Composition and Orientation of the Core Region of Novel HIV-1 Entry Inhibitors Influences Metabolic Stability.

Fostemsavir/temsavir is an investigational HIV-1 entry inhibitor currently in late-stage clinical trials. Although it holds promise to be a first-in-class Env-targeted entry inhibitor for the clinic, issues with bioavailability relegate its use to salvage therapies only. As such, the development of a small molecule HIV-1 entry inhibitor that can be used in standard combination antiretroviral therapy (cART) remains a longstanding goal for the field. We previously demonstrated the ability of extending the chemotypes available to this class of inhibitor as the first step towards this overarching goal. In addition to poor solubility, metabolic stability is a crucial determinant of bioavailability. Therefore, in this short communication, we assess the metabolic stabilities of five of our novel chemotype entry inhibitors. We found that changing the piperazine core region of temsavir alters the stability of the compound in human liver microsome assays. Moreover, we identified an entry inhibitor with more than twice the metabolic stability of temsavir and demonstrated that the orientation of the core replacement is critical for this increase. This work further demonstrates the feasibility of our long-term goal-to design an entry inhibitor with improved drug-like qualities-and warrants expanded studies to achieve this.

Influence of Ethanol on Darunavir Hepatic Clearance and Intracellular PK/PD in HIV-Infected Monocytes, and CYP3A4-Darunavir Interactions Using Inhibition and in Silico Binding Studies.

PURPOSE: Although the prevalence of alcohol consumption is higher in HIV+ people than general public, limited information is available on how alcohol affects the metabolism and bioavailability of darunavir (DRV). METHODS: DRV was quantified by using LC-MS/MS method. All in vitro experiments were performed using human liver microsomes and HIV-infected monocytic cells. CYP3A4 and DRV/Ritonavir (RTV) docking was performed using GOLD suite 5.8. RESULTS: Ethanol (20 mM) significantly decreased apparent half-life and increased degradation rate constant of RTV-boosted DRV but not for DRV alone. Similarly, ethanol exposure increased hepatic intrinsic clearance for RTV-boosted DRV with no significant influence on DRV alone. Ethanol showed a limited influence on intracellular total DRV exposure in the presence of RTV without altering maximum concentration (Cmax) values in HIV-infected monocytic cells. Ethanol alone elevated HIV replication but this effect was nullified with the addition of DRV or DRV + RTV. Additionally, inhibitory potency of DRV was significantly reduced in the presence of ethanol. Our docking results projected that ethanol increases the average distance between DRV and CYP3A4 heme, and alter the orientation of DRV-CYP3A4 binding. CONCLUSIONS: Collectively these findings suggest that DRV metabolism is primarily influenced by ethanol in the liver, but has minor effect in HIV-residing monocytes.

C-3 benzoic acid derivatives of C-3 deoxybetulinic acid and deoxybetulin as HIV-1 maturation inhibitors.

A series of C-3 phenyl- and heterocycle-substituted derivatives of C-3 deoxybetulinic acid and C-3 deoxybetulin was designed and synthesized as HIV-1 maturation inhibitors (MIs) and evaluated for their antiviral activity and cytotoxicity in cell culture. A 4-subsituted benzoic acid moiety was identified as an advantageous replacement for the 3'3'-dimethylsuccinate moiety present in previously disclosed MIs that illuminates new aspects of the topography of the pharmacophore. The new analogs exhibit excellent in vitro antiviral activity against wild-type (wt) virus and a lower serum shift when compared with the prototypical HIV-1 MI bevirimat (1, BVM), the first MI to be evaluated in clinical studies. Compound 9a exhibits comparable cell culture potency toward wt virus as 1 (WT EC50=16 nM for 9a compared to 10nM for 1). However, the potency of 9a is less affected by the presence of human serum, while the compound displays a similar pharmacokinetic profile in rats to 1. Hence 9a, the 4-benzoic acid derivative of deoxybetulinic acid, represents a new starting point from which to explore the design of a 2nd generation MI.

Discovery of anthranilamides as a novel class of inhibitors of neurotropic alphavirus replication.

