Hepatitis C virus/HCV NS5B protein (IC50 < 28 nM); Beclabuvir targets the NS5B RNA-dependent RNA polymerase of the hepatitis C virus (HCV). It is a non-nucleoside allosteric inhibitor that specifically binds to thumb site 1 of the NS5B polymerase . This binding site is located in the "thumb" domain of the polymerase structure; inhibitor binding primarily blocks the initiation step of viral RNA replication rather than the elongation step . Beclabuvir inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5, with half-maximal inhibitory concentrations (IC50) below 28 nM .

Beclabuvir exhibits synergistic or additive antiviral effectiveness when combined with pegIFN/RBV and in 2- or 3-drug combinations with other DAAs, including nucleoside NS5B inhibitors, NS5A inhibitors, and HCV NS3 protease inhibitors[2].

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Beclabuvir is a potent, non-nucleoside inhibitor of the HCV NS5B RNA polymerase, with nanomolar activity against HCV genotypes 1, 3, 4, 5 and 6 in vitro. Beclabuvir (formerly BMS-791325) is a potent, non-nucleoside NS5B polymerase inhibitor that binds the NS5B thumb pocket 1 allosteric site and shows nanomolar activity against HCV GTs 1, 3, 4, 5 and 6, with 50% effective concentration (EC50) of 3 and 6 nm, respectively, for GT1a and GT1b 11. In vitro, Beclabuvir demonstrated additive or synergistic antiviral activity with pegIFN/RBV and in 2- or 3-drug combinations with a range of DAAs, such as HCV NS3 protease inhibitors, NS5A inhibitors' and/or nucleoside NS5B inhibitors [2].

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Beclabuvir is an allosteric inhibitor of HCV NS5B RNA-dependent RNA polymerase. The known allosteric inhibitors of HCV NS5B target four distinct sites on the polymerase. The enzyme structure resembles a hand and sites I and II are found in the ‘thumb’ domain, sites III and IV in the ‘palm’ domain. Allosteric inhibitors that target different binding sites are non-cross-resistant each-others. Therefore, these inhibitors might be used with other allosteric inhibitors of NS5B interacting in a synergistic or non-antagonistic way. Beclabuvir is a thumb site 1-NS5B polymerase ligand. In general, such compounds inhibit the initiation step of RNA replication and not the elongation step. Beclabuvir has been shown to equally inhibit de novo and primer dependent synthesis, 5-75 fold more potently than previously studied compounds, thus resulting the most effective thumb site 1 inhibitor of genotype 1 (GT-1) NS5B polymerase to our knowledge. After its binding, beclabuvir inhibits NS5B activity in a time-dependent manner, and thus prevents the formation of active replication complexes[1].

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In vitro, Beclabuvir is able to inhibit recombinant NS5B proteins derived from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC50) with nanomolar potency. In cell culture, beclabuvir impedes replication of HCV subgenomic replicons from genotypes 1a and 1b at 50% effective concentrations (EC50s) of 3 nM and 6 nM, respectively. Similar values (3 to 18 nM) are observed for genotypes 3a, 4a, and 5a. With regard to genotype 6a, EC50 values shows more variability (9 to 125 nM) while potency was weaker against genotype 2 (EC50, 87 to 925 nM). The absence of activity (EC50s of > 4 μM) against a panel of mammalian viruses (pestivirus, bovine viral diarrhea virus) or human DNA polymerases α,β,γ (IC50 values > 25 µM) is indicative of its specificity, along with cytotoxic concentrations (50%) > 3000-fold above the HCV EC50. Moreover, beclabuvir shows additive synergistic effects on replicon inhibition when in combination with other HCV inhibitors such as daclatasvir, asunaprevir, and human lambda 1 IFN. Moreover, a recent in vitro study has shown that the addition of Beclabuvir and sofosbuvir in a quadruple combination regimen (daclatasvir/asunaprevir/beclabuvir/sofosbuvir) efficiently cleared daclatasvir/asunaprevir-resistant replicons from cells in 5 days of treatment[1].

Researchers analysed the efficacy of DCV/ASV/BCV (Beclabuvir) treatment for HCV-infected mice and chronic hepatitis patients. Human hepatocyte chimaeric mice were injected with serum samples obtained from either a DAA-naïve patient or a DCV/ASV treatment failure and were then treated with DCV/ASV alone or in combination with BCV for 4 weeks. DCV/ASV treatment successfully eliminated the virus in DAA-naïve-patient HCV-infected mice. DCV/ASV treatment failure HCV-infected mice developed viral breakthrough during DCV/ASV treatment, with the emergence of NS5A-L31V/Y93H HCV resistance-associated variants (RAVs) being observed by direct sequencing. DCV/ASV/BCV treatment inhibited viral breakthrough in NS5A-L31V/Y93H-mutated HCV-infected mice, but HCV relapsed with the emergence of NS5B-P495S variants after the cessation of the treatment. The efficacy of the triple therapy was also analysed in HCV-infected patients; one DAA-naïve patient and four prior DAA treatment failures were treated with 12 weeks of DCV/ASV/BCV therapy. Sustained virological response was achieved in a DAA-naïve patient and one of the DCV/ASV treatment failures through DCV/ASV/BCV therapy; however, HCV relapse occurred in the other patients with prior DCV/ASV and/or sofosbuvir/ledipasvir treatment failures. DCV/ASV/BCV therapy seems to have limited efficacy for patients with NS5A RAVs for whom prior DAA treatment has failed. [3]

