HCV NS3 protease(Ki=14 nM)
Boceprevir (EBP-520; SCH-503034): Targets hepatitis C virus (HCV) NS3 protease; it shows potent inhibitory activity with a Ki value of 14 nM in enzyme assay and an EC₉₀ value of 0.35 μM in cell-based replicon assay. It also exhibits high selectivity, with a 2200-fold higher inhibitory effect against HCV NS3 protease compared to human neutrophil elastase (HNE) [1]
- Boceprevir (EBP-520; SCH-503034): Targets HCV NS3/4A protease; it can inhibit the activity of NS3/4A protease in transgenic mouse models, thereby reducing the secretion of Gaussia luciferase (Gluc) into the plasma [2]

The potency of boceprevir (SCH 503034) in the HCV NS3 protease continuous assay is 14 nM (Ki) on average over a high number of runs. The EC50 and EC90 values in the HuH-7 cells bicistronic subgenomic cell-based replicon assay are found to be 0.20 µM and 0.35 µM, respectively. The assay lasts for 72 hours. Additionally, it is discovered that boceprevir has a selectivity of 2200 and is a very weak inhibitor of HNE (Ki=26 µM)[1].

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Enzyme and cell-based inhibitory activity: Boceprevir (EBP-520; SCH-503034) is a potent inhibitor of HCV NS3 protease. In the enzyme assay, its Ki value is 14 nM, indicating strong binding affinity to the target enzyme. In the cell-based replicon assay, it exhibits significant antiviral activity with an EC₉₀ value of 0.35 μM. Moreover, it shows high selectivity for HCV NS3 protease, being 2200 times more potent against this viral protease than against human neutrophil elastase (HNE), which helps reduce potential off-target effects [1]
- Verification in cell transfection experiments: The pBI-NS3/4A plasmid (carrying NS3/4A gene) was co-transfected into CHO cells along with pTet-On-rtTA and pBI-Cre plasmids, with pBI-NS3/4A alone as a control. At 6 hours after transfection, the cells were treated with doxycycline (Dox, 1 μg/mL) and harvested 48 hours later. The functionality of the system was verified by detecting Firefly luciferase (Fluc) activity in cell lysates and Gluc activity in the culture medium. Subsequently, when Boceprevir (EBP-520; SCH-503034) was applied in this in vitro system, it could inhibit the activity of NS3/4A protease, as reflected by the reduction in Gluc activity in the culture medium, confirming its inhibitory effect on NS3/4A protease in vitro [2]

An HCV protease inhibitor called boceprevir is used to treat hepatitis C virus infection. Boceprevir's pharmacokinetic profile is assessed in a variety of animal species. After being given orally, rats (10 mg/kg), dogs (3 mg/kg), and monkeys (3 mg/kg) absorb boceprevir moderately. The mean absorption times (MAT) of mice (10 mg/kg), rats, and monkeys show that while absorption is relatively fast in dogs, it is slower in these animals. In dogs and rats, the AUC is good; in mice, it is moderate; and in monkeys, it is low. In mice, rats, and dogs (26-34%), the absolute oral bioavailability is moderate, but in monkeys (4%) it is low[1]. Triple-transgenic mice treated with boceprevir (100 mg/kg) showed reduced HCV NS3/4A protease activity[2].

