Brian A. Carr

COMPARISON OF IMMORTALIZED Fa2N-4 CELLS AND HUMAN HEPATOCYTES AS IN VITRO MODELS FOR CYTOCHROME P450 INDUCTION

Fa2N-4 cells have been proposed as a tool to identify CYP3A4 inducers. To evaluate whether Fa2N-4 cells are a reliable surrogate for cryopreserved human hepatocytes, we assessed the basal mRNA expression of 64 drug disposition genes in Fa2N-4 cells. Significant differences were found in the expression of major drug-metabolizing enzymes, nuclear receptors, and transporters between both cell types. Importantly, the expression of constitutive androstane receptor (CAR) and several hepatic uptake transporters was significantly lower (>50-fold) in Fa2N-4 cells, whereas the expression of pregnane X-receptor (PXR) and aryl hydrocarbon receptor (AhR) was similar between Fa2N-4 cells and human hepatocytes. By using an optimized induction assay for Fa2N-4 cells, CYP3A4 induction by rifampicin, the prototypical PXR activator, increased from 1.5- to 7-fold at the level of functional activity. With nine selected compounds, which are known inducers of CYP3A4 either via activation of PXR, CAR, or both, we evaluated CYP3A4 and CYP2B6 mRNA induction using Fa2N-4 cells and human hepatocytes. No response was observed in Fa2N-4 cells treated with the selective CAR activators 6-(4-chlorophenyl)imidazo[2,1-b][1,3]-thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime and artemisinin. CYP3A4 and CYP2B6 induction in Fa2N-4 cells were also low for phenytoin, phenobarbital, and efavirenz, which are dual activators of PXR/CAR. This finding was in agreement with the lack of expression of CAR. The EC50 value for rifampicin-mediated CYP3A4 induction was 10-fold higher than that in human hepatocytes. This result could be attributed to the low expression of hepatic organic anion-transporting polypeptides OATP1B1 and OATP1B3 in Fa2N-4 cells. In summary, our findings identify limitations of Fa2N-4 cells as a predictive induction model.

Characterization of the Rhesus Monkey CYP3A64 Enzyme: Species Comparisons of CYP3A Substrate Specificity and Kinetics Using Baculovirus-Expressed Recombinant Enzymes

The rhesus monkey (Macaca mulatta) is a primate species used extensively as a preclinical safety species in drug development. In this report, we describe the cloning, expression, and characterization of CYP3A64 (AY334551), a CYP3A4 homolog expressed in rhesus liver. The deduced amino acid sequence was found to be 93% similar to human CYP3A4, 83% similar to human CYP3A5, and identical to the previously reported cynomolgus monkey CYP3A8 (Komori et al., 1992). The substrate specificity of CYP3A64 for testosterone (0–250 μM), midazolam (0–200 μM), nifedipine (0–200 μM), and 7-benzoxy-4-trifluoromethylcoumarin (0–200 μM) were compared with recombinant enzymes from rat (CYP3A1, CYP3A2), dog (CYP3A12, CYP3A26), rabbit (CYP3A6), and human (CYP3A4, CYP3A5). Immunoinhibition and chemical inhibition of CYP3A64 was demonstrated using the inhibitory monoclonal antibody (MAb) 10-1-1 (anti-3A4) and ketoconazole (0–10 μM). The utility of CYP3A64 to be used as a standard in monkey induction assays was shown and the concentration of CYP3A64 protein in rhesus liver microsomes was estimated to be 72 pmol/mg protein. In summary, these results support the utilization of rhesus monkey CYP3A64 for in vitro drug metabolism studies and provide a more complete understanding of CYP3A substrate specificities and species differences in metabolic capabilities.

In Vitro and in Vivo CYP3A64 Induction and Inhibition Studies in Rhesus Monkeys: A Preclinical Approach for CYP3A-Mediated Drug Interaction Studies

