Cytochrome P450 3A Induction Predicts P-glycoprotein Induction; Part 1: Establishing Induction Relationships Using Ascending Dose Rifampin.

Drug transporter and cytochrome P450 expression is regulated by shared nuclear receptors and, hence, an inducer should induce both, although the magnitude may differ. The objective of this study was to establish relative induction relationships between CYP3A and drug transporters (P-glycoprotein (P-gp), organic anion transporting polypeptide (OATP), and breast cancer resistance protein (BCRP)) or other P450s (CYP2C9 and CYP1A2) using ascending doses of the prototypical pregnane xenobiotic receptor (PXR) agonist, rifampin, to elicit weak, moderate, and strong PXR agonism. Healthy subjects received dabigatran etexilate, pravastatin, rosuvastatin, and a midazolam/tolbutamide/caffeine cocktail before and after rifampin 2, 10, 75, or 600 mg q.d. Unlike CYP3A, only moderate induction of P-gp, OATP, and CYP2C9 was observed and dose-dependent induction of P-gp, OATP, and CYP2C9 was always one drug-drug interaction category lower than observed for CYP3A, even when correcting for probe drug sensitivity. Data from this study establish proof-of-concept that P450 induction data can be leveraged to inform on the effect on transporters.

Cytochrome P450 3A Induction Predicts P-glycoprotein Induction; Part 2: Prediction of Decreased Substrate Exposure After Rifabutin or Carbamazepine.

Rifampin demonstrated dose-dependent relative induction between cytochrome P (CYP)3A and P-glycoprotein (P-gp), organic anion transporting polypeptides (OATPs), or CYP2C9; P-gp, OATP, and CYP2C9 induction was one drug-drug interaction (DDI) category lower than that observed for CYP3A across a wide range of pregnane X receptor (PXR) agonism. The objective of this study was to determine if these relationships could be utilized to predict transporter induction by other CYP3A inducers (rifabutin and carbamazepine) and of another P-gp substrate, sofosbuvir. Healthy subjects received sofosbuvir and a six-probe drug cassette before and after 300 mg q.d. rifabutin or 300 mg b.i.d. carbamazepine. Induction of P-gp, CYP2C9, and decreased sofosbuvir exposure were successfully predicted by observed CYP3A induction. Carbamazepine induction of OATP was underpredicted, likely due to reported additional non-PXR agonism. The results demonstrate that the effect of a PXR agonist on CYP3A can be leveraged to inform on induction liability for other primarily PXR-regulated P450s/transporters, allowing for prioritization of targeted DDI assessments during new drug development.

Antiviral response and resistance analysis of treatment-naïve HCV infected patients receiving multiple doses of the NS3 protease inhibitor GS-9256.

The NS3 protease inhibitor (PI) GS-9256 has demonstrated antiviral activity in a monotherapy study and in combination with other DAAs for treatment of chronic hepatitis C virus (HCV) infection. The resistance profile of GS-9256 was investigated in a phase 1 monotherapy study of patients with HCV genotype (GT) 1 infection. No PI resistance associated substitutions (RASs) at positions 36, 155, 156, 168 and 170 were observed at baseline by population sequencing (15% cutoff) in the 54 patients enrolled in the study, however the PI RAS Q80K were detected in 41% of patients at baseline. In patients who received 75 mg of the investigational protease inhibitor (PI) GS-9256 BID, 300 mg of GS-9256 QD and 200 mg of GS-9256 BID for three days, NS3 RASs (A156V, R155K, D168G/E/N/V) were observed in 9/21, 3/7 and 8/8 post-treatment, respectively. Q80K was not selected in any patients post-treatment. The mean maximal viral load response was -3.0 ± 0.42 log10 IU/mL HCV RNA in the 200 mg BID cohort. In more than 50% of the patients with RASs detected at Day 4, mutations were no longer detectable by population sequencing at Day 14. One patient had the R155K mutation persist to Week 24. Phenotypic analyses showed that substitutions at R155, A156 and D168 significantly reduced susceptibility to GS-9256. In conclusion, NS3 PI RASs were rapidly selected in the majority of patients receiving GS-9256 as monotherapy, despite undetectable levels at baseline. The R155, A156 and D168 substitutions identified in patients confer reduced susceptibility to GS-9256 and other PIs in vitro.

Clinical Resistance to Velpatasvir (GS-5816), a Novel Pan-Genotypic Inhibitor of the Hepatitis C Virus NS5A Protein.

