Effectiveness and safety of glecaprevir/pibrentasvir for 8 weeks in the treatment of patients with acute hepatitis C: A single-arm retrospective study.

BACKGROUND AND AIMS: No direct-acting antiviral is currently approved for acute HCV infection, delaying treatment. We investigated the effectiveness and safety of 8-week glecaprevir/pibrentasvir (G/P) in patients with acute HCV infection. APPROACH AND RESULTS: This noninterventional, single-arm, retrospective chart review was designed to enroll adults/adolescents with acute HCV infection. Analyses were conducted on a full analysis set (FAS; all enrolled) and modified FAS (FAS excluding nonvirologic failures). The primary end point (modified FAS) was sustained virologic response at posttreatment week 12 (SVR12) with superiority to 92.6% threshold determined by historic chronic HCV G/P SVR12 rates. Secondary end points (FAS) included SVR12, on-treatment virologic failure, posttreatment relapse, and reinfection. Adverse events and safety laboratory values were assessed.Overall, 202 adults were enrolled; in the modified FAS, 150/151 (99.3%; 95% CI: 96.3-99.9) achieved SVR12, demonstrating superiority to efficacy threshold. In the FAS, the SVR12 rate was 74.3% and the on-treatment virologic failure rate was 0%. Relapse and reinfection rates after the final treatment visit (FAS) were 0.5% and 3%, respectively; 39 patients had missing SVR12 data. No on-treatment alanine aminotransferase elevations > 3 × upper limit of normal with total bilirubin > 2 × upper limit of normal were reported. All 53 patients with alanine aminotransferase Grade ≥ 2 at baseline improved to Grade 0/1 on treatment. No adverse eventss of hepatic decompensation/failure or leading to G/P discontinuation occurred. Two patients had serious adverse events unrelated to G/P. CONCLUSIONS: Eight-week G/P therapy was effective and well-tolerated in patients with acute HCV infection. Data support further investigation of G/P in acute HCV to shorten care cascades, reduce transmission, and support HCV elimination.

Assessment of Drug-Drug Interaction Risk Between Intravenous Fentanyl and the Glecaprevir/Pibrentasvir Combination Regimen in Hepatitis C Patients Using Physiologically Based Pharmacokinetic Modeling and Simulations.

INTRODUCTION: An unsafe injection practice is one of the major contributors to new hepatitis C virus (HCV) infections; thus, people who inject drugs are a key population to prioritize to achieve HCV elimination. The introduction of highly effective and well-tolerated pangenotypic direct-acting antivirals, including glecaprevir/pibrentasvir (GLE/PIB), has revolutionized the HCV treatment landscape. Glecaprevir is a weak cytochrome P450 3A4 (CYP3A4) inhibitor, so there is the potential for drug-drug interactions (DDIs) with some opioids metabolized by CYP3A4, such as fentanyl. This study estimated the impact of GLE/PIB on the pharmacokinetics of intravenous fentanyl by building a physiologically based pharmacokinetic (PBPK) model. METHODS: A PBPK model was developed for intravenous fentanyl by incorporating published information on fentanyl metabolism, distribution, and elimination in healthy individuals. Three clinical DDI studies were used to verify DDIs within the fentanyl PBPK model. This model was integrated with a previously developed GLE/PIB PBPK model. After model validation, DDI simulations were conducted by coadministering GLE 300 mg + PIB 120 mg with a single dose of intravenous fentanyl (0.5 µg/kg). RESULTS: The predicted maximum plasma concentration ratio between GLE/PIB + fentanyl and fentanyl alone was 1.00, and the predicted area under the curve ratio was 1.04, suggesting an increase of only 4% in fentanyl exposure. CONCLUSION: The administration of a therapeutic dose of GLE/PIB has very little effect on the pharmacokinetics of intravenous fentanyl. This negligible increase would not be expected to increase the risk of fentanyl overdose beyond the inherent risks related to the amount and purity of the fentanyl received during recreational use.

Reaching people receiving opioid agonist therapy at community pharmacies with hepatitis C virus: an international randomised controlled trial.