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Activity in triple-transgenic mouse model: A conditional inducible NS3/4A/Lap/LC-1 triple-transgenic mouse model was established. The mice were induced with Dox (1 mg/mL Dox and 50 g/L sugar dissolved in drinking water) for 3 days to activate NS3/4A protease expression, resulting in a 70-fold increase in plasma Gluc activity compared to non-induced mice. When Boceprevir (EBP-520; SCH-503034) was administered orally to these Dox-induced triple-transgenic mice at a dose of 100 mg/kg twice daily for 7 days (starting from the 3rd day of Dox induction), plasma Gluc activity was reduced by 65% compared to the DMSO-treated control group. This result demonstrates that Boceprevir can effectively inhibit NS3/4A protease activity in the liver of transgenic mice in vivo [2]
- Efficacy in liver transplant recipients with HCV recurrence: In five liver transplant patients with genotype 1 HCV recurrence, Boceprevir (EBP-520; SCH-503034) was administered at a dose of 800 mg three times a day after a 4-week lead-in phase, in combination with pegylated interferon (PegIFN) and ribavirin (RBV), and concomitantly used with immunosuppressive agents (cyclosporine in 3 patients, tacrolimus in 2 patients, everolimus in 1 patient). During a mean follow-up period of 14.8 ± 3.1 weeks, all patients achieved a virological response: the mean baseline HCV viral load (HVL) was 6.87 ± 0.70 log₁₀ IU/mL, and by week 12, the mean HVL decrease was 6.64 ± 0.35 log₁₀ IU/mL, showing significant in vivo antiviral efficacy in this specific patient population [3]
- Efficacy in different populations: In the treatment of genotype 1 chronic HCV infection, the combination of Boceprevir (EBP-520; SCH-503034) with PegIFN and RBV (triple therapy) has significantly improved sustained virological response (SVR) rates compared to the standard dual therapy (PegIFN + RBV). The standard dual therapy has an SVR rate of only 40%-50%, while the triple therapy with Boceprevir increases the SVR rate to up to 70%. However, in African American patients with genotype 1 chronic HCV, clinical trials show that their SVR rates with Boceprevir-based triple therapy are lower than those in white patients, and African American patients may require a longer duration of therapy to achieve the same SVR rate as white patients [4, 5]

Verification of the functionality of the pBI-NS3/4A transgene in vitro[2]
Chinese hamster ovary (CHO) cells were cultivated in RPMI-1640 media supplemented with 10% fetal bovine serum. The functionality of pBI-NS3/4A was tested through co-transfection along with the pTet-On and pBI-G//Cre plasmids. To accomplish this, CHO cells were transfected using Lipofectamine 2000 reagent according to the manufacturer’s protocol. After co-transfection for 6 h, the culture medium was replaced with fresh medium containing 1 μg/mL Dox, and induction proceeded for 48 h. Subsequently, the cell medium and cells were separately collected to assay luciferase activity via bioluminescent imaging (BLI) and to assay luciferase expression via western blot analysis.

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HCV NS3 protease activity assay:
1. Preparation of enzyme and substrate: Isolate and purify HCV NS3 protease to obtain the active enzyme. Prepare peptide substrates that mimic the natural cleavage site of NS3 protease, which may be labeled with a detectable group (such as a fluorescent group) to facilitate activity detection.
2. Reaction setup: Mix the purified NS3 protease with different concentrations of Boceprevir (EBP-520; SCH-503034) in a suitable reaction buffer, and incubate at a specific temperature (e.g., 37°C) for a certain period to allow the inhibitor to bind to the enzyme.
3. Substrate addition and detection: Add the prepared peptide substrate to the reaction system and continue incubation. Monitor the cleavage of the substrate by detecting the change in the signal of the labeled group (e.g., fluorescent intensity) over time using a detection instrument (such as a fluorescence microplate reader).
4. Data analysis: Calculate the enzyme activity under different Boceprevir concentrations based on the detected signal changes. Use appropriate kinetic models to fit the data and determine the Ki value (14 nM) of Boceprevir for NS3 protease, evaluating its inhibitory potency against the enzyme [1]
- Selectivity assay against human neutrophil elastase (HNE):
1. Preparation of HNE and substrate: Obtain human neutrophil elastase and prepare a specific substrate for HNE (also labeled with a detectable group for activity measurement).
2. Reaction and detection: Set up reaction systems containing HNE and different concentrations of Boceprevir (EBP-520; SCH-503034). After incubation, add the HNE substrate and detect the enzyme activity by measuring the signal change of the substrate's labeled group, following the same detection method as in the NS3 protease assay.
3. Selectivity calculation: Compare the inhibitory concentrations of Boceprevir against HCV NS3 protease and HNE. Calculate the selectivity ratio (2200-fold) by determining the ratio of the concentration required to inhibit 50% of HNE activity to that required to inhibit 50% of NS3 protease activity, confirming the high selectivity of Boceprevir for the viral protease [1]