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In patients infected with HCV genotype 1, the most prevalent genotype globally, the combination of Beclabuvir, asunaprevir, and daclatasvir provides very high rates of viral eradication (approximately 90%)[1].

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Trials in monotherapy [1]
In a double-blind, placebo-controlled study, a single ascending daily dose of Beclabuvir (100, 300, 600, or 900 mg) or placebo was administered to 24 patients (randomized 5:1 in each dosing cohort) with chronic HCV genotype 1 infection, both IFN naive and experienced. Peak beclabuvir concentrations were observed between 2 and 4 h, and t1/2 was 6.8 to 9.4 h. Although t1/2 appears to be below 10 h, achievable exposures at 24 h greatly exceeded effective concentrations established in vitro. Maximum antiviral response correlated with plasma drug exposure and was medially achieved 24 h post-dose (range, 12 -48 h) by 12 of the 20 patients who received the drug. At all doses of the drug, a robust reduction of viral RNA (between 1 log10 and over 3 log10 IU/ml) was observed. The highest observed maximum reduction of HCV RNA was reached by two patients, one in the 600-mg cohort (pre-dose baseline, 6.6 log10 IU/ml) and one in the 900-mg cohort (pre-dose baseline, 6.5 log10 IU/ml) whose viral load declined by 3.4 log10 UI/mL 24 h after the dose of the drug.

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IFN-free combination trials [1]
The efficacy and safety of the all-oral fixed-dose combination of 3 DAAs, the NS5A inhibitor daclatasvir (DCV), the NS3/4 protease inhibitor asunaprevir (ASV) and the non-nucleoside NS5B polymerase inhibitor Beclabuvir (BCV), given to patients with chronic HCV infection were evaluated in one Phase II study and in two Phase III studies: i) AI443-014 which was a Phase II study carried out on treatment-naive GT-1 and GT-4 patients, and pegIFN/RBV (ribavirin) prior null-responders GT-1 patients. This study explored safety and efficacy of the DCV TRIO therapy (DCV - ASV - BCV) administered for 12 or 24 weeks; ii) UNITY-1 (AI443-102) which was a Phase III study carried out on treatment-naive and treatment-experienced GT-1 patients without cirrhosis. This study assessed safety and efficacy of DCV TRIO therapy administered for 12 weeks; iii) UNITY-2 (AI443-113) which was a Phase III study conducted on treatment-naive and treatment-experienced GT-1 patients with compensated cirrhosis. This study evaluated the DCV TRIO therapy plus RBV administered for 12 weeks.

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This study evaluated the efficacy and safety of Beclabuvir, in combination with peginterferon alfa-2a (pegIFN) and ribavirin (RBV), in HCV genotype 1. In this randomized (1:1:1), double-blinded, placebo-controlled, dose-ranging phase 2a study, 39 treatment-naive patients chronically infected with HCV genotype 1 were treated for 48 weeks with beclabuvir (75 mg or 150 mg) plus pegIFN (180 μg) and RBV (1000 mg/day [<75 kg] or 1200 mg/day [≥ 75 kg]) vs pegIFN/RBV alone. The primary efficacy endpoint of extended rapid virologic response (undetectable HCV RNA at treatment weeks 4 and 12) was achieved by 76.9% (10/13) of patients receiving beclabuvir 75 mg and 38.5% (5/13) receiving beclabuvir 150 mg vs 0% receiving pegIFN/RBV alone. Higher response rates were observed among patients receiving beclabuvir 75 mg for all secondary efficacy endpoints, including sustained virologic response at follow-up weeks 12 or 24. Three patients experienced virologic breakthrough on treatment, all in the beclabuvir 150-mg treatment group. Beclabuvir was well tolerated at both doses, with the most commonly observed adverse events (headache, fatigue, nausea, decreased appetite, irritability, depression and insomnia) consistent with those observed with pegIFN/RBV. In conclusion, beclabuvir was both effective and well tolerated when administered in combination with pegIFN/RBV for the treatment of chronic HCV GT 1, supporting the study of beclabuvir as part of an all-oral regimen for HCV GT1. [2]

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Overall, 69 patients were screened and 39 randomized; of these, 29 patients completed the study (Fig. S1). Baseline characteristics were generally balanced across the three groups, except for a lower proportion of males, and higher proportions of white patients and patients with IL28B (rs12979860) CC genotypes in the Beclabuvir 75-mg arm compared with the other arms (Table S1).

