Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.

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This article is available online at http://dmd.aspetjournals.orgThe cytochrome P450 enzymes are involved in the biotransformation of both xenobiotic and endobiotic hydrophobic compounds, implicated in the bioactivation of certain procarcinogens (e.g., benzo-[a]pyrene), and responsible for many metabolism-based drug-drug interactions (Wrighton and Stevens, 1992). Consequently, the goal of drug metabolism and toxicology labs is to not only to determine the P450 isoform contribution to the metabolism of a given compound but also to understand the various factors that effect the activity and behavior of these isoforms. Classically, metabolism of a particular compound is described kinetically using the Michaelis-Menten equation, which yields a hyperbolic rate profile (Fig. 1A) and estimates of maximal reaction velocity (V max ) and apparent K m . It is evident, however, that for some drugs the kinetic profile is better described by a non-Michaelis-Menten or atypical kinetic model.

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AimsThe present study was conducted to evaluate metabolism of the enantiomers of verapamil and norverapamil using a broad range of cytochrome P450 isoforms and measure the kinetic parameters of these processes. Methods Cytochrome P450 cDNA-expressed cells and microsomes from a P450-expressed lymphoblastoid cell line were incubated with 40 mm concentrations of R-or S-verapamil and R-or S-norverapamil and metabolite formation measured by h.p.l.c. as an initial screening. Those isoforms exhibiting substantial activity were then studied over a range of substrate concentrations (2.5-450 mm) to estimate the kinetic parameters for metabolite formation. Results P450s 3A4, 3A5, 2C8 and to a minor extent 2E1 were involved in the metabolism of the enantiomers of verapamil. Estimated K m values for the production of D-617 and norverapamil by P450 s 3A4 and 3A5 were similar (range= 60-127 mm) regardless of the enantiomer of verapamil studied while the V max estimates were also similar (range=4-8 pmol min −1 pmol −1 P450). Only nominal production of D-620 by these isoforms was noted. Interestingly, P450 2C8 readily metabolized both S-and R-verapamil to D-617, norverapamil and PR-22 with only slightly higher K m values than noted for P450s 3A4 and 3A5. However, the V max estimates for P450 2C8 metabolism of S-and R-verapamil were in general greater (range=8-15 pmol min −1 pmol −1 P450) than those noted for P450 s 3A4 and 3A5 with preference noted for metabolism of the S-enantiomer. Similarly, P450 s 3A4, 3A5 and 2C8 also mediated the metabolism of the enantiomers of norverapamil with minor contributions by P450 s 2D6 and 2E1. P450s 3A4 and 3A5 readily formed the D-620 metabolite with generally a lower K m and higher V max for Snorverapamil than for the R-enantiomer. In contrast, P450 2C8 produced both the D-620 and PR-22 metabolites from the enantiomers of norverapamil, again with stereoselective preference seen for the S-enantiomer. Conclusions These results confirm that P450s 3A4, 3A5 and 2C8 play a major role in verapamil metabolism and demonstrate that norverapamil can also be further metabolized by the P450s.

ABSTRACT:The visible spectrum of a ligand-bound cytochrome P450 is often used to determine the nature of the interaction between the ligand and the P450. One particularly characteristic form of spectra arises from the coordination of nitrogen-containing ligands to the P450 heme iron. These type II ligands tend to be inhibitors because they stabilize the low reduction potential P450 and prevent oxygen binding to the heme. Yet, several type II ligands containing aniline, imidazole, and triazole moieties are also known to be substrates of P450, although P450 binding spectra are not often scrutinized to make this distinction. Therefore, the three nitrogenous ligands aniline, imidazole, and triazole were used as binding spectra standards with purified human CYP3A4 and CYP2C9, because their small size should not present any steric limitations in their accessing the heme prosthetic group. Next, the spectra of P450 with drugs containing the three nitrogenous groups were collected for comparison. The absolute spectra demonstrated that the red-shift of the low-spin Soret band is mostly dependent on the electronic properties of the nitrogen ligand since they tended to match their respective standards, aniline, imidazole, and triazole. On the other hand, difference spectra seemed to be more sensitive to the steric properties of the ligand because they facilitated comparison of the spectral amplitudes achieved with the drugs versus those with the standard nitrogen ligands. Therefore, difference spectra may help reveal "weak" coordination to the heme that results from suboptimal orientation or ligand binding to more remote locations within the P450 active sites.When studying substrate binding to the cytochromes P450 (P450s), the heme prosthetic group often serves as a very useful chromophore that can be exploited for purposes of characterization. With P450s, the heme is bound as in a b-type cytochrome except that the iron atom is liganded to a single Cys side chain as found in only a few other heme-containing proteins (e.g., chloroperoxidase, nitric oxide-synthase, and prostacyclin synthase). Because oxidation of P450-bound substrate occurs at the heme, certain aspects of substrate binding can be monitored readily with a spectrophotometer. Most commonly, ligand titration experiments are carried out to determine a spectral dissociation constant (K S ) for a P450 substrate or inhibitor, but little attention is paid to the signature manners in which the P450 heme spectra are altered (reviewed by Jefcoate, 1978). In particular, there seems to be an oversimplification regarding coordination of P450 substrates to the heme iron. This interaction stabilizes the low-spin iron configuration and thus is assumed to prevent catalysis since it is the high-spin iron that is more conducive to reduction by P450 reductase. [More specifically, P450 isoform differences arise because the rate of reduction is more dependent on reduction potential than spin state (Ost et al., 2003).]Generally, nitrogen-containing heme ligands, which bind more avidl...

