Michael Sinz

Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole

Individual variation in drug metabolism is a major cause of unpredictable side effects during therapy. Drug metabolism is controlled by a class of orphan nuclear receptors (NRs), which regulate expression of genes such as CYP (cytochrome)3A4 and MDR-1 (multi-drug resistance-1), that are involved in this process. We have found that xenobiotic-mediated induction of CYP3A4 and MDR-1 gene transcription was inhibited by ketoconazole, a commonly used antifungal drug. Ketoconazole mediated its effect by inhibiting the activation of NRs, human pregnenolone X receptor and constitutive androstene receptor, involved in regulation of CYP3A4 and MDR-1. The effect of ketoconazole was specific to the group of NRs that control xenobiotic metabolism. Ketoconazole disrupted the interaction of the xenobiotic receptor PXR with the co-activator steroid receptor co-activator-1. Ketoconazole treatment resulted in delayed metabolism of tribromoethanol anesthetic in mice, which was correlated to the inhibition of PXR activation and downmodulation of cyp3a11 and mdr-1 genes and proteins. These studies demonstrate for the first time that ketoconazole represses the coordinated activation of genes involved in drug metabolism, by blocking activation of a specific subset of NRs. Our results suggest that ketoconazole can be used as a pan-antagonist of NRs involved in xenobiotic metabolism in vivo, which may lead to novel strategies that improve drug effect and tolerance.

Evaluation of 170 Xenobiotics as Transactivators of Human Pregnane X Receptor (hPXR) and Correlation to Known CYP3A4 Drug Interactions

The human transcription factor pregnane X receptor (hPXR) is a key regulator of enzyme expression, especially cytochrome P450 3A4 (CYP3A4). Due to the prominence of CYP3A4 in the elimination of many drugs, the development of high throughput in vitro models to predict the effect of drugs on CYP3A4 expression have increased. To better interpret and predict potential drug-drug interactions due to CYP3A4 enzyme induction, we evaluated 170 xenobiotics in a hPXR transactivation assay and compared these results to known clinical drug-drug interactions. Of the 170 xenobiotics tested, 54% of them demonstrated some level of hPXR transactivation. By taking into consideration cell culture conditions (solubility, cytotoxicity, appropriate drug concentration in media), as well as in vivo pharmacokinetics (therapeutic plasma C(max), distribution, route of administration, dosing regimen, liver exposure, potential to inhibit CYP3A4), the risk potential of CYP3A4 enzyme induction for most compounds reduced dramatically. By employing this overall interpretation strategy, the final percentage of compounds predicted to significantly induce CYP3A4 reduced to 5%, all of which are known to cause drug-drug interactions. Also, this is the first report that identifies several potent compounds that have the ability to transactivate hPXR that previously have not been identified, such as terbinafine, diclofenac, sildenafil, glimepiride, montelukast, and ticlopidine.

Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032)

ABSTRACT Asunaprevir (ASV; BMS-650032) is a hepatitis C virus (HCV) NS3 protease inhibitor that has demonstrated efficacy in patients chronically infected with HCV genotype 1 when combined with alfa interferon and/or the NS5A replication complex inhibitor daclatasvir. ASV competitively binds to the NS3/4A protease complex, with Ki values of 0.4 and 0.24 nM against recombinant enzymes representing genotypes 1a (H77) and 1b (J4L6S), respectively. Selectivity was demonstrated by the absence of any significant activity against the closely related GB virus-B NS3 protease and a panel of human serine or cysteine proteases. In cell culture, ASV inhibited replication of HCV replicons representing genotypes 1 and 4, with 50% effective concentrations (EC50s) ranging from 1 to 4 nM, and had weaker activity against genotypes 2 and 3 (EC50, 67 to 1,162 nM). Selectivity was again demonstrated by the absence of activity (EC50, >12 μM) against a panel of other RNA viruses. ASV exhibited additive or synergistic activity in combination studies with alfa interferon, ribavirin, and/or inhibitors specifically targeting NS5A or NS5B. Plasma and tissue exposures in vivo in several animal species indicated that ASV displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC50s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients.