The primary efficacy endpoint of eRVR was achieved by a higher proportion of patients in the Beclabuvir 75-mg (76.9%) and 150-mg (38.5%) groups than in the pegIFN/RBV group (0%) (Fig. 1). Patients in the beclabuvir 75-mg group demonstrated the highest response rates for the secondary efficacy endpoints RVR, cEVR, SVR12 and SVR24 (Fig. 1). Similar results were obtained when patients with missing measurements were excluded from the analysis (on-treatment analysis; Fig. S2). Response rates were also generally higher in the beclabuvir treatment arms compared with placebo for both IL28B CC and non-CC genotypes (Fig. 1), although numbers were small. The higher response rates with beclabuvir 75 mg vs 150 mg may be explained by 2 factors: (i) a lower proportion of patients with IL28B (rs1297860) CC genotypes – known to be associated with higher response rates to pegIFN/RBV-based regimes than non-CC genotypes 14-16 – in the 150-mg group (15%) than in the 75-mg group (46%), and (ii) a higher proportion of patients in the 150-mg arm with missing HCV RNA data at post-treatment week 24. When missing data were accounted for, SVR24 rates in the 150-mg treatment arm improved, with observed values of SVR24 rates in both the beclabuvir 150-mg and the placebo arms of 71% (5/7) and 90% (9/10) in the beclabuvir 75-mg arm.

In the Beclabuvir 75-mg group, no patient experienced virologic breakthrough or relapse post-treatment; among the four patients without SVR24, one had detectable HCV RNA at the end of treatment, whereas the remaining 3 (all with undetectable HCV RNA prior to post-treatment follow-up week 24) had missing week 24 post-treatment data, were lost to follow-up, or discontinued due to other reasons (incarceration) prior to this time point. In the beclabuvir 150-mg group, among the eight patients without SVR24, three had virologic breakthrough and one had detectable HCV RNA at end of treatment; no post-treatment relapse was observed. The remaining four patients (all with undetectable HCV RNA prior to post-treatment week 24) either had missing RNA data at post-treatment week 24 (n = 1) or were lost to follow-up (n = 3). In the placebo group, among the eight patients without SVR24, two had detectable HCV RNA at end of treatment, three experienced post-treatment relapse, whereas the remaining three patients (all with undetectable HCV RNA prior to post-treatment week 24) had missing RNA data or withdrew consent.

Among the three patients with virologic breakthrough (all Beclabuvir 150 mg), all had GT1a infection, were IL28B non-CC genotype and had evidence of the NS5B substitutions A421V and P495A/L/S at breakthrough; one patient also had the NS5B substitution M426L at baseline. The observation that all patients with virologic breakthrough had GT1a and NS5B variants at A421 and P495 is consistent with prior data for beclabuvir and other thumb pocket 1 NS5B inhibitors. Variants at P495 are signature resistance-associated substitutions for thumb pocket-1 inhibitors, and A421V has previously been observed following in vitro passage of GT1a replicon cells in the presence of beclabuvir alone and when combined with the NS3 inhibitor asunaprevir [2].

In in vitro studies, binding assays with NS5B genetic variants (wild type, L30S and P495L) revealed a two-step, slow-binding mechanism of the drug. In detail, resistance substitutions selected by Beclabuvir/BCV in genotype 1-based replicons mostly mapped to the NS5B amino acid 495 (P495A/S/L/T), which therefore can be considered the only clinically relevant resistance variant. For P495, the rate of initial complex formation and dissociation is similar to wild type, but the kinetics of the second step is significantly faster. This is associated with a shortened residence time and finally translates into a decrease in inhibitor potency. In contrast, the potency of BCV on L30S is roughly equal to wild-type polymerase. Therefore the detection of L30S mutation is not considered clinically relevant.
With regard to genotype-specific resistance profiles, GT1a has the highest resistance barrier versus Beclabuvir/BCV while GT6a has the lowest. At least in genotype 3 to 6-based replicon systems, substitutions in the thumb domain of NS5B at residues 494 and 495 conferred different levels of resistance to BCV. However, no cross-resistance to NS5A or NS3 protease inhibitors was observed. Regarding subtype 6a, it is noteworthy that A494 polymorphism, which confers reduced potency to BCV, is present in 21% of sequences in the European HCV database[1].

The in vitro cellular activity of Beclabuvir is typically evaluated using HCV subgenomic replicon cell models. The protocol is as follows: Huh-7 cells stably harboring an HCV subgenomic replicon (containing a luciferase reporter gene or assessed by qPCR for HCV RNA levels) are seeded into culture plates and allowed to attach overnight. Subsequently, serial dilutions of Beclabuvir are added to the culture medium, and the cells are incubated for an additional 72 hours at 37°C in a 5% CO₂ incubator. After the incubation period, cells are lysed, and the level of HCV RNA replication is measured by luciferase activity or real-time quantitative PCR (RT-qPCR) to calculate the half-maximal effective concentration (EC50) for inhibition of viral replication. In this system, Beclabuvir exhibits EC50 values of 3 nM against genotype 1a replicons and 6 nM against genotype 1b replicons . Concurrent cytotoxicity control experiments should be performed using methods such as MTT or CellTiter-Glo to assess drug-induced cytotoxicity, ensuring that the observed antiviral effects are not due to reduced cell viability. Studies have shown that the half-maximal cytotoxic concentration (CC50) of Beclabuvir is >3000-fold above its antiviral EC50, indicating a high selectivity index .