In this study, induction and inhibition of rhesus monkey CYP3A64 versus human CYP3A4 were characterized in vitro, and the corresponding pharmacokinetic consequences were evaluated in rhesus monkeys. In monkey hepatocytes, rifampin markedly induced CYP3A64 mRNA (EC50 = 0.5 μM; Emax = 6-fold) and midazolam (MDZ) 1′-hydroxylase activity (EC50 = 0.2 μM; Emax = 2-fold). Compound A (N-[2(R)-hydroxy-1(S)-indanyl-5-[2(S)-(1,1-dimethylethylaminocarbonyl)-4-[(furo[2,3-b]pyridin-5-yl)-methyl]piperazin-1-yl]-4(S)-hydroxy-2(R)-phenylmethylpentanamide), a known potent and mechanism-based inhibitor of CYP3A4, strongly inhibited the formation of 1′-hydroxy MDZ by recombinant CYP3A64 in a concentration- and time-dependent manner (KI = 0.25 μM; kinact = 0.4 min–1). Similar corresponding results also were obtained with human CYP3A4 in the presence of rifampin or compound A. In rhesus monkeys, MDZ exhibited a relatively high metabolic clearance (primarily via 1′-hydroxylation followed by glucuronidation) and a low hepatic availability (Fh = 16%). Consistent with the induction of hepatic metabolism of a high-clearance compound, pretreatment with rifampin (18 mg/kg p.o. for 5 days) did not significantly affect the i.v. kinetics of MDZ, but caused a pronounced reduction (∼10-fold) in the systemic exposure to MDZ and, consequently, its Fh following intrahepatic portal vein administration (i.pv.) of MDZ. A comparable extent of the pharmacokinetic interaction also was obtained after a 1.8 mg/kg rifampin dose. Also consistent with the in vitro CYP3A64 inhibition finding, compound A (6 mg/kg i.v.) markedly increased (10-fold) the i.pv. administered MDZ exposure. At the doses studied, plasma concentrations of rifampin or compound A reached or exceeded their respective in vitro EC50 or KI values. These findings suggest the potential applicability of the in vitro-in vivo relationship approach in rhesus monkeys for studying CYP3A-mediated interactions in humans.

HETEROTROPIC MODULATION OF SULFOTRANSFERASE 2A1 ACTIVITY BY CELECOXIB: PRODUCT RATIO SWITCHING OF ETHYNYLESTRADIOL SULFATION

This work had two separate aims: to evaluate different modeling techniques and to make a detailed structural characterization of CYP2C9. To achieve these goals, the consensus principal component analysis (CPCA) technique and distance measurements were used to explore available crystal structures, newly built homology models, and repeated molecular dynamics simulations. The CPCA was based on molecular interaction fields focused on the active site regions of the proteins and include detailed amino acid analysis. The comparison of the CYP2C9 and CYP2C5 crystal structures revealed differences in the flexible regions such as the B-C and F-G loop and the N and C termini. Cross homology models of CYP2C9 and CYP2C5, using their respective crystal structures as templates, indicated that such models were more similar to their templates than to their target proteins. Inclusion of multiple templates slightly improved the similarity to the crystal target in some cases and could be recommended even though it requires a careful manual alignment process. The application of molecular dynamics simulations to highly flexible proteins such as cytochromes P450 is also explored and the information is extracted by the CPCA. Advantages and drawbacks are presented for the different modeling techniques. Despite the varying modeling success, the models give insight and understanding by the mutual forming and discarding of hypotheses. This is a dynamic process since the crystal structures are improving with time and, therefore, the answers to the models are also changing accordingly.

Rifampin Induces the In Vitro Oxidative Metabolism, but Not the In Vivo Clearance of Diclofenac in Rhesus Monkeys

Effects of rifampin on in vitro oxidative metabolism and in vivo pharmacokinetics of diclofenac (DF), a prototypic CYP2C9 marker substrate, were investigated in rhesus monkeys. In monkey hepatocytes, rifampin markedly induced DF 4′-hydroxylase activity, with values for EC50 of 0.2 to 0.4 μM and Emax of 2- to 5-fold over control. However, pretreatment with rifampin did not alter the pharmacokinetics of DF obtained after either i.v. or intrahepatic portal vein (i.pv.) administration of DF to monkeys. At the dose studied, plasma concentrations of rifampin reached 10 μM, far exceeding the in vitro EC50 values. Under similar treatment conditions, rifampin was previously shown to induce midazolam (MDZ) 1′-hydroxylation in rhesus monkey hepatocytes (EC50 and Emax values ∼0.2 μM and ∼2- to 3-fold, respectively), and markedly affected the in vivo pharmacokinetics of MDZ (>10-fold decreases in the i.pv. MDZ systemic exposure and its hepatic availability, Fh) in this animal species. In monkey liver microsomes, DF underwent, predominantly, glucuronidation, and, modestly, oxidation; the intrinsic clearance (CLint = Vmax/Km) value for the glucuronidation pathway accounted for >95% (versus about 75% in human liver microsomes) of the total (glucuronidation + hydroxylation) intrinsic clearance value. In monkey hepatocytes, the hydroxylation also was a minor component (≤10%) relative to the glucuronidation, supporting the liver microsomal finding. Collectively, our results suggest that the oxidative metabolism is not the major in vivo clearance mechanism of DF in either untreated or rifampin-treated monkeys and, conceivably, also in humans, raising a question about the utility of DF as an in vivo CYP2C9 probe.