HCV replicon cell assay:
1. Cell culture: Cultivate HCV replicon cells (cells stably containing the HCV replicon, which can replicate independently in the cell) in a suitable medium supplemented with necessary nutrients and antibiotics to maintain cell growth and replicon stability.
2. Drug treatment: Seed the replicon cells into 96-well plates at an appropriate density. After the cells adhere to the plate, add different concentrations of Boceprevir (EBP-520; SCH-503034) to the culture medium, with a vehicle control group (e.g., DMSO) set up simultaneously. Incubate the cells at 37°C in a 5% CO₂ incubator for a specific period (e.g., 72 hours) to allow the drug to exert its effect.
3. Viral replication detection: After incubation, extract the total RNA from the cells using a suitable RNA extraction method. Use quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to detect the level of HCV RNA in the cells, with a housekeeping gene (e.g., GAPDH) as an internal reference to normalize the RNA loading amount.
4. Data analysis: Calculate the relative HCV RNA levels in each Boceprevir-treated group compared to the control group. Determine the concentration of Boceprevir that inhibits 90% of viral replication (EC₉₀ = 0.35 μM) by fitting the dose-response curve, evaluating its in vitro antiviral activity [1]
- CHO cell co-transfection assay for NS3/4A protease activity:
1. Cell preparation: Culture CHO cells in a complete medium until they reach the logarithmic growth phase. Adjust the cell density to an appropriate concentration (e.g., 1×10⁵ cells/mL) for transfection.
2. Transfection: Co-transfect the pBI-NS3/4A plasmid (carrying NS3/4A gene and Gluc reporter gene with NS3/4A cleavage site) into CHO cells together with pTet-On-rtTA (expressing rtTA protein) and pBI-Cre (expressing Cre recombinase) plasmids using a transfection reagent. Set up a control group transfected with pBI-NS3/4A alone.
3. Induction and drug treatment: At 6 hours after transfection, add Dox (1 μg/mL) to the culture medium to induce the expression of Cre and NS3/4A proteins. Simultaneously, add different concentrations of Boceprevir (EBP-520; SCH-503034) to the experimental groups. Incubate the cells for 48 hours.
4. Activity detection: Collect the cell culture medium to measure Gluc activity using a Gluc detection kit and a luminometer. Lyse the cells to prepare cell lysates, and detect Fluc activity to verify the efficiency of Cre-mediated recombination. Perform Western blot analysis on the cell lysates using anti-NS3/4A antibody to confirm NS3/4A protein expression, and on the concentrated culture medium using anti-Gluc antibody to detect Gluc secretion. Analyze the effect of Boceprevir on NS3/4A protease activity by comparing Gluc activity in the drug-treated and control groups [2]

Mice
Triple-transgenic mice (n = 5 per group) are induced with Doxycycline (Dox) for 10 days in order to assess the impact of Boceprevir. The mice receive oral gavage twice daily for seven days with either Boceprevir (100 mg/kg) or DMSO after plasma Gluc activity peaks on the third day following Dox induction. Every day during this time, blood is drawn from the caudal vein in order to measure plasma Gluc activity.

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Using triple-transgenic mice to evaluate the effects of NS3/4A inhibitors[2]
Telaprevir and boceprevir were used. To evaluate the effect of telaprevir, triple-transgenic mice were randomized into two groups (n = 5 per group) and administered either telaprevir (200 mg/kg) or vehicle (dimethyl sulfoxide, DMSO) via oral gavage twice daily for 10 days. At the same time, the mice were continuously induced with Dox (1 mg/mL Dox and 50 g/L sugar were dissolved in their drinking water).
To evaluate the effect of boceprevir, triple-transgenic mice were induced with Dox for 10 days (n = 5 per group). On the third day after Dox induction, when plasma Gluc activity reached its peak, the mice were administered either boceprevir (100 mg/kg) or DMSO via oral gavage twice daily for 7 days. During this period, blood was collected from the caudal vein daily to detect plasma Gluc activity.[2]