Neurotropic alphaviruses are debilitating pathogens that infect the central nervous system (CNS) and are transmitted to humans via mosquitoes. There exist no effective human vaccines against these viruses, underlining the need for effective antivirals, but no antiviral drugs are available for treating infection once the viruses have invaded the CNS. Previously, we reported the development of novel indole-2-carboxamide-based inhibitors of alphavirus replication that demonstrate significant reduction of viral titer and achieve measurable brain permeation in a pharmacokinetic mouse model. Herein we report our continued efforts to improve physicochemical properties predictive of in vivo blood-brain barrier (BBB) permeability through reduction of overall molecular weight, replacing the indole core with a variety of aromatic and non-aromatic monocyclics. These studies culminated in the identification of simple anthranilamides that retain excellent potency with improved metabolic stability and significantly greater aqueous solubility. Furthermore, in a live virus study, we showed that two new compounds were capable of reducing viral titer by two orders of magnitude and that these compounds likely exert their effects through a mechanism similar to that of our indole-2-carboxamide inhibitors.

Significantly decreased and more variable expression of major CYPs and UGTs in liver microsomes prepared from HBV-positive human hepatocellular carcinoma and matched pericarcinomatous tissues determined using an isotope label-free UPLC-MS/MS method.

PURPOSE: To determine the liver expression of cytochrome P450 (CYPs) and uridine 5'-diphosphate-glucuronosyltransferases (UGTs), the major phase I and II metabolism enzymes responsible for clearance and detoxification of drugs, xenobiotic and endogenous substances. METHODS: A validated isotope label-free method was established for absolute and simultaneous quantification of 9 CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D, 2E1 and 3A4) and 5 UGTs (1A1, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes using LC-MS/MS. RESULTS: The LC-MS/MS method displayed excellent dynamic range (at least 250-fold) and high sensitivity for each of the signature peptides with acceptable recovery, accuracy and precision. The protein expression profile of CYP and UGT isoforms were then determined in match microsomes samples prepared from patients with HBV-positive human hepatocellular carcinoma (HCC). In the tumor microsomes, the average absolute amounts of 8 major CYP isoforms (except CYP2C19) and 3 UGT isoforms (UGT1A1, UGT1A4 and UGT2B7) were decreased significantly (p < 0.05), whereas UGT1A6 and UGT1A9 levels were unchanged (p > 0.05). In addition, among isoforms with altered expression, 6 of 8 CYP isoforms and all three UGT isoforms were much more variable in tumor microsomes. Lastly, the importance of CYP3A4 was greatly diminished whereas the importance of UGT1A6 was enhanced in tumor microsomes. CONCLUSION: The use of an isotope label-free absolute quantification method for the simultaneous determination of 9 CYPs and 5 UGTs in human liver microsomes reveals that expression levels of CYPs and UGTs in human liver are severely impact by HCC, which could impact drug metabolism, disposition and pharmacotherapy.

Effect of hepatitis C virus infection on the mRNA expression of drug transporters and cytochrome p450 enzymes in chimeric mice with humanized liver.

The expression of drug transporters and metabolizing enzymes is a primary determinant of drug disposition. Chimeric mice with humanized liver, including PXB mice, are an available model that is permissive to the in vivo infection of hepatitis C virus (HCV), thus being a promising tool for investigational studies in development of new antiviral molecules. To investigate the potential of HCV infection to alter the pharmacokinetics of small molecule antiviral therapeutic agents in PXB mice, we have comprehensively determined the mRNA expression profiles of human ATP-binding cassette (ABC) transporters, solute carrier (SLC) transporters, and cytochrome P450 (P450) enzymes in the livers of these mice under noninfected and HCV-infected conditions. Infection of PXB mice with HCV resulted in an increase in the mRNA expression levels of a series of interferon-stimulated genes in the liver. For the majority of genes involved in drug disposition, minor differences in the mRNA expression of ABC and SLC transporters as well as P450s between the noninfected and HCV-infected groups were observed. The exceptions were statistically significantly higher expression of multidrug resistance-associated protein 4 and organic anion-transporting polypeptide 2B1 and lower expression of organic cation transporter 1 and CYP2D6 in HCV-infected mice. Furthermore, the enzymatic activities of the major human P450s were, in general, comparable in the two experimental groups. These data suggest that the pharmacokinetic properties of small molecule antiviral therapies in HCV-infected PXB mice are likely to be similar to those in noninfected PXB mice. However, caution is needed in the translation of this relationship to HCV-infected patients as the PXB mouse model does not accurately reflect the pathology of patients with chronic HCV infection.