BACKGROUND: Conventional healthcare models struggle to engage those at risk of hepatitis C virus (HCV) infection. This international study evaluated point-of-care (PoC) HCV RNA diagnostic outreach and direct-acting antiviral (DAA) treatment for individuals receiving opioid agonist therapy (OAT) in community pharmacies. AIMS: We assessed the effectiveness of a roving nurse-led pathway offering PoC HCV RNA testing to OAT clients in community pharmacies relative to conventional care. METHODS: Pharmacies in Scotland, Wales, and Australia were randomised to provide PoC HCV RNA testing or conventional referral. Pharmacists directed OAT clients to on-site nurses (intervention) or local clinics (control). Infected participants were treated with DAAs, alongside OAT. Primary outcome was the number of participants with sustained virologic response at 12 weeks (SVR) and analysed using mixed effects logistic regression in the intention-to-treat (ITT) population. RESULTS: Forty pharmacies were randomised. The ITT population contained 1410 OAT clients. In the conventional arm (n = 648), 62 (10%) agreed to testing, 17 (27%) were tested, 6 (35%) were positive and 5 (83%) initiated treatment. In the intervention arm (n = 762), 148 (19%) agreed to testing, 144 (97%) were tested, 23 (16%) were positive and 22 (96%) initiated treatment. SVR was obtained by 2 (40%; conventional) and 18 (82%; intervention). Intervention arm participants had higher odds of testing, OR 16.95 (7.07-40.64, p < 0.001); treatment, OR 4.29 (1.43-12.92, p = 0.010); and SVR, OR 8.64 (1.82-40.91, p = 0.007). CONCLUSIONS: Nurse-led PoC diagnosis in pharmacies made HCV care more accessible for OAT clients relative to conventional care. However, strategies to improve testing uptake are required. TRIAL REGISTRATION: NCT03935906.

Efficacy and Pharmacokinetics of Glecaprevir and Pibrentasvir With Concurrent Use of Acid-Reducing Agents in Patients With Chronic HCV Infection.

BACKGROUND & AIMS: Proton pump inhibitors (PPIs) are commonly prescribed to treat acid-related disorders. Some direct-acting antiviral regimens for chronic hepatitis C virus (HCV) infection have reduced efficacy in patients taking concomitant acid-reducing agents, including PPIs, due to interactions between drugs. We analyzed data from 9 multicenter, phase 2 and 3 trials to determine the efficacy and pharmacokinetics of an HCV therapeutic regimen comprising glecaprevir and pibrentasvir (glecaprevir/pibrentasvir) in patients taking concomitant acid-reducing agents. METHODS: We analyzed data from 2369 patients infected with HCV genotypes 1-6 and compensated liver disease treated with an all-oral regimen of glecaprevir/pibrentasvir for 8-16 weeks. We compared efficacy and pharmacokinetics among patients receiving at least 1 dose of an acid-reducing agent (a PPI, an H2 blocker, or antacid). High-dose PPI was defined as daily dose greater than 20 mg omeprazole dose equivalent. The objectives were to evaluate rate of sustained virologic response 12 weeks post-treatment (SVR12) and to assess steady-state glecaprevir and pibrentasvir exposures in patients on acid-reducing agents. RESULTS: Of the 401 patients (17%) who reported use of acid-reducing agents, 263 took PPIs (11%; 109 patients took a high-dose PPI and 154 patients took a low-dose PPI). Rates of SVR12 were 97.0% among patients who used acid-reducing agents and 97.5% among those not using acid-reducing agents (P = .6). An SVR12 was achieved in 96.3% taking a high-dose PPI and 97.4% taking a low-dose PPI, with no virologic failures in those receiving a high-dose PPI (P = .7). Glecaprevir, but not pibrentasvir, bioavailability was affected; its exposure decreased by 41% in patients taking a high-dose PPI. CONCLUSIONS: In an analysis of data from 9 clinical trials, we observed a high rate of SVR12 (approximately 97%) among patients treated with glecaprevir/pibrentasvir for HCV infection-even among patients taking concomitant ARA or high-dose PPI. This was despite decreased glecaprevir exposures in patients when on high-dose PPIs. ClinicalTrials.gov numbers, NCT02243280 (SURVEYOR-I), NCT02243293 (SURVEYOR-II), NCT02604017 (ENDURANCE-1), NCT02640482 (ENDURANCE-2), NCT02640157 (ENDURANCE-3), NCT02636595 (ENDURANCE-4), NCT02642432 (EXPEDITION-1), NCT02651194 (EXPEDITION-4), NCT02446717 (MAGELLAN-I).

A microRNA screen to identify modulators of sensitivity to BCL2 inhibitor ABT-263 (navitoclax).

