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BackgroundConventional dendritic cells subtype 1 (cDC1) play a vital role in the priming and expansion of tumor specific CD8+ T cells and their recruitment to tumor microenvironment (TME). However, cDC1s are often underrepresented in the TME. Systemic administration of Fms-like tyrosine kinase 3 ligand (FLT3L), a hematopoietic growth factor that binds to FLT3 on myeloid and lymphoid progenitor cells, leads to expansion of cDC1s in the periphery which can then be recruited into the TME. FLT3 pathway stimulation using GS-3583, a novel FLT3 agonistic Fc fusion protein, has the potential to promote T cell mediated anti-tumor activity. We sought to evaluate the pharmacodynamic (PD) effect of a single dose of GS-3583 in healthy volunteers alongside its safety. Herein, we present the updated results of the study.MethodsThis was a first-in-human, placebo-controlled study of GS-3583 in healthy volunteers to evaluate the safety, pharmacokinetics (PK), and PD of escalating single doses (ranging from 75 micrograms to 2000 micrograms) of GS-3583. The study was blinded to the subjects and the investigator. Each dose cohort enrolled 8–12 healthy subjects who received GS-3583 or placebo as single IV infusion at 3:1 ratio. Subjects were observed in the phase 1 unit for 15 days and then for 12 weeks as outpatients. As part of the PD evaluation, we investigated the changes in the number of cDC1 and cDC2 cells.ResultsAs of 2nd July 2021, selected safety, PK and PD data from all 4 cohorts were available. GS-3583 was well tolerated and all subjects had been discharged. To date, there have been no serious or grade 3 or higher adverse events. Preliminary PK analysis suggested dose-dependent increase in GS-3583 exposure (AUC and Cmax). Preliminary PD analysis shows that administration of GS-3583 resulted in temporary, dose-dependent increases in cDC1/cDC2 cells that peaked between days 5–11 (higher doses resulted in later peaks) and returned to baseline within 3 weeks of drug administration (table 1, figure 1).Abstract 380 Table 1Selected subject characteristics and pharmacodynamic resultsAbstract 380 Figure 1A) Comparison of cDC1 cell quantitative changes in cohorts 1–4; B) Comparison of cDC2 cell quantitative changes in cohorts 1–4ConclusionsGS-3583 infusion was well tolerated and induced dose dependent expansion of dendritic cells in the periphery in healthy volunteers. In patients with cancer, this increase in dendritic cells can be utilized to enhance anti-tumor therapeutic effects of immuno-oncology therapies.AcknowledgementsFunding provided by Gilead Sciences, Inc.Ethics ApprovalThe study received study site IRB/Ethics Committee approval prior to enrollment of subjects.

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.

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.

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.

2559 Background: Conventional dendritic cells subtype 1 (cDC1) play a vital role in the priming and expansion of tumor specific CD8+ T cells and their recruitment to tumor microenvironment (TME). However, cDC1s are often underrepresented in the TME. Systemic administration of Fms-like tyrosine kinase 3 ligand (FLT3L), a hematopoietic growth factor that binds to FLT3 on myeloid and lymphoid progenitor cells, leads to expansion of cDC1s in the periphery which can then be recruited into the TME. We hypothesize that FLT3 pathway stimulation using GS-3583, a FLT3 agonist Fc fusion protein, has the potential to promote T cell mediated anti-tumor activity. Methods: This was a first-in-human placebo-controlled study of GS-3583 in healthy volunteers to evaluate the safety, PK, and PD of escalating single doses (ranging from 75μg to 2000μg) of GS-3583. The study was blinded to the subjects and the investigator. Each dose cohort enrolled 8-12 healthy subjects who received GS-3583 or placebo as single IV infusion at 3:1 ratio. Subjects were observed in the phase 1 unit for 15 days and then for 12 weeks as outpatients. As part of the PD evaluation, we investigated the changes in the number of cDC1s and cDC subtype 2 (cDC2) cells. Results: As of 8th Feb 2021, selected safety, PK and PD data from the first 3 cohorts were available. GS-3583 was well tolerated and all subjects had been discharged. To date, there have been no serious or grade 3 or higher adverse events. Preliminary PK analysis suggested dose-dependent increase in GS-3583 exposure (AUC and Cmax). Preliminary PD analysis shows that administration of GS-3583 resulted in dose-dependent increases in cDC1/cDC2 cells that peaked at day 5 or day 8 and returned to baseline within three weeks of drug administration. Conclusions: GS-3583 was safe and well tolerated and induced dose dependent expansion of dendritic cells in the periphery. In patients with cancer, this increase in dendritic cells can be utilized to enhance anti-tumor responses to immuno-oncology therapies.[Table: see text]