Patients and Methods [2]
This was a randomized (1:1:1), double-blinded, placebo-controlled, dose-ranging phase 2a study (AI443-012; ClinicalTrials.gov: NCT 01193361) evaluating the safety and efficacy of Beclabuvir combined with pegIFN/RBV in treatment-naive adults chronically infected with HCV GT1. Eligible patients received 48 weeks of twice-daily oral beclabuvir at 75 mg, beclabuvir at 150 mg, or placebo, each administered in combination with once-weekly subcutaneous pegIFN (180 μg) and twice-daily oral RBV (weight-based dosing of 1000 mg/day [<75 kg] or 1200 mg/day [≥75 kg]). The duration of post-treatment follow-up was 24 weeks (in patients with undetectable HCV RNA at end of treatment) or 48 weeks (in patients with detectable HCV RNA at end of treatment or relapse).

Patients were required to have HCV RNA ≥10–5 IU/mL (COBAS TaqMan HCV Test 2.0; Roche Molecular Diagnostics, Pleasanton, California; lower limit of quantitation [LLOQ] 25 IU/mL) at screening, with no evidence of cirrhosis by liver biopsy within 24 months of randomization. Key exclusion criteria included >4 weeks of prior treatment with interferon or RBV within 6 months prior to randomization; alanine aminotransferase (ALT) ≥5 × upper limit of normal (ULN); total bilirubin ≥34 μmol/L (≥2 mg/dL) or direct bilirubin >ULN; international normalization ratio (INR) ≥1.7; confirmed creatinine clearance ≤50 mL/min; or concurrent diagnosis of chronic hepatitis B infection, HIV infection, hepatocellular carcinoma or other non-HCV liver disease.

The primary safety endpoints were the incidence of serious adverse events (SAEs) and discontinuations of study therapy for AEs. The primary efficacy endpoint was the proportion of patients with extended rapid virologic response (eRVR), defined as undetectable (Beclabuvir and its metabolite BMS-794712, and associations between antiviral activity or safety of beclabuvir and host IL28B genotype or beclabuvir exposure.

Population sequencing of NS5B was performed at baseline, and for all Beclabuvir-treated patients experiencing futility (defined as [i] virologic breakthrough [increase in HCV RNA >1 log10 IU/mL above nadir, or HCV RNA ≥LLOQ following a confirmed undetectable measurement on treatment]; [ii] <1 log10 decrease in HCV RNA at week 4; [iii] failure to achieve EVR [defined as <2 log10 IU/mL decrease in HCV RNA at week 12]; or [iv] detectable HCV RNA at week 12 and ≥LLOQ at week 24) or relapse (undetectable HCV RNA at end of treatment followed by confirmed detectable HCV RNA at any post-treatment visit) and with HCV RNA ≥1000 IU/mL.