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In vivo efficacy evaluation in triple-transgenic mice:
1. Mouse preparation: Use NS3/4A/Lap/LC-1 triple-transgenic mice (containing rtTA, Cre, and NS3/4A transgenes). Maintain the mice under specific pathogen-free (SPF) conditions with a 12-hour light/dark cycle and free access to food and water.
2. Dox induction: Dissolve Dox (1 mg/mL) and sugar (50 g/L) in the drinking water of the triple-transgenic mice to induce NS3/4A protease expression. Continue the induction for 3 days, with non-induced triple-transgenic mice and wild-type (WT) mice as controls.
3. Drug administration: On the 3rd day of Dox induction, divide the induced triple-transgenic mice into two groups: the Boceprevir (EBP-520; SCH-503034) treatment group and the DMSO control group. Administer Boceprevir orally via gavage at a dose of 100 mg/kg twice daily for 7 days; the control group receives the same volume of DMSO. During the drug treatment period, continue the Dox induction by providing Dox-containing drinking water.
4. Sample collection and detection: Collect blood samples from the caudal vein of the mice daily to separate plasma. Measure the Gluc activity in the plasma using a Gluc detection kit and a luminometer to monitor the inhibitory effect of Boceprevir on NS3/4A protease activity in real time. After the treatment, sacrifice the mice, collect liver tissue, and perform Western blot analysis with anti-NS3 antibody to confirm NS3/4A protein expression, and histological examination to assess liver tissue changes [2]

Absorption, Distribution and Excretion
Boceprevirin reaches peak plasma concentration 2 hours after administration. Absolute bioavailability has not been determined. Exposure can increase to 65% when taken with food. Boceprevirin capsules consist of two diastereomers in a 1:1 ratio. In plasma, this ratio changes to 2:1, with the active diastereomer being dominant. Boceprevirin is primarily excreted in feces (79%), with a small amount excreted in urine (9%). Approximately 8% and 3% are excreted unchanged in feces and urine, respectively. The mean apparent volume of distribution of Boceprevirin at steady state is 772 L. The mean systemic clearance of Boceprevirin is 161 L/hour. In healthy subjects, the drug exposure of 800 mg Boceprevirin three times daily, administered alone, was characterized by an AUC(T) of 5408 ng·hr/mL (n=71), a Cmax of 1723 ng/mL (n=71), and a Cmin of 88 ng/mL (n=71). Pharmacokinetic results were similar in healthy and HCV-infected subjects. The median time to peak absorption (Tmax) after oral administration of Boceprevirin was 2 hours. The increases in steady-state AUC, Cmax, and Cmin were less than dose-proportional, and there was significant overlap in individual exposures at 800 mg and 1200 mg doses, suggesting reduced absorption at higher doses. Drug accumulation was extremely low (0.8–1.5 times), and pharmacokinetic steady state was reached approximately one day after three daily doses. Boceprevirin should be taken with food. Compared to a fasting state, food can increase the exposure of bocepivir at three times daily doses of 800 mg by up to 65%. Bioavailability of bocepivir is similar regardless of meal type (e.g., high-fat vs. low-fat meals) or timing of administration (5 minutes before, during, or immediately after a meal). Therefore, the type and timing of meals are not important considerations when taking bocepivir. In healthy subjects, the mean apparent volume of distribution (Vd/F) of bocepivir at steady state is approximately 772 liters. For more complete data on absorption, distribution, and excretion of bocepivir (10 items in total), please visit the HSDB records page. Metabolism/Metabolites Bocepivir is primarily metabolized via an aldehyde-ketone reductase-mediated pathway, producing a mixture of diastereomers, with exposure levels up to four times that of the parent compound. Bocepivir is also metabolized oxidatively via CYP3A4/5, but to a lesser extent.
In vitro studies have shown that bocepivir is primarily metabolized to ketone reductase (AKR)-mediated metabolites, which are inactive against HCV. Following a single oral dose of 800 mg (14)C-bocepivir, the most abundant circulating metabolite was a mixture of diastereomers of the ketone reductase, with an average exposure approximately four times that of bocepivir. Bocepivir is also metabolized via CYP3A4/5-mediated oxidative metabolism, but to a lesser extent.
Biological Half-Life
The mean elimination half-life of bocepivir is 3.4 hours.
The mean plasma half-life (t1/2) of bocepivir is approximately 3.4 hours.