Evasion of apoptosis is a known feature of cancer cells. One mechanism of deregulating the apoptotic pathway is through overexpression of antiapoptotic BCL2 family members. ABT-263 (navitoclax) is a first-in-class BCL2 family inhibitor that restores the ability of cancer cells to undergo apoptosis. However, many cancer cells are resistant to ABT-263 due to high levels of a BCL2 family member, MCL1, which is not targeted by the drug. MCL1 expression is regulated transcriptionally, translationally, and through proteasome-mediated degradation. Recently, MCL1 expression was shown to be affected by microRNAs (miRNA). To identify miRNAs that modulate the sensitivity of cancer cells to ABT-263, we screened a library of 810 human miRNA mimics in HCT-116 cells in the presence of ABT-263. The screen revealed 19 miRNAs that sensitize HCT-116 cells to ABT-263. Fifteen of these miRNAs were also shown to sensitize CHL1 melanoma cells to the same agent. We further evaluated 12 of the strongest sensitizers in these cell lines. We found that these sensitizers induced apoptosis only in the presence of ABT-263. In addition, whereas all 12 of these miRNAs reduced MCL1 protein expression, only 10 of them targeted MCL1 through direct binding to the 3'-untranslated region of the gene, raising the possibility that other resistance regulators of MCL1 expression may be identified using our method. Finally, because sensitizing miRNA expression is lower in tumors compared with normal tissues, our data can facilitate the design of miRNA replacement therapies to increase sensitivity to BCL2 antagonists.

An algorithm for classifying tumors based on genomic aberrations and selecting representative tumor models.

BACKGROUND: Cancer is a heterogeneous disease caused by genomic aberrations and characterized by significant variability in clinical outcomes and response to therapies. Several subtypes of common cancers have been identified based on alterations of individual cancer genes, such as HER2, EGFR, and others. However, cancer is a complex disease driven by the interaction of multiple genes, so the copy number status of individual genes is not sufficient to define cancer subtypes and predict responses to treatments. A classification based on genome-wide copy number patterns would be better suited for this purpose. METHOD: To develop a more comprehensive cancer taxonomy based on genome-wide patterns of copy number abnormalities, we designed an unsupervised classification algorithm that identifies genomic subgroups of tumors. This algorithm is based on a modified genomic Non-negative Matrix Factorization (gNMF) algorithm and includes several additional components, namely a pilot hierarchical clustering procedure to determine the number of clusters, a multiple random initiation scheme, a new stop criterion for the core gNMF, as well as a 10-fold cross-validation stability test for quality assessment. RESULT: We applied our algorithm to identify genomic subgroups of three major cancer types: non-small cell lung carcinoma (NSCLC), colorectal cancer (CRC), and malignant melanoma. High-density SNP array datasets for patient tumors and established cell lines were used to define genomic subclasses of the diseases and identify cell lines representative of each genomic subtype. The algorithm was compared with several traditional clustering methods and showed improved performance. To validate our genomic taxonomy of NSCLC, we correlated the genomic classification with disease outcomes. Overall survival time and time to recurrence were shown to differ significantly between the genomic subtypes. CONCLUSIONS: We developed an algorithm for cancer classification based on genome-wide patterns of copy number aberrations and demonstrated its superiority to existing clustering methods. The algorithm was applied to define genomic subgroups of three cancer types and identify cell lines representative of these subgroups. Our data enabled the assembly of representative cell line panels for testing drug candidates.

Acquired resistance to combination treatment with temozolomide and ABT-888 is mediated by both base excision repair and homologous recombination DNA repair pathways.

Many established cancer therapies involve DNA-damaging chemotherapy or radiotherapy. Gain of DNA repair capacity of the tumor represents a common mechanism used by cancer cells to survive DNA-damaging therapy. Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that is activated by DNA damage and plays a critical role in base excision repair. Inhibition of PARP represents an attractive approach for the treatment of cancer. Previously, we have described the discovery and characterization of a potent PARP inhibitor, ABT-888. ABT-888 potentiates the activity of DNA-damaging agents such as temozolomide (TMZ) in a variety of preclinical models. We report here the generation of HCT116 cells resistant to treatment with TMZ and ABT-888 (HCT116R cells). HCT116R cells exhibit decreased H2AX phosphorylation in response to treatment with TMZ and ABT-888 relative to parental HCT116 cells. Microarray and Western blot studies indicate that HCT116R cells have decreased PARP-1 and elevated Rad51 expression levels. HCT116R cells are dependent on Rad51 for proliferation and survival, as shown by inhibition of proliferation and induction of apoptosis upon treatment with Rad51 small interfering RNA. In addition, HCT116R cells are more resistant to radiation than the parental HCT116 cells. Our study suggests that cancer cells upregulate the homologous recombination DNA repair pathway to compensate for the loss of base excision repair, which may account for the observed resistance to treatment with TMZ and ABT-888.