Pharmacokinetics and Metabolism [1] Optimization of the properties of early NS5B polymerase inhibitor compounds led to a promising class of alkyl-bridged piperazine carboxamide antiviral compounds, and Beclabuvir was discovered and preclinically characterized. In vitro, researchers studied Beclabuvir in liver microsomes of humans, rats, dogs, and cynomolgus monkeys. In human and monkey microsomes, the half-life (t1/2) of Beclabuvir was 53 min and 23 min, respectively, while in rat and dog experiments, the t1/2 was greater than 200 min. Based on these data, researchers conducted a 24-hour pharmacokinetic study of the compound in rats. In this study, the oral bioavailability was 66% and the volume of distribution was 2.7 L/kg when administered with PEG-400 solution; after intravenous administration, the plasma clearance was 3.5 mL/min/kg and the plasma half-life was estimated to be 8.3 h.
In multiple animal groups (rats, dogs, and monkeys), plasma and liver exposures following oral administration indicated that Beclabuvir exhibits hepatic tropism (liver/plasma ratios ranging from 1.6 to 60 times across different species). Twenty-four hours after administration, liver exposures in all tested species were greater than or equal to 10 times the EC50 values of HCV genotypes 1, 3, and 5 replicons; greater than 6.7 to 40 times the EC50 value of the GT 4 replicon; and 1.5 to 14 times higher than the EC50 value of the GT 6 replicon.
Beclabuvir exposure, as measured by maximum drug concentration (Cmax) and area under the plasma concentration-time curve (AUCinf), was dose-dependent and directly proportional to the dose. The point estimate and 90% confidence interval for Cmax were approximately 1.1 (0.99 to 1.22), and the point estimate and 90% confidence interval for AUCinf were approximately 1.18 (0.99 to 1.36), consistent with the expectation of once- or twice-daily dosing. In replicon cultures, at all doses, protein-corrected EC90 values were lower than plasma concentrations (52 ng/ml) in all patients within 1 hour and 24 hours after administration. Therefore, even repeated dosing at the lowest test dose (100 mg) provided satisfactory antiviral efficacy, while single doses exceeding 300 mg offered little additional antiviral benefit.
Furthermore, Beclabuvir is metabolized into an equivalent compound (BMS-794712) with similar pharmacokinetic profiles and approximately 22% of the parent drug's plasma exposure, thus significantly contributing to overall antiviral activity. Beclabuvir has been used in combination with daclatasvir and asunavir in several clinical trials (see below). Since all these drugs are CYP3A4 substrates, OATP1B1 inhibitors, and P-glycoprotein inhibitors, researchers investigated potential drug interactions of the triplet combination in a pharmacokinetic sub-study of the AI443014 trial. In this sub-study, 32 treatment-naïve non-cirrhotic patients with HCV GT1 infection received 12 or 24 weeks of daclatasvir (60 mg qd), asunavir (200 mg bis in die [bid]), and two doses of beclabuvir (75 mg bid or 150 mg bid). Adding beclabuvir to daclatasvir and asunavir did not reveal any clinically significant interactions. Although daclatasvir (DCV), asunavir (ASV), and beclabuvir/BCV are primarily excreted in feces (renal excretion accounts for <10% of total clearance), there are concerns that indirect mechanisms due to chronic kidney disease may also affect the non-renal clearance of these drugs. Therefore, the open-label, multi-dose AI443110 study evaluated the pharmacokinetics and safety of DCV, ASN, and BCV in 41 subjects not infected with hepatitis C virus (HCV) (33 patients with varying degrees of renal impairment and 8 healthy controls with normal renal function). In subjects with moderate to severe renal impairment, the mean concentrations of DCV, ASN, beclabuvir/BCV, and BMS-794712 were higher than in subjects with normal renal function. For patients with end-stage renal disease (ESRD) undergoing hemodialysis (HD), the mean concentrations of DCV, BCV, and BMS-794712 were comparable to those in patients with normal renal function, while the mean concentration of ASV was lower. In patients with impaired renal function, both Cmax and AUCtau of the drugs were higher than in patients with normal renal function, particularly in those with severe renal impairment. Due to hemodialysis, drug exposure in ESRD patients was generally comparable to that in patients with normal renal function, and the free pharmacokinetic parameters of DCV were lower after hemodialysis compared to healthy subjects. In the primary regression analysis (excluding patients undergoing hemodialysis), the Cmax and AUCtau of DCV, ASV, BCV, and BMS-794712 increased with decreasing creatinine clearance, primarily in subjects with severe renal impairment (range 42% to 105%). The median time (Tmax) to reach maximum plasma concentrations for Beclabuvir and its metabolite BMS-794712 was 2 hours at each dose (Figure S3). At week 12 of treatment, exposures to both Beclabuvir and BMS-794712 were above dose proportions, with AUC ratios of metabolite to parent drug of approximately 0.23–0.25, consistent with single-dose data (100–900 mg) from a previous Phase I Beclabuvir study (sup>12). No association was observed between the composite trough concentration of Beclabuvir and virological response (eRVR, SVR24, RVR, cEVR, SVR12; Figure S4a and data not shown), the incidence of serious adverse events (SAEs), or discontinuation due to adverse events (AEs) (Figure S4a). There was no sustained association between drug exposure and baseline changes in specific clinical laboratory endpoints, including total bilirubin (Figure S4b), ALT, hemoglobin, or absolute neutrophil count (data not shown); however, it should be noted that trough concentrations are an indirect indicator of peak exposure. [2]