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Effect on oral immunosuppressant clearance: In liver transplant patients with HCV relapse, when bocepivir (EBP-520; SCH-503034) (800 mg, three times daily) is used in combination with immunosuppressants (ITs), bocepivir inhibits cytochrome P450 3A enzymes, thereby reducing the oral clearance of the immunosuppressants. Compared with intrathecal administration alone, the mean estimated oral clearance of cyclosporine was reduced by about 50%, tacrolimus by up to 80%, and everolimus by 52%. This suggests that bocepivir affects the metabolism of these immunosuppressants by inhibiting related metabolic enzymes [3].

Hepatotoxicity
In large randomized controlled trials, triple therapy with bocepivir, pegylated interferon, and ribavirin has been associated with a higher incidence of adverse events, often requiring dose adjustments and leading to premature discontinuation in 5% to 20% of patients. However, elevated serum ALT and clinically significant liver injury are generally not cited as adverse events. Exceptions have occurred in patients with pre-existing cirrhosis, where some treated patients have experienced new-onset, seemingly spontaneous liver decompensation. The cause of this decompensation is unclear, and the independent role of bocepivir in relation to pegylated interferon and ribavirin, as well as the possibility of it occurring even without treatment, remains difficult to define. However, in post-marketing studies of triple therapy for chronic hepatitis C with cirrhosis, 3% to 8% of patients reported decompensation, and 1% to 3% died of liver failure.
Potential score for combined use of Boceprevirin, pegylated interferon, and ribavirin: B (may lead to liver damage and decompensation in patients with a history of cirrhosis or advanced fibrosis).

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Use during pregnancy and lactation
◉ Overview of use during lactation
Boceprevirin has been withdrawn from the US market. No studies have been conducted in breastfeeding women receiving treatment for hepatitis C. Because it must be used in combination with ribavirin and pegylated interferon alfa, its use during lactation is not recommended. Until more data are available, especially during the breastfeeding of newborns or premature infants, alternative medications may be preferred.
Hepatitis C is not transmitted through breast milk, and breast milk has been shown to inactivate the hepatitis C virus (HCV). However, the US Centers for Disease Control and Prevention (CDC) recommends that mothers infected with HCV should consider discontinuing breastfeeding if they experience cracked or bleeding nipples. It is unclear whether this warning applies to mothers receiving treatment for hepatitis C. Infants born to mothers infected with HCV should be tested for HCV; nucleic acid testing is recommended because maternal antibodies are present in the infant for the first 18 months after birth and before the infant develops an immune response. ◉ Effects on breastfed infants: No published information found as of the revision date. ◉ Effects on lactation and breast milk: No published information found as of the revision date. Protein binding: After a single dose, bosapivir binds to approximately 75% of human plasma proteins. Hematologic toxicity in liver transplant patients: In 5 liver transplant patients treated with bosapivir (EBP-520; SCH-503034) in combination with pegylated interferon and ribavirin, all patients developed anemia. The mean hemoglobin level at baseline was 13.18 ± 1.18 g/dL, which decreased to 10.4 ± 1.5 g/dL at week 12, a mean decrease of 3.12 ± 2.27 g/dL. All five patients required β-erythropoietin to alleviate anemia; three patients required a reduction in ribavirin dose (33%-75%), and one patient required a transfusion due to a drop in hemoglobin level below 8 g/dL. This suggests that combination therapy containing Boceprevirin may induce hematologic toxicity, primarily manifested as anemia [3]
- Drug Interactions: Boceprevirin (EBP-520; SCH-503034) inhibits the cytochrome P450 3A enzyme, and therefore drug interactions can occur when used in combination with immunosuppressants metabolized by this enzyme (such as cyclosporine, tacrolimus, everolimus). As a result, the plasma concentrations of these immunosuppressants may increase due to reduced oral clearance, potentially increasing the risk of immunosuppressant-related adverse reactions. Therefore, when used in combination with Boceprevirin, the dosage of the immunosuppressant needs to be adjusted [3]

[1]. Challenges in modern drug discovery: a case study of boceprevir, an HCV protease inhibitor for the treatment of hepatitis C virus infection. Acc Chem Res. 2008 Jan;41(1):50-9.

