HCV NS3 protease(Ki=14 nM)

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].

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].

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.

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]

Absorption, Distribution and Excretion
Boceprevir reaches peak plasma concentration 2 hours after administration. Absolute bioavailability has not been determined. When taken with food exposure increases up to 65%. In capsule, Boceprevir consists of two diaseromers in a 1:1 ratio. In plasma this ratio changes to 2:1 favoring the active diastereomer.
Boceprevir is mainly eliminated in the feces (79%) with a small amount eliminated in the urine (9%). Approximately 8% and 3% is excreted as the parent compound in the feces and urine respectively.
The mean apparent volume of distribution for Bocepravir is 772 litres at steady state.
Boceprevir has a mean total body clearance of 161 liters per hour.
In healthy subjects who received 800 mg three times daily alone, boceprevir drug exposure was characterized by AUC(T) of 5408 ng. hr per mL (n=71), Cmax of 1723 ng per mL (n=71), and Cmin of 88 ng per mL (n=71). Pharmacokinetic results were similar between healthy subjects and HCV-infected subjects.
Boceprevir was absorbed following oral administration with a median Tmax of 2 hours. Steady state AUC, Cmax, and Cmin increased in a less-than-dose-proportional manner and individual exposures overlapped substantially at 800 mg and 1200 mg, suggesting diminished absorption at higher doses. Accumulation is minimal (0.8- to 1.5-fold) and pharmacokinetic steady state is achieved after approximately 1 day of three times daily dosing.
Boceprevir should be administered with food. Food enhanced the exposure of boceprevir by up to 65% at the 800 mg three times daily dose, relative to the fasting state. The bioavailability of boceprevir was similar regardless of meal type (e.g., high-fat vs. low-fat) or whether taken 5 minutes prior to eating, during a meal, or immediately following completion of the meal. Therefore, boceprevir may be taken without regard to either meal type or timing of the meal.
Boceprevir has a mean apparent volume of distribution (Vd/F) of approximately 772 L at steady state in healthy subjects.
For more Absorption, Distribution and Excretion (Complete) data for Boceprevir (10 total), please visit the HSDB record page.

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Metabolism / Metabolites
Bocepravir is primarily metabolized via the aldo-ketoreductase-mediated pathway producing a diastereomeric mix of metabolites at a 4 fold greater exposure than the parent compound. Boceprevir also undergoes oxidative metabolism via CYP3A4/5, although to a lesser extent.
Studies in vitro indicate that boceprevir primarily undergoes metabolism through the aldo-ketoreductase (AKR)-mediated pathway to ketone-reduced metabolites that are inactive against HCV. After a single 800-mg oral dose of (14)C-boceprevir, the most abundant circulating metabolites were a diasteriomeric mixture of ketone-reduced metabolites with a mean exposure approximately 4-fold greater than that of boceprevir. Boceprevir also undergoes, to a lesser extent, oxidative metabolism mediated by CYP3A4/5.

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Biological Half-Life
Boceprevir has a mean half-life of elimination of 3.4 hours.
Boceprevir is eliminated with a mean plasma half-life (t1/2) of approximately 3.4 hours.

Hepatotoxicity
In large randomized controlled trials, triple therapy with boceprevir, peginterferon and ribavirin was associated with a high rate of adverse events that often required dose adjustments and led to early discontinuation in 5% to 20% of patients. However, serum ALT elevations and clinically apparent liver injury were not generally mentioned as adverse events of therapy. The exception to this occurred in patients with preexisting cirrhosis in whom de novo, seemingly spontaneous hepatic decompensation occurred in a proportion of treated subjects. The cause of the decompensation was not clear and the separate role of boceprevir from peginterferon and ribavirin and from what might happen even without therapy could not be easily defined. Nevertheless, in postmarketing studies of triple therapy of chronic hepatitis C with cirrhosis, decompensation was reported in 3% to 8% of patients and deaths from hepatic failure in 1% to 3%.
Likelihood score for the combination of boceprevir, peginterferon and ribavirin: B (likely cause of liver injury and hepatic decompensation in patients with preexisting cirrhosis or advanced fibrosis).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Boceprevir has been removed from the US market. It has not been studied in nursing mothers being treated for hepatitis C infection. Because it must be used with ribavirin and peginterferon alfa, it is not considered a good choice during breastfeeding. Until more data become available, an alternate drug may be preferred, especially while nursing a newborn or preterm infant.
Hepatitis C is not transmitted through breastmilk and breastmilk has been shown to inactivate hepatitis C virus (HCV). However, the Centers for Disease Control recommends that mothers with HCV infection should consider abstaining from breastfeeding if their nipples are cracked or bleeding. It is not clear if this warning would apply to mothers who are being treated for hepatitis C.
Infants born to mothers with HCV infection should be tested for HCV infection; because maternal antibody is present for the first 18 months of life and before the infant mounts an immunologic response, nucleic acid testing is recommended.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Bocepravir is approximately 75% bound to human plasma proteins following a single dose.