Velpatasvir (VEL, GS-5816) is a novel pan-genotypic hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor with activity against genotype 1 (GT1) to GT6 HCV replicons. In a phase 1b 3-day monotherapy study, patients treated with a 150-mg dose of GS-5816 had a mean maximal HCV RNA decline of ≥3.3 log10 IU/ml in GT1a, -1b, -2, -3, and -4. This report characterizes virologic resistance to VEL in these patients. NS5A resistance-associated substitutions (RASs) were detected by deep sequencing (1% cutoff) pretreatment in 22/70 patients, i.e., 10/35 (29%) patients with GT1a, 1/8 (13%) with GT1b, 4/8 (50.0%) with GT2, 5/17 (29.4%) with GT3, and 2/2 (100.0%) with GT4. In GT1a and GT3 patients, pretreatment RASs were associated with a slightly reduced HCV RNA response compared to that of patients without pretreatment RASs; among patients with GT1b, GT2, and GT4, no significant difference in response was observed in those with or without pretreatment RASs. Following treatment, the pattern of emergent RASs was more complex for GT1a than for the other genotypes. In GT1a, substitutions emerged at positions M28, Q30, L31, P32, H58, E92, and Y93, with the most prevalent substitutions at positions Y93, M28, and L31. RASs were observed at two positions in GT1b and GT2 (Y93 and L31), three positions in GT3 (Y93, L31, and E92), and four positions in GT4 (L28, M31, P32L, and Y93). RASs that were present pretreatment persisted through the 48-week follow-up period; however, RASs emerging during treatment were more likely to decline both in prevalence and in frequency within the viral population during follow-up. (This study has been registered at ClinicalTrials.gov under registration no. NCT01740791.).

Characterization of Hepatitis C virus resistance from a multiple-dose clinical trial of the novel NS5A inhibitor GS-5885.

GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log10 HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at ≥3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30- and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilities in vitro. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT01193478.).

Peginesatide and erythropoietin stimulate similar erythropoietin receptor-mediated signal transduction and gene induction events.

Peginesatide is a synthetic, PEGylated, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. Peginesatide has a unique structure that consists of a synthetic peptide dimer (with no sequence similarity to erythropoietin) conjugated to a 40-kDa PEG moiety. Peginesatide is being developed for the treatment of anemia associated with chronic kidney disease in dialysis patients. To compare signaling effects of peginesatide to recombinant human erythropoietin (rHuEPO), dose-dependent effects on protein phosphorylation and gene expression were evaluated using phosphoproteomics, quantitative signal transduction analyses, and gene profiling. After stimulation with peginesatide or rHuEPO, cell lysates were prepared from UT-7/EPO cells. Liquid chromatography-tandem mass spectrometry and MesoScale arrays were used to quantify phosphorylation events. Transcriptional changes were analyzed using microarrays and quantitative reverse transcription polymerase chain reaction. Peginesatide and rHuEPO were found to regulate the tyrosine phosphorylation of an essentially equivalent set of protein substrates, and modulate the expression of a similar set of target genes. Consistent with their roles in stimulating erythropoiesis, peginesatide and rHuEPO regulate similar cellular pathways.

Susceptibility of treatment-naive hepatitis C virus (HCV) clinical isolates to HCV protease inhibitors.

In order to assess the natural variation in susceptibility to hepatitis C virus (HCV) NS3 protease inhibitors (PIs) among untreated HCV patient samples, the susceptibilities of 39 baseline clinical isolates were determined using a transient-replication assay on a panel of HCV PIs, including two α-ketoamides (VX-950 and SCH-503034) and three macrocyclic inhibitors (MK-7009, ITMN-191, and TMC-435350). Some natural variation in susceptibility to all HCV PIs tested was observed among the baseline clinical isolates. The susceptibility to VX-950 correlated strongly with the susceptibility to SCH-503034. A moderate correlation was observed between the susceptibilities to ITMN-191 and MK-7009. In contrast, the phenotypic correlations between the α-ketoamides and macrocyclic inhibitors were significantly lower. This difference is partly attributable to reduced susceptibility of the HCV variants containing the NS3 polymorphism Q80K (existing in 47% of genotype 1a isolates) to the macrocyclic compounds but no change in the sensitivity of the same variants to the α-ketoamides tested. Our results suggest that the natural variation in baseline susceptibility may contribute to different degrees of antiviral response among patients in vivo, particularly at lower doses.