[2]. Conditional Inducible Triple-Transgenic Mouse Model for Rapid Real-Time Detection of HCV NS3/4A ProteaseActivity. PLoS One. 2016 Mar 4;11(3):e0150894.

[3]. Practical management of boceprevir and immunosuppressive therapy in liver transplant recipients with hepatitis C virus recurrence. Antimicrob Agents Chemother. 2012 Nov;56(11):5728-34.

[4]. New developments in the management of hepatitis C virus infection: focus on boceprevir. Biologics. 2012;6:249-56.

[5]. Telaprevir and boceprevir in african americans with genotype 1 chronic hepatitis C: implications for patients and providers. South Med J. 2012 Aug;105(8):431-6.

Boceprevir is a synthetic tripeptide composed of N-(tert-butylcarbamoyl)-3-methyl-L-valine, cyclopropyl-fused prolyl, and 3-amino-4-cyclobutyl-2-oxobutyramide residues linked in sequence. It is used to treat chronic hepatitis C virus type 1 infection. It is a hepatitis C protease inhibitor, peptide mimic, and antiviral drug. It is a tripeptide belonging to the urea class of drugs. Boceprevir is a direct-acting antiviral drug used in combination with other drugs to treat chronic hepatitis C, an infectious liver disease caused by hepatitis C virus (HCV) infection. HCV is a single-stranded RNA virus with nine different genotypes, of which genotype 1 is the most common in the United States, affecting 72% of chronic HCV infections. Since 2011, significant progress has been made in treatment options for chronic hepatitis C with the development of direct-acting antiviral agents (DAAs) such as Boceprevir. Boceprevir is an NS3/4A inhibitor. NS3/4A are serine proteases encoded by HCV genotypes 1 and 4. These enzymes are crucial for viral replication, cleaving viral-encoded polyproteins into mature proteins such as NS4A, NS4B, NS5A, and NS5B. Compared to another class of DAAs—NS5B inhibitors—NS3/4A inhibitors have a lower barrier to resistance. Amino acid substitutions at positions 155, 156, or 168 are known to lead to resistance. Amino acid substitutions in the enzyme catalytic triad composed of H58, D82, and S139 may also alter the drug's affinity for NS3/4A or the enzyme's activity itself. Despite these drawbacks, Boceprevir remains effective against hepatitis C virus (HCV) when used in combination with [DB00811], [DB00008], and [DB00022]. In a 2016 joint guideline published by the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA), the American Society for the Study of Liver Diseases (AASLD) did not recommend the combination of boceprevir with [DB00811], [DB00008], and [DB00022] as a first-line treatment for hepatitis C. The goal of using boceprevir in combination with [DB00811], [DB00008], and [DB00022] is to cure hepatitis C or achieve sustained virological response (SVR) after 48 weeks of daily treatment. SVR and eradication of hepatitis C virus infection are associated with significant long-term health benefits, including reduced liver-related damage, improved quality of life, reduced incidence of hepatocellular carcinoma, and reduced all-cause mortality. Boceprevir is a fixed-dose formulation (brand name Victrelis) used to treat chronic hepatitis C. Victrelis was approved by the FDA in May 2011 for use in combination with [DB00811], [DB00008], and [DB00022] for the treatment of HCV genotype 1 infection. Due to the advent of interferon-free therapies, Victrelis is no longer widely used. Bosepiril is a hepatitis C virus NS3/4A protease inhibitor. Bosepiril's mechanism of action is as an HCV NS3/4A protease inhibitor, a cytochrome P450 3A4 inhibitor, and a cytochrome P450 3A5 inhibitor. Bosepiril is an oral, direct-acting hepatitis C virus (HCV) protease inhibitor that was previously used in combination with pegylated interferon and ribavirin for the treatment of genotype 1 chronic hepatitis C. It was initially approved in 2012 but was withdrawn from the market in 2015 due to the emergence of more effective and better-tolerated all-oral direct-acting antiviral regimens. Bosapivir was not associated with cases of acute liver injury during treatment, but when used in combination with pegylated interferon and ribavirin, it was associated with cases of liver decompensation in patients with pre-existing cirrhosis. Bosapivir is a highly bioavailable, orally administered synthetic tripeptide inhibitor that inhibits the non-structural protein 3 and 4A complex (NS3/NS4A) and has potential activity against hepatitis C virus (HCV) genotype 1. After administration, bosapivir reversibly binds to the active site of HCV NS3/NS4A and prevents NS3/NS4A protease-mediated polyprotein maturation. This disrupts viral protein processing and the formation of viral replication complexes, thereby inhibiting viral replication in HCV genotype 1 infected host cells. NS3 is a serine protease essential for proteolytic cleavage within the HCV polyprotein and plays a key role in HCV viral RNA replication. NS4A is an activator of NS3. HCV is a small, enveloped, single-stranded RNA virus belonging to the Flaviviridae family.