Integrative genomic analysis of small-cell lung carcinoma reveals correlates of sensitivity to bcl-2 antagonists and uncovers novel chromosomal gains.

Cancer is a highly heterogeneous disease in terms of the genetic profile and the response to therapeutics. An early identification of a genomic marker in drug discovery may help select patients that would respond to treatment in clinical trials. Here we suggest coupling compound screening with comparative genomic hybridization analysis of the model systems for early discovery of genomic biomarkers. A Bcl-2 antagonist, ABT-737, has recently been discovered and shown to induce regression of solid tumors, but its activity is limited to a fraction of small-cell lung carcinoma (SCLC) models tested. We used comparative genomic hybridization on high-density single-nucleotide polymorphism genotyping arrays to carry out a genome-wide analysis of 23 SCLC cell lines sensitive and resistant to ABT-737. The screen revealed a number of novel recurrent gene copy number abnormalities, which were also found in an independent data set of 19 SCLC tumors and confirmed by real-time quantitative PCR. A previously unknown amplification was identified on 18q and associated with the sensitivity of SCLC cell lines to ABT-737 and another Bcl-2 antagonist. The region of gain contains Bcl-2 and NOXA, two apoptosis-related genes. Expression microarray profiling showed that the genes residing in the amplified region of 18q are also overexpressed in the sensitive lines relative to the resistant lines. Fluorescence in situ hybridization analysis of tumors revealed that Bcl-2 gain is a frequent event in SCLC. Our findings suggest that 18q21-23 copy number will be a clinically relevant predictor for sensitivity of SCLC to Bcl-2 family inhibitors. The 18q21-23 genomic marker may have a broader application in cancer because Bcl-2 is associated with apoptosis evasion and chemoresistance.

Endothelin signaling in osteoblasts: global genome view and implication of the calcineurin/NFAT pathway.

Patients with prostate cancer develop osteoblastic metastases when tumor cells arrive in the bone and stimulate osteoblasts by secreting growth-promoting factors. Endothelin 1 (ET-1) is believed to be a key factor in promoting osteoblastic metastasis. Selective blockade of the ET(A) receptor is an established strategy in the development of cancer therapeutics. However, the molecular mechanisms whereby prostate cancer promotes abnormal bone growth are not fully understood. In this study, we have applied genomic approaches to elucidate the molecular mechanism of stimulation of osteoblasts by ET-1. To examine the ET-1 axis, we generated genomic signatures for osteoblasts treated with ET-1, in the presence and absence of a selective ET(A) antagonist (ABT-627). The ET-1 signature was comprised of several motifs, such as osteoblastic differentiation, invasion, and suppression of apoptosis. The signature also pointed at possible activation of the calcineurin/NFAT pathway. We showed that ET-1 activates calcineurin and causes nuclear translocation of NFATc1, implicating the pathway in the ET-1-mediated stimulation of osteoblasts. We also showed that ET-1 inhibits apoptosis in osteoblasts, implying that the suppression of apoptosis may be an important factor in the promotion of osteoblastic growth by ET-1.

Evaluating hypoxia-inducible factor-1alpha as a cancer therapeutic target via inducible RNA interference in vivo.

Validating potential targets is an important step in the drug discovery process. In this study, we tested the feasibility of using inducible RNA interference (RNAi) in vivo to obtain an unbiased evaluation on the efficacy of inhibiting hypoxia-inducible factor-1alpha (HIF-1alpha) in established tumors. We showed that HIF-1alpha inhibition resulted in transient tumor stasis or tumor regression, and inhibiting HIF-1alpha in early-stage tumors was found to be more efficacious than inhibiting HIF-1alpha in more established tumors. A differential requirement of HIF-1alpha for tumor growth was also observed among different tumor types. Examination of tumors resistant to HIF-1alpha inhibition suggested that the resistance might result from a less hypoxic tumor environment and the level of HIF-1alpha expression in tumors may be a useful marker for predicting tumor response to HIF-1 inhibition. This study shows that inducible RNAi is a versatile tool for evaluating cancer targets in vivo. In addition to broad implications on in vivo validation of cancer targets, results from this study will also be instructive for practical applications of HIF-1-based cancer therapeutics.

