Several small-molecule Bruton tyrosine kinase (BTK) inhibitors are in development for B cell malignancies and autoimmune disorders, each characterized by distinct potency and selectivity patterns. Herein we describe the pharmacologic characterization of BTK inhibitor acalabrutinib [compound 1, ACP-196 (4-[8-amino-3-[(2)-1-but-2-ynoylpyrrolidin-2-yl]imidazo[1,5-]pyrazin-1-yl]--(2-pyridyl)benzamide)]. Acalabrutinib possesses a reactive butynamide group that binds covalently to Cys481 in BTK. Relative to the other BTK inhibitors described here, the reduced intrinsic reactivity of acalabrutinib helps to limit inhibition of off-target kinases having cysteine-mediated covalent binding potential. Acalabrutinib demonstrated higher biochemical and cellular selectivity than ibrutinib and spebrutinib (compounds 2 and 3, respectively). Importantly, off-target kinases, such as epidermal growth factor receptor (EGFR) and interleukin 2-inducible T cell kinase (ITK), were not inhibited. Determination of the inhibitory potential of anti-immunoglobulin M-induced CD69 expression in human peripheral blood mononuclear cells and whole blood demonstrated that acalabrutinib is a potent functional BTK inhibitor. In vivo evaluation in mice revealed that acalabrutinib is more potent than ibrutinib and spebrutinib. Preclinical and clinical studies showed that the level and duration of BTK occupancy correlates with in vivo efficacy. Evaluation of the pharmacokinetic properties of acalabrutinib in healthy adult volunteers demonstrated rapid absorption and fast elimination. In these healthy individuals, a single oral dose of 100 mg showed approximately 99% median target coverage at 3 and 12 hours and around 90% at 24 hours in peripheral B cells. In conclusion, acalabrutinib is a BTK inhibitor with key pharmacologic differentiators versus ibrutinib and spebrutinib and is currently being evaluated in clinical trials.

The increasing incidence of resistance to current HIV-1 therapy underscores the need to develop antiretroviral agents with new mechanisms of action. Integrase, one of three viral enzymes essential for HIV-1 replication, presents an important yet unexploited opportunity for drug development. We describe here the identification and characterization of L-870,810, a small-molecule inhibitor of HIV-1 integrase with potent antiviral activity in cell culture and good pharmacokinetic properties. L-870,810 is an inhibitor with an 8-hydroxy-(1,6)-naphthyridine-7-carboxamide pharmacophore. The compound inhibits HIV-1 integrase-mediated strand transfer, and its antiviral activity in vitro is a direct consequence of this ascribed effect on integration. L-870,810 is mechanistically identical to previously described inhibitors from the diketo acid series; however, viruses selected for resistance to L-870,810 contain mutations (integrase residues 72, 121, and 125) that uniquely confer resistance to the naphthyridine. Conversely, mutations associated with resistance to the diketo acid do not engender naphthyridine resistance. Importantly, the mutations associated with resistance to each of these inhibitors map to distinct regions within the integrase active site. Therefore, we propose a model of the two inhibitors that is consistent with this observation and suggests specific interactions with discrete binding sites for each ligand. These studies provide a structural basis and rationale for developing integrase inhibitors with the potential for unique and nonoverlapping resistance profiles.A gents for the treatment of HIV-1 infection target two of the three virally encoded enzymes and belong to three mechanistic classes known as nucleoside reverse transcriptase, nonnucleoside reverse transcriptase (NNRTI), and protease inhibitors. Although treatment regimens comprising combinations of these agents have significantly reduced AIDS-related morbidity and mortality, it is estimated that 78% of treatment-naive patients harbor viruses that have evolved resistance to at least one of these drug classes (1, 2). The emergence of HIV-1 strains resistant to reverse transcriptase and protease inhibitors highlights the need to develop antiviral agents with novel mechanisms of action.Integrase (3, 4), one of the three virally encoded enzymes required for HIV-1 replication, catalyses the integration of viral DNA into the genome of the host cell. The integration reaction requires three discrete steps: assembly of a stable preintegration complex at the termini of the viral DNA and two sequential transesterification reactions. In the first reaction, 3Ј-end processing, endonucleolytic cleavage of the two 3Ј nucleotides at each DNA end generates 3Ј-hydroxyl groups that function as nucleophiles in the second reaction. The strand breakage of the cellular DNA and concomitant covalent linkage to the viral DNA is a consequence of the second transesterification reaction, strand transfer.The discovery of a series of diketo acids containing HIV-1 integrase i...