In Vitro and in Vivo Induction of Cytochrome P450: A Survey of the Current Practices and Recommendations: A Pharmaceutical Research and Manufacturers of America Perspective

Cytochrome P450 (P450) induction is one of the factors that can affect the pharmacokinetics of a drug molecule upon multiple dosing, and it can result in pharmacokinetic drug-drug interactions with coadministered drugs causing potential therapeutic failures. In recent years, various in vitro assays have been developed and used routinely to assess the potential for drug-drug interactions due to P450 induction. There is a desire from the pharmaceutical industry and regulatory agencies to harmonize assay methodologies, data interpretation, and the design of clinical drug-drug interaction studies. In this article, a team of 10 scientists from nine Pharmaceutical Research and Manufacturers of America (PhRMA) member companies conducted an anonymous survey among PhRMA companies to query current practices with regards to the conduct of in vitro induction assays, data interpretation, and clinical induction study practices. The results of the survey are presented in this article, along with reviews of current methodologies of in vitro assays and in vivo studies, including modeling efforts in this area. A consensus recommendation regarding common practices for the conduct of P450 induction studies is included.

Human Pregnane X Receptor Antagonists and Agonists Define Molecular Requirements for Different Binding Sites

The pregnane X receptor (PXR) is an important transcriptional regulator of the expression of xenobiotic metabolism and transporter genes. The receptor is promiscuous, binding many structural classes of molecules that act as agonists at the ligand-binding domain, triggering up-regulation of genes, increasing the metabolism and excretion of therapeutic agents, and causing drug-drug interactions. It has been suggested that human PXR antagonists represent a means to counteract such interactions. Several azoles have been hypothesized to bind the activation function-2 (AF-2) surface on the exterior of PXR when agonists are concurrently bound in the ligand-binding domain. In the present study, we have derived novel computational models for PXR agonists using different series of imidazoles, steroids, and a set of diverse molecules with experimental PXR agonist binding data. We have additionally defined a novel pharmacophore for the steroidal agonist site. All agonist pharmacophores showed that hydrophobic features are predominant. In contrast, a qualitative comparison with the corresponding PXR antagonist pharmacophore models using azoles and biphenyls showed that they are smaller and hydrophobic with increased emphasis on hydrogen bonding features. Azole antagonists were docked into a proposed hydrophobic binding pocket on the outer surface at the AF-2 site and fitted comfortably, making interactions with key amino acids involved in charge clamping. Combining computational and experimental data for different classes of molecules provided strong evidence for agonists and antagonists binding distinct regions on PXR. These observations bear significant implications for future discovery of molecules that are more selective and potent antagonists.

Pharmacokinetic Drug Interactions Involving 17α-Ethinylestradiol

Abstract17α-Ethinylestradiol (EE) is widely used as the estrogenic component of oral contraceptives (OC). In vitro and in vivo metabolism studies indicate that EE is extensively metabolised, primarily via intestinal sulfation and hepatic oxidation, glucuronidation and sulfation. Cytochrome P450 (CYP)3A4-mediated EE 2-hydroxylation is the major pathway of oxidative metabolism of EE. For some time it has been known that inducers of drug-metabolising enzymes (such as the CYP3A4 inducer rifampicin [rifampin]) can lead to breakthrough bleeding and contraceptive failure. Conversely, inhibitors of drug-metabolising enzymes can give rise to elevated EE plasma concentrations and increased risks of vascular disease and hypertension. In vitro studies have also shown that EE inhibits a number of human CYP enzymes, such as CYP2C19, CYP3A4 and CYP2B6. Consequently, there are numerous reports in the literature describing EE-containing OC formulations as perpetrators of pharmacokinetic drug interactions. Because EE may participate in multiple pharmacokinetic drug interactions as either a victim or perpetrator, pharmaceutical companies routinely conduct clinical drug interaction studies with EE-containing OCs when evaluating new chemical entities in development. It is therefore critical to understand the mechanisms underlying these drug interactions. Such an understanding can enable the interpretation of clinical data and lead to a greater appreciation of the profile of the drug by physicians, clinicians and regulators. This article summarises what is known of the drug-metabolising enzymes and transporters governing the metabolism, disposition and excretion of EE. An effort is made to relate this information to known clinical drug-drug interactions. The inhibition and induction of drug-metabolising enzymes by EE is also reviewed.

