 μmol · l−1) and tolbutamide (apparent Ki=128 μmol · l−1). No effect was observed when α-naphthoflavone and troleandomycin were employed as inhibitors, but reaction with furafylline produced, on average, a maximum inhibition of 23%. At a naproxen concentration of 150 μmol · l−1, formation of desmethylnaproxen was observed in cells expressing P450 1A2, 2C8, 2C9 and its allelic variant 2C9R144C. To further characterize these reactions, saturation kinetics experiments were conducted for the P450s 1A2, 2C8 and 2C9. The kM and Vmax for P450 1A2 were 189.5 μmol · l−1 and 7.3 pmol · min−1 · pmol−1 P450, respectively. Likewise, estimates of kM and Vmax for P450 2C9 were 340.5 μmol · l−1 and 41.4 pmol · min−1 · pmol−1 P450, respectively. Reliable estimates of kM and Vmax could not be made for P450 2C8 due to the nonsaturable nature of the process over the concentration range studied. Conclusion: Multiple cytochrome P450 isoforms (P450 1A2, 2C8 and 2C9) appear to be involved in naproxen demethylation, although 2C9 appears to be the predominant form.