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Drug Indications
Bosepiramisu, in combination with [DB00811], [DB00008], and [DB00022], is indicated for the treatment of chronic HCV genotype 1 infection in adults.
FDA Label
Victrelis is indicated for the treatment of compensated liver disease in adults with chronic hepatitis C (CHC) genotype 1 infection who have not received prior treatment or have failed prior treatment, in combination with pegylated interferon-alpha and ribavirin.
Treatment of Chronic Hepatitis C

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Mechanism of Action
Bosepiramisu is an NS3/4a protease inhibitor used to inhibit HCV viral replication. NS3/4a proteases are an important component of viral replication, mediating the cleavage of viral-encoded polyproteins into mature proteins (NS4A, NS4B, NS5A, and NS5B). Bosapivir binds covalently but reversibly to a serine residue (S139) at its active site via its α-ketoamide functional group. This inhibits the proteolytic activity of HCV 1a and 1b-encoded enzymes. Bosapivir is a selective inhibitor of the non-structural (NS) 3/4A protease of hepatitis C virus (HCV). It is a direct-acting antiviral drug active against HCV. Bosapivir contains an α-ketoamide functional group that selectively, covalently, and reversibly binds to the active serine site of the HCV NS3 protease. By blocking the proteolytic cleavage of NS4A, NS4B, NS5A, and NS5B in the HCV-encoded polyprotein, the drug inhibits HCV replication in host cells. Bosapivir exhibits in vitro activity against HCV genotypes 1a and 1b, but lower activity against genotypes 2, 2a, and 3a. Bosapivir is an inhibitor of the HCV NS3/4A protease, which is crucial for the hydrolytic cleavage of the HCV-encoded polyprotein into mature NS4A, NS4B, NS5A, and NS5B proteins. Bosapivir inhibits viral replication in host cells by covalently but reversibly binding its α-ketoamide functional group to the serine residue (S139) at the active site of the NS3 protease. In biochemical assays, bosapivir inhibited the activity of recombinant HCV genotypes 1a and 1b NS3/4A proteases, with a Ki value of 14 nM for each subtype. …Bosapivir is a ketoamide protease inhibitor that reversibly binds to the active site of the HCV non-structural protein NS3 protease, thereby inhibiting intracellular viral replication. Phase III clinical studies have shown that bosapivir, in combination with current standard of care, significantly improves sustained virological response rates in both treatment-naïve and previously treated patients with genotype 1 chronic hepatitis C (CHC). ...