ABSTRACT:St. John's wort extract (SJW) (Hypericum perforatum L.) is among the most commonly used herbal medications in the United States. The predominance of clinical reports indicates that SJW increases the activity of cytochrome P450 3A4 (CYP3A4) enzyme and reduces plasma concentrations of certain drugs. Although the inductive effect of SJW on CYP3A4 is clear, other reports indicate that SJW constituents may have, to a small degree, some enzyme inhibitory effects. Therefore, we sought to study the induction and inhibition effects of the constituents of SJW on CYP3A4 in the human hepatocyte model. Moreover, most research has focused on the induction of CYP3A4 by SJW with little attention paid to other prominent drug-metabolizing enzymes such as CYP1A2, CYP2C9, and CYP2D6. To examine the effects of SJW on CYP1A2, CYP2C9, CYP2D6, as well as CYP3A4, hepatocytes were exposed to hyperforin and hypericin, the primary constituents of SJW extract. Hepatocytes treated with hypericin or hyperforin were exposed to probe substrates to determine enzyme activity and protein and RNA harvested. Hyperforin treatment resulted in significant increases in mRNA, protein, and activity of CYP3A4 and CYP2C9, but had no effect on CYP1A2 or CYP2D6. Acute administration of hyperforin at 5 and 10 M 1 h before and along with probe substrate inhibited CYP3A4 activity. Hypericin had no effect on any of the enzymes tested. These results demonstrate that with chronic exposure, the inductive effect of SJW on drug-metabolizing enzymes predominates, and human hepatocyte cultures are a versatile in vitro tool for screening the effect of herbal products on cytochrome P450 enzymes.In 2002, sales of botanical supplements in the United States reached nearly $293 million dollars. St. John's wort accounted for 15 million U.S. dollars in sales, making it the fourth highest grossing botanical supplement (Blumenthal, 2003). Several clinical studies have demonstrated the effectiveness of St. John's wort compared with conventional therapy in the treatment of mild to moderate depression (Linde et al., 1996;Wheatley, 1997).Marketed St. John's wort, an extract of the flowering portion of the plant Hypericum perforatum L., is a mixture of a number of biologically active, complex compounds. At 0.3 mg per capsule, the naphthodianthrone hypericin is used as a means of standardization of the marketed product. The phloroglucinol hyperforin, the most plentiful lipophilic compound in the extract, is a potent reuptake inhibitor of serotonin, norepinephrine, and dopamine (Muller et al., 1998).Several recent reports have documented decreased blood/plasma levels of cytochrome P450 3A4 (CYP3A4) substrates, such as indinavir and cyclosporin A, in patients concomitantly taking St. John's wort (Piscitelli et al., 2000;Ahmed et al., 2001). Similar observations have been documented for digoxin, a substrate of the intestinal transporter P-glycoprotein (P-gp 4 ). Additional in vivo evidence has demonstrated that St. John's wort increased CYP3A4 and P-gp protein levels in rats (Dur...

ABSTRACT: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 (K i ) 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.Drug-drug interactions (DDIs) are one of the primary causes of serious adverse events occurring in clinical practice (Dambro and Kallgren, 1988). The most commonly observed DDIs result from inhibition of target-drug metabolism by a coadministered drug. The cytochrome P450 enzymes are the primary family of oxidative drugmetabolizing enzymes and, as such, are implicated in a substantial number of DDIs. Cytochrome P450 2C is the second most abundant subfamily of P450 enzymes and is responsible for metabolism of almost 20% of the drugs currently available in the market (Rendic and Di Carlo, 1997). CYP2C9 is an important member of the subfamily, serving as the primary metabolic pathway of the narrow therapeutic index drugs warfarin and phenytoin as well as numerous other therapeutic entities (Rettie and Jones, 2005). Interactions with warfarin or phenytoin metabolism are of substantial clinical concern and can result in serious adverse events.With the increased mechanistic knowledge of P450 enzyme function and the role of P450s in drug metabolism, a more systematic approach has been taken by investigators and the pharmaceutical industry for predicting drug-drug interactions. The U.S. Food and Drug Administration as well as the Pharmaceutical Research Manufacturer's Association has defined guidelines for preclinical in vitro and in vivo studies for the prediction of DDIs (Bjornsson et al., 2003; http:/...