Therapy with the oral antidiabetic agent troglitazone (Rezulin) has been associated with cases of severe hepatotoxicity and drug-induced liver failure, which led to the recent withdrawal of the product from the U.S. market. While the mechanism of this toxicity remains unknown, it is possible that chemically reactive metabolites of the drug play a causative role. In an effort to address this possibility, this study was undertaken to determine whether troglitazone undergoes metabolism in human liver microsomal preparations to electrophilic intermediates. Following incubation of troglitazone with human liver microsomes and with cDNA-expressed cytochrome P450 isoforms in the presence of glutathione (GSH), a total of five GSH conjugates (M1-M5) were detected and identified tentatively by LC-MS/MS analysis. In two cases (M1 and M5), the structures of the adducts were confirmed by NMR spectroscopy and/or by comparison with an authentic standard prepared by synthesis. The formation of GSH conjugates M1-M5 revealed the operation of two distinct metabolic activation pathways for troglitazone, one of which involves oxidation of the substituted chromane ring system to a reactive o-quinone methide derivative, while the second involves a novel oxidative cleavage of the thiazolidinedione (TZD) ring, potentially generating highly electrophilic alpha-ketoisocyanate and sulfenic acid intermediates. When troglitazone was administered orally to a rat, samples of bile were found to contain GSH conjugates which reflected the operation of these same metabolic pathways in vivo. The finding that metabolism of the TZD ring of troglitazone was catalyzed selectively by P450 3A enzymes is significant in light of the recent report that troglitazone is an inducer of this isoform in human hepatocytes. The implications of these results are discussed in the context of the potential for troglitazone to covalently modify hepatic proteins and to cause oxidative stress through redox cycling processes, either of which may play a role in drug-induced liver injury.

ABSTRACT:The active forms of all marketed hydroxymethylglutaryl (HMG)-CoA reductase inhibitors share a common dihydroxy heptanoic or heptenoic acid side chain. In this study, we present evidence for the formation of acyl glucuronide conjugates of the hydroxy acid forms of simvastatin (SVA), atorvastatin (AVA), and cerivastatin (CVA) in rat, dog, and human liver preparations in vitro and for the excretion of the acyl glucuronide of SVA in dog bile and urine. Upon incubation of each statin (SVA, CVA or AVA) with liver microsomal preparations supplemented with UDP-glucuronic acid, two major products were detected. Based on analysis by high-pressure liquid chromatography, UV spectroscopy, and/or liquid chromatography (LC)-mass spectrometry analysis, these metabolites were identified as a glucuronide conjugate of the hydroxy acid form of the statin and the corresponding ␦-lactone.

A series of studies were conducted to explore the mechanism of the pharmacokinetic interaction between simvastatin (SV) and gemfibrozil (GFZ) reported recently in human subjects. After administration of a single dose of SV (4 mg/kg p.o.) to dogs pretreated with GFZ (75 mg/kg p.o., twice daily for 5 days), there was an increase (ϳ4-fold) in systemic exposure to simvastatin hydroxy acid (SVA), but not to SV, similar to the observation in humans. GFZ pretreatment did not increase the ex vivo hydrolysis of SV to SVA in dog plasma. In dog and human liver microsomes, GFZ exerted a minimal inhibitory effect on CYP3A-mediated SVA oxidation, but did inhibit SVA glucuronidation. After i.v. administration of [14 C]SVA to dogs, GFZ treatment significantly reduced (2-3-fold) the plasma clearance of SVA and the biliary excretion of SVA glucuronide (together with its cyclization product SV), but not the excretion of a major oxidative metabolite of SVA, consistent with the in vitro findings in dogs. Among six human UGT isozymes tested, UGT1A1 and 1A3 were capable of catalyzing the glucuronidation of both GFZ and SVA. Further studies conducted in human liver microsomes with atorvastatin (AVA) showed that, as with SVA, GFZ was a less potent inhibitor of the CYP3A4-mediated oxidation of this drug than its glucuronidation. However, with cerivastatin (CVA), the glucuronidation as well as the CYP2C8-and CYP3A4-mediated oxidation pathways were much more susceptible to inhibition by GFZ than was observed with SVA or AVA. Collectively, the results of these studies provide metabolic insight into the nature of drug-drug interaction between GFZ and statins, and a possible explanation for the enhanced susceptibility of CVA to interactions with GFZ.