Clinical Trials: Tolerability [1]
In monotherapy studies of Beclabuvir/BCV at escalating doses, the drug was well tolerated and safe. No deaths, serious adverse events (SAEs), or discontinuation due to adverse events (AEs) were reported. All recorded adverse events were mild, but two moderate gastrointestinal events occurred in the 900 mg dose group, which may have been at least partly attributable to the higher capsule dosage (18 capsules). Overall, the most common adverse events observed during treatment were nausea, vomiting, and headache (all three symptoms occurred in 2 of 29 patients).
In interferon-based treatment regimens, Beclabuvir/BCV was well tolerated at both doses (75 mg and 150 mg twice daily), and the most common adverse events (headache, fatigue, nausea, decreased appetite, irritability, depression, and insomnia) were consistent with those observed with pegylated interferon/ribavirin (pegIFN/RBV) alone.
In trials evaluating the combination of this drug with daclatasvir (DCV) and aspirin (ASV), 2, 3, and 1 patients, respectively, discontinued treatment due to adverse events in the AI443-014, UNITY-1, and UNITY-2 trials.
In the AI443-014 trial, 6 serious adverse events (SAEs) occurred in genotype 1 patients, and no SAEs occurred in genotype 4 patients; there were no deaths. Among previously untreated GT1 patients, 3 serious adverse events (SAEs) occurred, 1 in the Beclabuvir/BCV 75 mg group (esophageal tumor, unrelated to combination therapy, leading to treatment discontinuation), and 2 in the Beclabuvir/BCV 150 mg group (1 of which was a throat constriction sensation, related to combination therapy, leading to treatment discontinuation). The most common adverse events (AEs) during treatment (≥ 10%) were, in descending order of frequency, headache, diarrhea, fatigue, and nausea. In previously unresponsive patients, 3 SAEs (cervical radiculopathy, syncope, and a mental disorder occurring 7 days after treatment) and 1 Grade 3 AE (syncope) occurred during the study period; all 4 events occurred during the 24-week treatment period and were considered unrelated to the study drug. No Grade 4 AEs were observed. The most common (≥ 10%) adverse events included headache, fatigue, pruritus, diarrhea, and upper respiratory tract infection. No serious adverse events, Grade 3/4 adverse events, or deaths were reported in GT-4 patients. The most common adverse events in GT-4 patients (≥ 10% in both groups) were headache (29% overall), insomnia (19%), nausea (14%), and pain (14%). In the UNITY-1 study, a total of 7 serious adverse events and 1 death (post-treatment) occurred; all events were considered unrelated to treatment. Headache, fatigue, diarrhea, and nausea were the most common adverse events (incidence >10%). Three patients (<1%) discontinued treatment due to adverse events. In the UNITY-2 study, a total of three treatment-related serious adverse events occurred, with no deaths. Fatigue, headache, nausea, diarrhea, insomnia, and pruritus were the most common reported adverse events (incidence ≥10%). In patients receiving three direct-acting antiviral agents (DAAs) in combination with ribavirin (RBV), 5% experienced hemoglobin levels <9 g/dL during treatment, compared to none in patients not receiving RBV. One patient discontinued DAA treatment due to an adverse event. In the open-label, multi-dose AI443110 study of uninfected hepatitis C virus (HCV) patients with normal or impaired renal function, no deaths or serious adverse events were reported after DCV TRIO administration. One patient discontinued the study drug due to an adverse event. One participant in the moderate renal impairment group reported a moderate increase in serum uric acid related to the study drug. During the study period, 29% of patients with renal impairment experienced one or more adverse events (AEs), most of which were mild and transient, while no adverse events were reported in healthy subjects. No clinically significant laboratory abnormalities were assessed as adverse events. In summary, DCV-TRIO was well tolerated in both patients with renal impairment and subjects with normal renal function. No dose adjustment was required for patients with renal impairment, but patients with severe renal disease not receiving hemodialysis should take DCV-TRIO once daily. Beclabuvir was well tolerated at both doses, with no unexpected safety events reported (Table 1). No deaths were reported. The nature and incidence of adverse events during treatment were similar in all three groups (≥92%), with the most common adverse events generally associated with pegylated interferon/ribavirin (pegIFN/RBV) treatment. The most common grade 3/4 laboratory abnormalities during treatment were hematological abnormalities, which are also expected adverse events with pegylated interferon/ribavirin treatment. Serious adverse events (SAEs) occurred in 3 patients during treatment and follow-up; of these, SAEs were considered unrelated to the study drug in 2 patients. A third patient (receiving 75 mg becrabbuvir) developed grade 3 anemia and grade 2 leukopenia at week 4 of follow-up after treatment; these adverse events were considered related to the study drug. Six patients discontinued study treatment due to adverse events (AEs) (1 receiving 75 mg becrabbuvir, 2 receiving 150 mg becrabbuvir, and 3 receiving placebo). Four patients discontinued treatment according to protocol due to confirmed conjugated hyperbilirubinemia (bilirubin ≥ 3 times baseline and > upper limit of normal) within the first 2 weeks of treatment (days 5-10) (1 patient in the becrabbuvir group, 1 patient in the placebo group, and 2 patients in the placebo group). All four events were mild (grade 1) or moderate (grade 2) in severity, with direct bilirubin levels ranging from 0.4 to 1.0 mg/dL (normal range 0-0.2 mg/dL). In three patients, bilirubin abnormalities completely resolved or returned to slightly above baseline levels after discontinuation of all study drugs, but in one patient in the placebo group, bilirubin abnormalities persisted after starting treatment with commercially available pegylated interferon/ribavirin. [2]

[1]. Beclabuvir for the treatment of hepatitis C. Expert Opin Investig Drugs. 2015;24(8):1111-21.

[2]. A randomized, placebo-controlled study of the NS5B inhibitor beclabuvir with peginterferon/ribavirin for HCV genotype 1. J Viral Hepat. 2015 Aug;22(8):658-64.