Current Industrial Practices in Assessing CYP450 Enzyme Induction: Preclinical and Clinical

Induction of drug metabolizing enzymes, such as the cytochromes P450 (CYP) is known to cause drug-drug interactions due to increased elimination of co-administered drugs. This increased elimination may lead to significant reduction or complete loss of efficacy of the co-administered drug. Due to the significance of such drug interactions, many pharmaceutical companies employ screening and characterization models which predict CYP enzyme induction to avoid or attenuate the potential for drug interactions with new drug candidates. The most common mechanism of CYP induction is transcriptional gene activation. Activation is mediated by nuclear receptors, such as AhR, CAR, and PXR that function as transcription factors. Early high throughput screening models utilize these nuclear hormone receptors in ligand binding or cell-based transactivation/reporter assays. In addition, immortalized hepatocyte cell lines can be used to assess enzyme induction of specific drug metabolizing enzymes. Cultured primary human hepatocytes, the best established in vitro model for predicting enzyme induction and most accepted by regulatory agencies, is the predominant assay used to evaluate induction of a wide variety of drug metabolizing enzymes. These in vitro models are able to appropriately predict enzyme induction in patients when compared to clinical drug-drug interactions. Finally, transgenic animal models and the cynomolgus monkey have also been shown to recapitulate human enzyme induction and may be appropriate in vivo animal models for predicting human drug interactions.

Cytochrome P450 reaction-phenotyping: an industrial perspective

It is now widely accepted that the fraction of the dose metabolized by a given drug-metabolizing enzyme is one of the major factors governing the magnitude of a drug interaction and the impact of a polymorphism on (total) drug clearance. Therefore, most pharmaceutical companies determine the enzymes involved in the metabolism of a new chemical entity (NCE) in vitro, in conjunction with human data on absorption, distribution, metabolism and excretion. This so called reaction-phenotyping, or isozyme-mapping, usually involves the use of multiple reagents (e.g., recombinant proteins, liver subcellular fractions, enzyme-selective chemical inhibitors and antibodies). For the human CYPs, reagents are readily available and in vitro reaction-phenotyping data are now routinely included in most regulatory documents. Ideally, the various metabolites have been definitively identified, incubation conditions have afforded robust kinetic analyses, and well characterized (high quality) reagents and human tissues have been employed. It is also important that the various in vitro data are consistent (e.g., scaled turnover with recombinant CYP proteins, CYP inhibition and correlation data with human liver microsomes) and enable an integrated in vitro CYP reaction-phenotype. Results of the in vitro CYP reaction-phenotyping are integrated with clinical data (e.g., human radiolabel and drug interaction studies) and a complete package is then submitted for regulatory review. If the NCE receives market approval, information on key routes of clearance and their associated potential for drug–drug interactions are included in the product label. The present review focuses on in vitro CYP reaction-phenotyping and the integration of data. Relatively simple strategies enabling the design and prioritization of follow up clinical studies are also discussed.

Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.

Expanding the Roles for Pregnane X Receptor in Cancer: Proliferation and Drug Resistance in Ovarian Cancer

Purpose: We examined the presence of the pregnane X receptor (PXR) and its effects on ovarian cancer cells after activation by its cognate ligand. Experimental Design: SKOV-3 and OVCAR-8 ovarian carcinoma cells were analyzed for expression of PXR by quantitative reverse transcription-PCR and Western blot. Human ovarian cancer tissue was also analyzed for PXR expression by immunochemistry. Ligand (agonist)–induced PXR target genes were analyzed in SKOV-3 cells by quantitative reverse transcription-PCR. SKOV-3 cell proliferation was assessed by MTT assay. In vivo confirmation of in vitro effects of PXR ligands were done in NOD.SCID mice carrying SKOV-3 xenografts. Results: PXR is expressed in ovarian cancer cells. In SKOV-3 cells, PXR is functional and its activation by cognate ligands induces PXR target genes (CYP2B6, CYP3A4, and UGT1A1) but not MDR1 and MRP2. PXR activation in SKOV-3 cells induces cell proliferation and drug resistance. In mice harboring SKOV-3 xenografts, rifampicin (PXR agonist) induces cell proliferation and tumor growth. Conclusion: PXR activation, regardless of the type of ligand agonist present, promotes the “malignant” phenotype of cancer cells. These data serve as the basis for finding novel nontoxic inhibitors of PXR activation as a method to control cell growth and prevent induction of drug resistance.