The study aimed to evaluate the effects of baseline hepatitis C virus (HCV) nonstructural protein 5A (NS5A) resistance-associated substitutions (RASs) on sustained virologic response to ledipasvir (LDV)-containing regimens in the absence of sofosbuvir (SOF) in patients with HCV genotype (GT) 1 infection across 6 phase 2 clinical studies. We analysed data from 1103 patients who received either LDV + vedroprevir (NS3 protease inhibitor) + tegobuvir (NS5B inhibitor) ± ribavirin or LDV + ribavirin + pegylated interferon. Population sequencing of HCV NS5A was performed at baseline and at virologic failure from patient plasma samples. Of 1045 patients with available baseline sequences, 747 (67.7%) had GT1a, and 298 (26.9%) had GT1b infection. The overall prevalence of NS5A RASs at baseline was 9.4%; 7.6% (57/747) and 13.8% (41/298) of patients with GT1a and GT1b infection, respectively. The majority of GT1a-infected patients with NS5A RASs at baseline had a single NS5A RAS (78.9%) at NS5A positions K24R, M28T, Q30H/L, L31M and Y93H/N/C/S. The spectrum of NS5A RASs detected in GT1b patients was much less diverse compared to GT1a patients, with all patients harbouring a single NS5A RAS either L31M or Y93H/C. For patients treated with LDV-containing regimens in the absence of SOF, the presence of baseline NS5A RASs was associated with low SVR rates. In patients with virologic failure, nearly all had either pre-existing and/or emergent NS5A RASs: 287/287 (100%) and 40/42 (95.2%) patients with GT1a and GT1b infection, respectively. Three novel NS5A substitutions were identified as emergent NS5A RASs: K26E and S38F in GT1a; and L31I in GT1b. In conclusion, the presence of NS5A RASs at baseline reduced the SVR rate in patients treated with LDV in combination vedroprevir + tegobuvir ± ribavirin or ribavirin + pegylated interferon. Virologic failure was associated with the detection of NS5A RASs in nearly all patients. These results suggest that the resistance barrier may differ depending on HCV drug combination and may be more important than that of the individual DAAs.

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 log 10 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.)

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 log 10 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.)

e14097Background: Entospletinib (ENTO), a Syk inhibitor, has shown clinical activity in R/R CLL subjects. In vitro, ENTO inhibited OATP1B1/1B3, BCRP, P-gp and UGT1A1; ENTO metabolism is mediated by CYP2C9 and CYP3A. These studies evaluate the drug interaction (DDI) profile and mass balance (MB) of ENTO. Methods: Healthy subjects received ENTO alone, or with cobicistat (COBI; CYP3A inhibitor), rifampin (RIF; CYP3A inducer), fluconazole (FLU; CYP2C9/CYP3A inhibitor), rosuvastatin (ROS; OATP1B1/1B3, BCRP substrate) or digoxin (DIG, P-gp substrate). For the MB study, healthy subjects received a single dose of ENTO containing [14C]-labeled ENTO. Results: DDI results are shown below. ENTO was extensively metabolized to 14 metabolites in the MB study; ~91% and ~2% of the dose was recovered in feces and urine, respectively. The majority of circulating radioactivity was associated with unchanged ENTO. ENTO was generally well tolerated in these studies. Conclusions: Co-administration with strong CYP3A inducers may ...

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.

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.

68 69 70 71 72 73 74 75 76 77 78 79 80 81 Corrigendum to ‘‘Peginesatide and erythropoietin stimulate similar erythropoietin receptor–mediated signal transduction and gene induction events’’ [Experimental Hematology 40 (2012) 575–587] Jennifer M. Green, Karen Leu, Angela Worth, Richard B. Mortensen, David K. Martinez, Peter J. Schatz, Don M. Wojchowski, and Peter R. Young Affymax, Inc., Palo Alto, CA, USA; Maine Medical Center Research Institute, Scarborough, ME, USA 82 83 84 85 86 87 88 The authors regret that an affiliation was left out of the Acknowledgments. Investigations were supported in part by National Institutes of Health (NIH) grants R01HL044491 and R01DK089439 (D.M. Wojchowski). The authors would like to apologize for any inconvenience caused. 89