[3]. Limitations of daclatasvir/asunaprevir plus beclabuvir treatment in cases of NS5A inhibitor treatment failure. J Gen Virol. 2018 Aug;99(8):1058-1065.

Beclabusvir has been used in clinical trials investigating the treatment of chronic hepatitis C. Beclabusvir is a non-nucleoside polymerase inhibitor that inhibits hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase with potential anti-HCV activity. After administration, beclabusvir is absorbed intracellularly and binds to the non-catalytic Thumb 1 site of HCV NS5B polymerase via allosteric binding, thereby reducing viral RNA synthesis and replication. The HCV NS5B protein is crucial for the replication of the HCV RNA genome. HCV is a small, enveloped, single-stranded RNA virus belonging to the Flaviviridae family. Introduction: Approximately 185 million people worldwide are chronically infected with hepatitis C virus (HCV). Currently, the most effective antiviral therapies for hepatitis C virus (HCV) infection reduce the risk of disease progression to advanced liver disease (cirrhosis, hepatocellular carcinoma, and death). However, in recent years, several new direct-acting anti-HCV drugs have been marketed or are in the late stages of clinical development.
This article reviews: This article focuses on the allosteric non-nucleotide inhibitor of HCV polymerase, Beclabuvir. The article covers its pharmacokinetics, mechanism of action, tolerability and safety, as well as resistance.
Expert opinion: Beclabuvir has very good pharmacokinetics, efficacy and tolerability, as well as resistance barriers. In particular, Beclabuvir, in combination with asunaprevir and daclatasvir, can achieve very high viral clearance (about 90%) in patients with HCV genotype 1 infection, which is the most common genotype worldwide. Therefore, Beclabuvir is a powerful weapon against hepatitis C virus (HCV) infection and should be considered an ideal choice in customized interferon-free combination therapy regimens. [1]

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The pharmacokinetic properties of Beclabuvir/BCV allow for twice-daily dosing. A 12-week combination of becrabbuvir/BCV with the protease inhibitor aspirin (ASV) and the NS5A inhibitor daclatasvir (DCV) achieved a sustained virological response (SVR) rate of approximately 90% in patients with HCV genotype 1 infection. While data on the efficacy of this combination regimen in patients with HCV genotype 4 infection are limited, they are very encouraging. Data for other non-genotype 1 infections are currently lacking. The drug's tolerability and safety profile are satisfactory. In conclusion, becrabbuvir/BCV is a powerful weapon against HCV infection and should be considered an ideal choice for customized, interferon-free combination therapy. The treatment of HCV infection is undergoing an exciting and rapid transformation. The emergence of multiple antiviral drug combinations makes it possible to cure patients with different genotypes and stages of disease caused by hepatitis C virus (HCV). However, the following aspects still need to be evaluated: i) the tolerability and safety of direct-acting antiviral agents (DAAs) in patients with advanced disease; ii) the actual impact of viral clearance on survival and quality of life if the virus is cleared in advanced disease. In other words, is there a critical point beyond which viral clearance no longer provides significant clinical benefit? iii) the optimal drug combination for each condition; iv) the availability of drugs at a reasonable price for all patients; and v) the potential impact of viral clearance on patients with mild hepatic impairment but with HCV-related extrahepatic damage. Regarding the first two points, it is worth noting that there are currently no studies on the efficacy and safety of Beclabuvir/BCV in treating decompensated cirrhosis. This may be because the combination therapy uses ASV, which is contraindicated in patients with advanced cirrhosis, as Child-Pugh B/C patients may have elevated ASV plasma concentrations due to abnormal ASV distribution in the liver. We recommend conducting clinical trials to evaluate the efficacy and safety of Beclabuvir/BCV in combination with other drugs to assess its true potential in treating patients with HCV infection and advanced liver disease. Regarding the selection of the most cost-effective combination regimen, we emphasize that most patients participating in clinical trials are infected with HCV genotype 1, the most common genotype globally. However, for this genotype, we have several combination regimens with similarly excellent SVR rates, such as sofosbuvir/ledipasvir, sofosbuvir/daclatasvir, sofosbuvir/simeprevir, obbitasvir-paretasvir-ritonavir, and dasabuvir. The primary advantage of this new drug and its various combinations is that it provides physicians with more options. On one hand, this allows physicians to develop personalized treatment plans based on the patient's specific situation, addressing potential drug interactions, resistance, or contraindications. On the other hand, the large number of drugs targeting hepatitis C virus (HCV) has intensified competition among pharmaceutical companies. A fundamental function of market competition is to reduce drug prices. This is particularly significant for developing countries that currently cannot afford the high prices of these drugs. Finally, it is common in clinical practice to manage patients with concomitant extrahepatic HCV diseases (such as mixed cryoglobulinemia or non-Hodgkin's lymphoma), which can be life-threatening and severely impair their quality of life [76-78]. However, in some cases, extrahepatic damage cannot be quantified. Indeed, HCV infection is associated with several metabolic-related diseases, such as atherosclerosis, diabetes, and overall cardiovascular risk. Therefore, it is not surprising that some data show a reduction in cardiovascular-related mortality among HCV-infected individuals who achieve sustained virological response (SVR) [79]. The use of beclabuvir (BCV) still needs to be evaluated, for example, its efficacy against non-type 1 genotypes. In fact, although in vitro experiments showed that it has pangenotypic activity, it has only been used in clinical practice on 21 patients with genotype 4. In addition, as a substrate of CYP3A4 and an inhibitor of OATP1B1 and P-glycoprotein, becrabuvir may have drug interactions. Therefore, people look forward to conducting pharmacokinetic studies on the combined use of becrabuvir with some commonly used drugs. In summary, becrabuvir is an effective drug with good tolerability and safety. When used in combination with other antiviral drugs, it can achieve the best efficacy in the compensatory phase of hepatitis C virus infection. [1] At each dose, the median time (Tmax) for becrabuvir and its metabolite BMS-794712 to reach maximum plasma concentration was 2 hours (Figure S3). At week 12 of treatment, exposures to both Beclabuvir and BMS-794712 were above dose-proportional, with AUC ratios of metabolites to parent drug of approximately 0.23–0.25, consistent with single-dose data (100–900 mg) from previous Phase I Beclabuvir studies. No association was observed between Beclabuvir composite trough concentrations and virological responses (eRVR, SVR24, RVR, cEVR, SVR12; Figure S4a and data not shown), the incidence of serious adverse events (SAEs), or discontinuation due to adverse events (AEs) (Figure S4a). No sustained correlation was found between drug exposure and baseline changes in specific clinical laboratory endpoints, including total bilirubin (Figure S4b), ALT, hemoglobin, or absolute neutrophil count (data not shown); however, it should be noted that trough concentrations are an indirect indicator of peak exposure. In summary, these data demonstrate that beclabuvir in combination with pegylated interferon/ribavirin is effective and well-tolerated for the treatment of chronic HCV genotype 1 infection and support the use of beclabuvir in an all-oral regimen. Based on these results, and similar data involving the direct-acting antiviral agents daclatasvir (DCV; an NS5A inhibitor) and asunavir (ASV), a phase IIb study for genotype 1 infection was initiated, employing a triple DAA combination of beclabuvir (75 mg or 150 mg), DCV (60 mg), and ASV (200 mg). In a pilot cohort study (N = 66), this all-oral combination therapy achieved SVR12 in 92% of patients after 12 or 24 weeks of treatment, and response rates appeared to be independent of IL28B genotype and duration of treatment. Subsequently, a larger cohort of patients (N = 166) reported similar efficacy and safety results, using 75 mg or 150 mg becrabbuvir for 12 weeks. A phase 3 clinical trial is currently underway for a triple therapy of becrabbuvir (75 mg dose) in combination with daclatasvir and aspirin, administered in a fixed-dose combination formulation, targeting both treatment-naïve and previously treated patients. [2]