Novel indication for cancer therapy: Chk1 inhibition sensitizes tumor cells to antimitotics.

Paclitaxel (Taxol) is the most-prescribed anti-mitotic agent for a variety of advanced metastatic cancers. It induces mitotic arrest leading to apoptosis through microtubule stabilization. Chk1 is the major cell-cycle checkpoint kinase mediating S- and G2-arrests in response to various DNA-damages. Chk1 inhibitor is anticipated and has been demonstrated to potentiate the cytotoxicity of DNA-damaging agents through abrogation of cell-cycle checkpoints. Paclitaxel does not, however, induce Chk1 activation, and Chk1 has not been shown to function in mitotic checkpoint. Thus, Chk1 inhibitor is not expected to enhance the toxicity of paclitaxel. Here we show that downregulation of Chk1 sensitizes tumor cells to the toxicity of paclitaxel in cell proliferation assay. Fluorescence microscopy showed that Chk1 knockdown augments mitotic catastrophe and apoptosis in paclitaxel-treated cancer cells. Further, we elucidated the mechanism of this sensitization. Chk1 inhibition facilitates paclitaxel-induced M-phase entry by activation of Cdc2 kinase and accumulation of cyclin B1, the required cofactor for Cdc2 kinase activity. Moreover, Chk1 downregulation inhibits M phase exit through induction of the anaphase inhibitor, securin/PDS1. Collectively, Chk1 elimination sustains a more effective mitotic arrest as demonstrated by the more efficient accumulation of M-phase marker phospho-histone H3. We show that Chk1 elimination attenuates the paclitaxel-induced activation of the anti-apoptotic p42/p44 (ERK1/2) MAP kinase pathway, additionally contributing to the sensitization. Our results suggest that in addition to its well-established role as an enforcer of S and G2-checkpoints in response to genotoxic stress, Chk1 also plays a protective role in mitotic checkpoint to lessen mitotic catastrophe and thereby limits cell-death. Therefore Chk1 downregulation can not only potentiate DNA-damaging agents, but also enhance the toxicity of anti-microtubule agents, which significantly broadens its therapeutic applications.

siRNA-mediated gene silencing: a global genome view.

The task of specific gene knockdown in vitro has been facilitated through the use of short interfering RNA (siRNA), which is now widely used for studying gene function, as well as for identifying and validating new drug targets. We explored the possibility of using siRNA for dissecting cellular pathways by siRNA-mediated gene silencing followed by gene expression profiling and systematic pathway analysis. We used siRNA to eliminate the Rb1 gene in human cells and determined the effects of Rb1 knockdown on the cell by using microarray-based gene expression profiling coupled with quantitative pathway analysis using the GenMapp and MappFinder software. Retinoblastoma protein is one of the key cell cycle regulators, which exerts its function through its interactions with E2F transcription factors. Rb1 knockdown affected G1/S and G2/M transitions of the cell cycle, DNA replication and repair, mitosis, and apoptosis, indicating that siRNA-mediated transient elimination of Rb1 mimics the control of cell cycle through Rb1 dissociation from E2F. Additionally, we observed significant effects on the processes of DNA damage response and epigenetic regulation of gene expression. Analysis of transcription factor binding sites was utilized to distinguish between putative direct targets and genes induced through other mechanisms. Our approach, which combines the use of siRNA-mediated gene silencing, mediated microarray screening and quantitative pathway analysis, can be used in functional genomics to elucidate the role of the target gene in intracellular pathways. The approach also holds significant promise for compound selection in drug discovery.

Specificity of short interfering RNA determined through gene expression signatures.

Short interfering RNA (siRNA) is widely used for studying gene function and holds great promise as a tool for validating drug targets and treating disease. A critical assumption in these applications is that the effect of siRNA on cells is specific, i.e., limited to the specific knockdown of the target gene. In this article, we characterize the specificity of siRNA by applying gene expression profiling. Several siRNAs were designed against different regions of the same target gene for three different targets. Their effects on cells were compared by using DNA microarrays to generate gene expression signatures. When the siRNA design and transfection conditions were optimized, the signatures for different siRNAs against the same target were shown to correlate very closely, whereas the signatures for different genes revealed no correlation. These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA-mediated gene silencing as a reliable approach for large-scale screening of gene function and drug target validation.