CYP2C75-Involved Autoinduction of Metabolism in Rhesus Monkeys of MK-0686, a Bradykinin B1 Receptor Antagonist

After oral treatment (once daily) for 4 weeks with the potent bradykinin B1 receptor antagonist methyl 3-chloro-3′-fluoro-4′-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)-amino]ethyl}-1,1′-biphenyl-2-carboxylate (MK-0686), rhesus monkeys (Macaca mulatta) exhibited significantly reduced systemic exposure of the compound in a dose-dependent manner, suggesting an occurrence of autoinduction of MK-0686 metabolism. This possibility is supported by two observations. 1) MK-0686 was primarily eliminated via biotransformation in rhesus monkeys, with oxidation on the chlorophenyl ring as one of the major metabolic pathways. This reaction led to appreciable formation of a dihydrodiol (M11) and a hydroxyl (M13) product in rhesus liver microsomes supplemented with NADPH. 2) The formation rate of these two metabolites determined in liver microsomes from MK-0686-treated groups was ≥2-fold greater than the value for a control group. Studies with recombinant rhesus P450s and monoclonal antibodies against human P450 enzymes suggested that CYP2C75 played an important role in the formation of M11 and M13. The induction of this enzyme by MK-0686 was further confirmed by a concentration-dependent increase of its mRNA in rhesus hepatocytes, and, more convincingly, the enhanced CYP2C proteins and catalytic activities toward CYP2C75 probe substrates in liver microsomes from MK-0686-treated animals. Furthermore, a good correlation was observed between the rates of M11 and M13 formation and hydroxylase activities toward probe substrates determined in a panel of liver microsomal preparations from control and MK-0686-treated animals. Therefore, MK-0686, both a substrate and inducer for CYP2C75, caused autoinduction of its own metabolism in rhesus monkeys by increasing the expression of this enzyme.

CYP2C75-Involved Autoinduction of Metabolism in Rhesus Monkeys of Methyl 3-Chloro-3′-fluoro-4′-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)amino]ethyl}-1,1′-biphenyl-2-carboxylate (MK-0686), a Bradykinin B1 Receptor Antagonist

After oral treatment (once daily) for 4 weeks with the potent bradykinin B1 receptor antagonist methyl 3-chloro-3′-fluoro-4′-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)-amino]ethyl}-1,1′-biphenyl-2-carboxylate (MK-0686), rhesus monkeys (Macaca mulatta) exhibited significantly reduced systemic exposure of the compound in a dose-dependent manner, suggesting an occurrence of autoinduction of MK-0686 metabolism. This possibility is supported by two observations. 1) MK-0686 was primarily eliminated via biotransformation in rhesus monkeys, with oxidation on the chlorophenyl ring as one of the major metabolic pathways. This reaction led to appreciable formation of a dihydrodiol (M11) and a hydroxyl (M13) product in rhesus liver microsomes supplemented with NADPH. 2) The formation rate of these two metabolites determined in liver microsomes from MK-0686-treated groups was ≥2-fold greater than the value for a control group. Studies with recombinant rhesus P450s and monoclonal antibodies against human P450 enzymes suggested that CYP2C75 played an important role in the formation of M11 and M13. The induction of this enzyme by MK-0686 was further confirmed by a concentration-dependent increase of its mRNA in rhesus hepatocytes, and, more convincingly, the enhanced CYP2C proteins and catalytic activities toward CYP2C75 probe substrates in liver microsomes from MK-0686-treated animals. Furthermore, a good correlation was observed between the rates of M11 and M13 formation and hydroxylase activities toward probe substrates determined in a panel of liver microsomal preparations from control and MK-0686-treated animals. Therefore, MK-0686, both a substrate and inducer for CYP2C75, caused autoinduction of its own metabolism in rhesus monkeys by increasing the expression of this enzyme.