The normal metabolism of drugs can generate metabolites that have intrinsic chemical reactivity towards cellular molecules, and therefore have the potential to alter biological function and initiate serious adverse drug reactions. Here, we present an assessment of the current approaches used for the evaluation of chemically reactive metabolites. We also describe how these approaches are being used within the pharmaceutical industry to assess and minimize the potential of drug candidates to cause toxicity. At early stages of drug discovery, iteration between medicinal chemistry and drug metabolism can eliminate perceived reactive metabolite-mediated chemical liabilities without compromising pharmacological activity or the need for extensive safety evaluation beyond standard practices. In the future, reactive metabolite evaluation may also be useful during clinical development for improving clinical risk assessment and risk management. Currently, there remains a huge gap in our understanding of the basic mechanisms that underlie chemical stress-mediated adverse reactions in humans. This review summarizes our views on this complex topic, and includes insights into practices considered by the pharmaceutical industry.
Aims Many substrates of cytochrome P450 (CYP) 3A4 are used for in vitro investigations of drug metabolism and potential drug-drug interactions. The aim of the present study was to determine the relationship between 10 commonly used CYP3A4 probes using modifiers with a range of inhibitory potency. Methods The effects of 34 compounds on CYP3A4-mediated metabolism were investigated in a recombinant CYP3A4 expression system. Inhibition of erythromycin, dextromethorphan and diazepam N-demethylation, testosterone 6b-hydroxylation, midazolam 1-hydroxylation, triazolam 4-hydroxylation, nifedipine oxidation, cyclosporin oxidation, terfenadine C-hydroxylation and N-dealkylation and benzyloxyresorufin O-dealkylation was evaluated at the apparent K m or S 50 (for substrates showing sigmoidicity) value for each substrate and at an inhibitor concentration of 30 mm. Results While all CYP3A4 probe substrates demonstrate some degree of similarity, examination of the coefficients of determination, together with difference and cluster analysis highlighted that seven substrates can be categorized into two distinct substrate groups. Erythromycin, cyclosporin and testosterone form the most closely related group and dextromethorphan, diazepam, midazolam and triazolam form a second group. Terfenadine can be equally well placed in either group, while nifedipine shows a distinctly different relationship. Benzyloxyresorufin shows the weakest correlation with all the other CYP3A4 probes. Modifiers that caused negligible inhibition or potent inhibition are generally comparable in all assays, however, the greatest variability is apparent with compounds causing, on average, intermediate inhibition. Modifiers of this type may cause substantial inhibition, no effect or even activation depending on the substrate employed. Conclusions It is recommended that multiple CYP3A4 probes, representing each substrate group, are used for the in vitro assessment of CYP3A4-mediated drug interactions.
ABSTRACT:The selection of appropriate substrates for investigating the potential inhibition of CYP3A4 is critical as the magnitude of effect is often substrate-dependent, and a weak correlation is often observed among different CYP3A4 substrates. This feature has been attributed to the existence of multiple binding sites and, therefore, relatively complex in vitro data modeling is required to avoid erroneous evaluation and to allow prediction of drug-drug interactions. This study, performed in lymphoblastexpressed CYP3A4 with oxidoreductase, provides a systematic comparison of the effects of quinidine (QUI) and haloperidol (HAL) as modifiers of CYP3A4 activity using a selection of To assess the in vivo significance of drug-drug interactions involving P450 1 inhibition from in vitro data, it is necessary to identify the particular P450 enzymes involved, estimate their contribution to the overall elimination of the drug, and characterize the inhibition effects (Ito et al., 1998;Rodrigues et al., 2001;Tucker et al., 2001). The latter is the most problematic factor, because it is dependent on the appropriate selection of both an inhibition model to derive a K i value and an inhibitor concentration at the enzyme active site.The selection of an appropriate inhibition model for CYP3A4 is particularly difficult because this enzyme frequently does not obey Michaelis-Menten kinetics, shows substrate-dependent effects (Kenworthy et al., 1999;Stresser et al., 2000;Wang et al., 2000;Lu et al., 2001), and is prone to activation (Shou et al., 1994;Tang et al., 1999;Kenworthy et al., 2001) To provide a mechanistic insight for atypical enzyme properties shown by CYP3A4, various approaches have been reported in recent years (Hosea et al., 2000;Tang and Stearns, 2001), involving either the simultaneous binding of two molecules (Korzekwa et al., 1998;Shou et al., 2001b) or the existence of a separate effector-binding site (Ueng et al., 1997;Kenworthy et al., 2001). Additional evidence for the existence of multiple binding sites is provided by site-directed mutagenesis studies (Harlow and Halpert, 1998;Domanski et al., 2001), indicating that CYP3A4 substrate and effector-binding sites are separate, but closely linked, and the residues involved in the binding of either substrate and/or effector depend on the molecule present.The clinical significance of an observed in vitro heteroactivation of CYP3A4 is still uncertain, because to date few confirmations in vivo have been reported. A decrease in diclofenac steady-state plasma concentrations, observed upon the coadministration of quinidine in rhesus monkeys, is consistent with an in vitro activation interaction 1 Abbreviations used are: P450, cytochrome P450; QUI, quinidine; HAL, haloperidol; MDZ, midazolam; TST, testosterone; NIF, nifedipine; FEL, felodipine; SV, simvastatin; 6-HTS; 6-hydroxytestosterone; OX NIF, oxidized nifedipine; FEL PYR, felodipine and pyridine metabolite; CYP3A4/OR, coexpressed CYP3A4 and NADPH-cytochrome P450 reductase.
Different signals in addition to the antigenic signal are required to initiate an immunological reaction. In the context of sulfamethoxazole allergy, the Ag is thought to be derived from its toxic nitroso metabolite, but little is known about the costimulatory signals, including those associated with dendritic cell maturation. In this study, we demonstrate increased CD40 expression, but not CD80, CD83, or CD86, with dendritic cell surfaces exposed to sulfamethoxazole (250–500 μM) and the protein-reactive metabolite nitroso sulfamethoxazole (1–10 μM). Increased CD40 expression was not associated with apoptosis or necrosis, or glutathione depletion. Covalently modified intracellular proteins were detected when sulfamethoxazole was incubated with dendritic cells. Importantly, the enzyme inhibitor 1-aminobenzotriazole prevented the increase in CD40 expression with sulfamethoxazole, but not with nitroso sulfamethoxazole or LPS. The enzymes CYP2C9, CYP2C8, and myeloperoxidase catalyzed the conversion of sulfamethoxazole to sulfamethoxazole hydroxylamine. Myeloperoxidase was expressed at high levels in dendritic cells. Nitroso sulfamethoxazole immunogenicity was inhibited in mice with a blocking anti-CD40L Ab. In addition, when a primary nitroso sulfamethoxazole-specific T cell response using drug-naive human cells was generated, the magnitude of the response was enhanced when cultures were exposed to a stimulatory anti-CD40 Ab. Finally, increased CD40 expression was 5-fold higher on nitroso sulfamethoxazole-treated dendritic cells from an HIV-positive allergic patient compared with volunteers. These data provide evidence of a link between localized metabolism, dendritic cell activation, and drug immunogenicity.
This article is available online at http://dmd.aspetjournals.org
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