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.

ABSTRACT:1-Aminobenzotriazole (1-ABT) is generally considered to be a nonselective mechanism-based inactivator of both human and nonhuman cytochrome P450 (P450) enzymes. Thus, 1-ABT is routinely used when conducting in vitro reaction phenotyping studies with new chemical entities in drug discovery to decipher P450 from non-P450-mediated metabolism. Experiments with pooled human liver microsomes (HLMs) demonstrated that carbon monoxide binding, although substantially reduced after a 30-min preincubation with 1-ABT, was still measurable. Thus, remaining activity of nine major human P450s (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) in HLMs was determined using established selective probe substrates after 30-min preincubation with either 1-ABT (1 mM), a positive control time-dependent inhibitor, or a competitive inhibitor. Whereas P450 2A6 and 3A4 activity was essentially eliminated upon 30-min pretreatment with 1-ABT, the other human P450s were less affected, with at least 20% activity remaining after pretreatment. In contrast, most of the known P450 selective timedependent inhibitors were more effective inactivators than 1-ABT at lower concentrations. A particularly interesting finding was that 1-ABT was quite ineffective at inactivating P450 2C9, with roughly 60% activity remaining after pretreatment, which suggests that 1-ABT is much less selective for certain human P450s. This collection of data clearly demonstrates that assuming 1-ABT is a nonselective P450 inhibitor in vitro is risky, and false conclusions regarding remaining metabolic activity being non-P450 mediated after 1-ABT pretreatment may be made.In vitro reaction phenotyping, where enzymes contributing to the biotransformation of new chemical entities are identified, is a routine practice in drug discovery (Williams et al., 2003;Zhang et al., 2007). This information is essential for understanding the relative contributions of metabolic pathways to overall clearance and, thus, the risk of pharmacokinetic drug-drug interactions and/or interpatient variability in drug exposure. One critical issue in conducting these in vitro studies is having the appropriate tool substrates and inhibitors of the various human cytochrome P450 (P450) enzymes. A particularly useful tool inhibitor historically has been 1-aminobenzotriazole (1-ABT), which is thought to inactivate P450 enzymes nonselectively by covalent modification of the heme prosthetic group following bioactivation (Ortiz de Montellano and Mathews, 1981). Thus, it has become a common in vitro practice to preincubate either human liver microsomes (HLMs) or hepatocytes with high concentrations of 1-ABT (ϳ1 mM) before the introduction of test compounds (substrate-depletion approach) to decipher P450 from non-P450 mediated-metabolism (Dalmadi et al., 2003;Williams et al., 2003;Kostrubsky et al., 2006). Although it is acknowledged that this approach is useful for determining P450-mediated metabolism, the characterization of the specific effects of 1-ABT on the major human P450 enzymes in vitro has been l...

Over the years, significant progress has been made in reducing metabolic instability due to cytochrome P450-mediated oxidation. High throughput metabolic stability screening has enabled advancement of compounds with little to no oxidative metabolism. Furthermore, high lipophilicity and low aqueous solubility of presently pursued chemotypes reduces the probability of renal excretion. As such, these low microsomal turnover compounds are often substrates for non CYP-mediated metabolism. UGTs, esterases and aldehyde oxidase are major enzymes involved in catalyzing such metabolism. Hepatocytes provide an excellent tool to identify such pathways including elucidation of major metabolites. To predict human PK parameters for P450-mediated metabolism, in vitro–in vivo extrapolation using hepatic microsomes, hepatocytes, and intestinal microsomes has been actively investigated. However, such methods have not been sufficiently evaluated for non-P450 enzymes. In addition to the involvement of liver, extra-hepatic enzymes (intestine, kidney, lung) are also likely to contribute to these pathways. While there has been considerable progress in predicting metabolic pathways and clearance primarily mediated by the liver, progress in characterizing extra-hepatic metabolism and prediction of clearance has been slow. Well-characterized in vitro systems or in vivo animal models to assess drug-drug interaction potential and inter-subject variability due to polymorphism are not available. Here we focus on the utility of appropriate in vitro studies to characterize non CYP-mediated metabolism; understand the enzymes involved followed by pharmacokinetic studies in the appropriately characterized surrogate species. The review will highlight progress made in establishing in vitro-in vivo correlation; predicting human clearance and avoid costly clinical failures when non-CYP mediated metabolic pathways are predominant.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Incubations with human liver and gut microsomes revealed that the antibiotic, clindamycin, is primarily oxidized to form clindamycin sulfoxide. In this report, evidence is presented that the Soxidation of clindamycin is primarily mediated by CYP3A. This conclusion is based upon several lines of in vitro evidence, including the following. 1) Incubations with clindamycin in hepatic microsomes from a panel of human donors showed that clindamycin sulfoxide formation correlated with CYP3A-catalyzed testosterone 6␤-hydroxylase activity; 2) coincubation with ketaconazole, a CYP3A4-specific inhibitor, markedly inhibited clindamycin S-oxidase activity; and 3) when clindamycin was incubated across a battery of recombinant heterologously expressed human cytochrome P450 (P450) enzymes, CYP3A4 possessed the highest clindamycin S-oxidase activity. A potential role for flavin-containing monooxygenases (FMOs) in clindamycin S-oxidation in human liver was also evaluated. Formation of clindamycin sulfoxide in human liver microsomes was unaffected either by heat pretreatment or by chemical inhibition (e.g., methimazole). Furthermore, incubations with recombinant FMO isoforms revealed no detectable activity toward the formation of clindamycin sulfoxide. Beyond identifying the drug-metabolizing enzyme responsible for clindamycin S-oxidation, the ability of clindamycin to inhibit six human P450 enzymes was also evaluated. Of the P450 enzymes examined, only the activity of CYP3A4 was inhibited (ϳ26%) by coincubation with clindamycin (100 M). Thus, it is concluded that CYP3A4 appears to account for the largest proportion of the observed P450 catalytic clindamycin S-oxidase activity in vitro, and this activity may be extrapolated to the in vivo condition.Clindamycin (methyl 7-chloro-6,7,8-trideoxy-6-[(2S,4R)-1-methyl-4-propylpyrrolidine-2-carboxamido]-1-thio-1-threo-D-galacto-octopyranoside monohydrochloride) is an antibiotic of the "lincosamide" class (Brodasky et al., 1968). The lincosamide antibiotics seem to be most useful against the bacteria classified as Gram-positive cocci. In addition, clindamycin is helpful against protozoans such as Toxoplasma and Mycoplasma as well as many anaerobic bacteria (Luft and Remington, 1988;Dannemann et al., 1991;Mazur et al., 1999).In humans, absorption of clindamycin is rapid and virtually complete (90%) following oral administration (DeHaan et al., 1972;Metzler et al., 1973). Concentrations of clindamycin in the serum increase linearly with increased dose, and levels exceed the minimum inhibitory concentration for most indicated organisms for at least 6 h following administration of the recommended dose. Clindamycin is widely distributed throughout the body and has an average biological half-life of 2.4 h. The major bioactive metabolites excreted in urine and feces are clindamycin sulfoxide and N-desmethylclindamycin (Seaberg et al., 1984;Flaherty et al., 1988;Gatti et al., 1998).To date, there are no published reports tha...