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Drug Discovery and Design Process: Boceprevir (EBP-520; SCH-503034) was developed using a structure-based drug design approach. Since initial screening failed to yield a lead compound, an α-ketoamide electrophilic agent was designed to covalently bind to the serine hydroxyl group of the HCV NS3 protease, thereby inhibiting enzyme activity. Early ketoamide inhibitors mimicked the structure of the NS3 protease peptide substrate. Using X-ray crystallography, the lead compound was progressively optimized: starting with an undecapeptide with a molecular weight of 1265 Da, it was progressively truncated to a tripeptide with a molecular weight of 500 Da. To reduce the peptide characteristics of the inhibitor, researchers explored various strategies, such as replacing the amide bond with hydrazine and macrocyclization at the P2-P4 and P1-P3 positions. Further optimization of the tripeptide inhibitor identified the optimal group for each site: P' site was primary ketoamide, P1 site was cyclobutylalanine, P2 site was dimethylcyclopropylproline, P3 site was tert-leucine, and the end capping group was tert-butylurea. The combination of these groups eventually led to the discovery of Boceprevirin and it entered the clinical development stage [1].
- Current status of clinical development: Boceprevirin (EBP-520; SCH-503034) was well tolerated in a phase I clinical trial and showed antiviral activity. As of the time of this publication, the drug is in a phase II clinical trial for the treatment of hepatitis C virus (HCV) infection [1]. Later, the U.S. Food and Drug Administration (FDA) approved its use in combination with pegylated interferon (PegIFN) and ribavirin (RBV) for the treatment of genotype 1 chronic hepatitis C virus (HCV) infection, marking a significant advancement in HCV treatment [4, 5]. Treatment Considerations: In the treatment of HCV relapse in liver transplant patients, Boceprevirin (EBP-520; SCH-503034) is administered at a dose of 800 mg three times daily, starting after a 4-week introductory period of pegylated interferon/ribavirin (PegIFN/RBV) treatment. Due to its interaction with immunosuppressants, plasma concentrations of immunosuppressants need to be closely monitored during combination therapy, and the doses of these drugs should be reduced (e.g., cyclosporine dose reduced by 50%, tacrolimus dose reduced by an even greater margin) to avoid excessive immunosuppression and its associated adverse effects [3].
- Efficacy in specific populations: In African American patients with genotype 1 chronic hepatitis C virus infection, although triple therapy based on bocepivir (EBP-520; SCH-503034) improved the sustained virological response rate (SVR) compared with standard dual therapy, the SVR rate was still lower than that in white patients. Clinical evidence suggests that African American patients may require a longer course of bocepivir-containing therapy to achieve the same SVR rate as white patients. However, due to the small number of African American patients included in early clinical trials, larger sample size studies are needed to accurately assess the efficacy of bocepivir in this population and provide more targeted treatment recommendations [5].

C27H45N5O5

519.68

519.342

C, 62.40; H, 8.73; N, 13.48; O, 15.39

394730-60-0

Boceprevir-d9;1256751-11-7

10324367

Off-white to pale yellow solid powder

1.2±0.1 g/cm3

1.533

2.05

4

5

10

37

959

4

O=C(N1[C@@H]([C@@]2([H])C(C)([C@]2(C1)[H])C)C(NC(C(C(N)=O)=O)CC3CCC3)=O)[C@@H](NC(NC(C)(C)C)=O)C(C)(C)C

LHHCSNFAOIFYRV-DOVBMPENSA-N

InChI=1S/C27H45N5O5/c1-25(2,3)20(30-24(37)31-26(4,5)6)23(36)32-13-15-17(27(15,7)8)18(32)22(35)29-16(19(33)21(28)34)12-14-10-9-11-14/h14-18,20H,9-13H2,1-8H3,(H2,28,34)(H,29,35)(H2,30,31,37)/t15-,16?,17-,18-,20+/m0/s1

(1R,2S,5S)-N-(4-amino-1-cyclobutyl-3,4-dioxobutan-2-yl)-3-((S)-2-(3-(tert-butyl)ureido)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

EBP 520; EBP-520; EBP520; SCH-503034; SCH503034; SCH 503034; trade name: Victrelis;

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)

DMSO : 16.67 ~100 mg/mL ( 32.08~192.42 mM )
H2O : < 0.1 mg/mL
Ethanol : ~100 mg/mL

Solubility in Formulation 1: ≥ 1.67 mg/mL (3.21 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 1.67 mg/mL (3.21 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 1.67 mg/mL (3.21 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 1.67 mg/mL (3.21 mM)

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