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.

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:Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (ϳ5% F) would have been fairly well predicted using simple hepatic extraction (f h ) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O 6 -benzylguanine was within ϳ80% of the observed total clearance in humans after intravenous administration (15 ml ⅐ min ؊1 ⅐ kg ؊1 ), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:CYP2C9 wild-type protein has been shown to exhibit atypical kinetic profiles of metabolism that may affect in vitro-in vivo predictions made during the drug development process. Previous work suggests a substrate-dependent effect of polymorphic variants of CYP2C9 on the rate of metabolism; however, it is hypothesized that these active site amino acid changes will affect the kinetic profile of a drug's metabolism as well. To this end, the kinetic profiles of three model CYP2C9 substrates (flurbiprofen, naproxen, and piroxicam) were studied using purified CYP2C9*1 (wild-type) and variants involving active site amino acid changes, including the naturally occurring variants CYP2C9*3 (Leu359) and CYP2C9*5 (Glu360) and the man-made mutant CYP2C9 F114L. CYP2C9*1 (wild-type) metabolized each of the three compounds with a distinctive profile reflective of typical hyperbolic (flurbiprofen), biphasic (naproxen), and substrate inhibition (piroxicam) kinetics. CYP2C9*3 metabolism was again hyperbolic for flurbiprofen, of a linear form for naproxen (no saturation noted), and exhibited substrate inhibition with piroxicam. CYP2C9*5-mediated metabolism was hyperbolic for flurbiprofen and piroxicam but linear with respect to naproxen turnover. The F114L mutant exhibited a hyperbolic kinetic profile for flurbiprofen metabolism, a linear profile for naproxen metabolism, and a substrate inhibition kinetic profile for piroxicam metabolism. In all cases except F114L-mediated piroxicam metabolism, turnover decreased and the K m generally increased for each allelic variant compared with wild-type enzyme. It seems that the kinetic profile of CYP2C9-mediated metabolism is dependent on both substrate and the CYP2C9 allelic variant, thus having potential ramifications on drug disposition predictions made during the development process.