C36H45N5O5S.HCL

696.299

695.29

C, 62.10; H, 6.66; Cl, 5.09; N, 10.06; O, 11.49; S, 4.60

958002-36-3

958002-36-3 (HCl);958002-33-0;

72722244

White to off-white solid powder

0

2

7

6

48

1320

4

Cl.CN(S(NC(C1C=CC2C(=C3N(C=2C=1)C[C@]1(C(N2[C@@H]4CC[C@H]2CN(C4)C)=O)[C@@H](C1)C1=C3C=CC(OC)=C1)C1CCCCC1)=O)(=O)=O)C

IHXVACFNNPBRLK-OZSFMWOHSA-N

InChI=1S/C36H45N5O5S.ClH/c1-38(2)47(44,45)37-34(42)23-10-14-28-31(16-23)40-21-36(35(43)41-24-11-12-25(41)20-39(3)19-24)18-30(36)29-17-26(46-4)13-15-27(29)33(40)32(28)22-8-6-5-7-9-22;/h10,13-17,22,24-25,30H,5-9,11-12,18-21H2,1-4H3,(H,37,42);1H/t24-,25+,30-,36-;/m0./s1

(4bS,5aR)-12-cyclohexyl-N-(N,N-dimethylsulfamoyl)-3-methoxy-5a-((1R,5S)-3-methyl-3,8-diazabicyclo[3.2.1]octane-8-carbonyl)-4b,5,5a,6-tetrahydrobenzo[3,4]cyclopropa[5,6]azepino[1,2-a]indole-9-carboxamide.

Beclabuvir; BMS791325; BMS-791325; Beclabuvir hydrochloride; BMS-791,325 hydrochloride; 958002-36-3; Beclabuvir hydrochloride [USAN]; UNII-3KU5345YJF; 3KU5345YJF; BMS-791325-08; BECLABUVIR HYDROCHLORIDE [MI]; BMS 791325.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)