[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 consisting of N-(tert-butylcarbamoyl)-3-methyl-L-valyl, a cyclopropyl-fused prolyl and 3-amino-4-cyclobutyl-2-oxobutanamide residues joined in sequence. Used for treatment of chronic hepatitis C virus genotype 1 infection. It has a role as a hepatitis C protease inhibitor, a peptidomimetic and an antiviral drug. It is a tripeptide and a member of ureas.
Boceprevir is a direct acting antiviral medication used as part of combination therapy to treat chronic Hepatitis C, an infectious liver disease caused by infection with Hepatitis C Virus (HCV). HCV is a single-stranded RNA virus that is categorized into nine distinct genotypes, with genotype 1 being the most common in the United States, and affecting 72% of all chronic HCV patients. Treatment options for chronic Hepatitis C have advanced significantly since 2011, with the development of Direct Acting Antivirals (DAAs) such as Boceprevir. Boceprevir is an inhibitor of NS3/4A, a serine protease enzyme, encoded by HCV genotypes 1 and 4 [synthesis]. These enzymes are essential for viral replication and serve to cleave the virally encoded polyprotein into mature proteins like NS4A, NS4B, NS5A and NS5B. The barrier for develoment of resistance to NS3/4A inhibitors is lower than that of NS5B inhibitors, another class of DAAs. Subtitutions at amino acid positions 155, 156, or 168 are known to confer resistance. The substitutions of the enzyme's catalytic triad consisting of H58, D82, and S139 are also likely to alter the affinity of the drug for NS3/4A or the activity of the enzyme itself. Despite this disadvantage Boceprevir is still effective against HCV when paired with [DB00811], [DB00008], and [DB00022]. In a joint recommendation published in 2016, the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA) do not reccomend Boceprevir in combination with [DB00811], [DB00008], and [DB00022] as first line therapy for Hepatitis C. Boceprevir, [DB00811], [DB00008], and [DB00022] are used with the intent to cure, or achieve a sustained virologic response (SVR), after 48 weeks of daily therapy. SVR and eradication of HCV infection is 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 available as a fixed dose product (tradename Victrelis) used for the treatment of chronic Hepatitis C. Approved in May 2011 by the FDA, Victrelis is indicated for the treatment of HCV genotype 1 in combination with [DB00811], [DB00008], and [DB00022]. Victrelis is no longer widely used as interferon-free therapies have been developed.
Boceprevir is a Hepatitis C Virus NS3/4A Protease Inhibitor. The mechanism of action of boceprevir is as a HCV NS3/4A Protease Inhibitor, and Cytochrome P450 3A4 Inhibitor, and Cytochrome P450 3A5 Inhibitor.
Boceprevir is an oral, direct acting hepatitis C virus (HCV) protease inhibitor that was used in combination with peginterferon and ribavirin in the treatment of chronic hepatitis C, genotype 1. Initially approved for use in 2012, it was withdrawn in 2015 because of the availability of more effective and better tolerated all oral regimens of direct acting antiviral agents. Boceprevir was not linked to instances of acute liver injury during therapy but, when combined with peginterferon and ribavirin, was associated with cases of hepatic decompensation in patients with preexisting cirrhosis.
Boceprevir is an orally bioavailable, synthetic tripeptide inhibitor of the nonstructural protein 3 and 4A complex (NS3/NS4A), with potential activity against hepatitis C virus (HCV) genotype 1. Upon administration, boceprevir reversibly binds to the active center of the HCV NS3/NS4A and prevents NS3/NS4A protease-mediated polyprotein maturation. This disrupts the processing of viral proteins and the formation of a viral replication complex, which inhibits viral replication in HCV genotrype 1-infected host cells. NS3, a serine protease, is essential for the proteolytic cleavages within the HCV polyprotein and plays a key role during HCV viral RNA replication. NS4A is an activating factor for NS3. HCV is a small, enveloped, single-stranded RNA virus belonging to the Flaviviridae family.

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Drug Indication
Boceprevir, when used in combination with [DB00811], [DB00008], and [DB00022] is indicated for use in the treatment of chronic HCV genotype 1 infection in adults.
FDA Label
Victrelis is indicated for the treatment of chronic hepatitis-C (CHC) genotype-1 infection, in combination with peginterferon alfa and ribavirin, in adult patients with compensated liver disease who are previously untreated or who have failed previous therapy.
Treatment of chronic hepatitis C

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Mechanism of Action
Boceprevir is a NS3/4a protease inhibitor used to inhibit viral HCV replication. NS3/4a protease is an integral part of viral replication and mediates the cleavage the virally encoded polyprotein to mature proteins (NS4A, NS4B, NS5A and NS5B). Boceprevir covalently but reversibly binds the serine (S139) resiude in the active site via a (α)-ketoamide functional group. This inhibits the proteolytic acitvity of the HCV 1a and 1b encoded enzyme.
Boceprevir is a selective hepatitis C virus (HCV) nonstructural (NS) 3/4A protease inhibitor. The drug is a direct-acting antiviral agent with activity against HCV. Boceprevir contains an alpha-ketoamide functional group that selectively, covalently, and reversibly binds the active serine site of HCV NS3 protease. By blocking proteolytic cleavage of NS4A, NS4B, NS5A, and NS5B from the HCV-encoded polyprotein, the drug inhibits HCV replication in host cells. Boceprevir has in vitro activity against HCV genotypes 1a and 1b, but is less active against genotypes 2, 2a, and 3a.
Boceprevir is an inhibitor of the HCV NS3/4A protease that is necessary for the proteolytic cleavage of the HCV encoded polyprotein into mature forms of the NS4A, NS4B, NS5A and NS5B proteins. Boceprevir covalently, yet reversibly, binds to the NS3 protease active site serine (S139) through an (alpha)-ketoamide functional group to inhibit viral replication in HCV-infected host cells. In a biochemical assay, boceprevir inhibited the activity of recombinant HCV genotype 1a and 1b NS3/4A protease enzymes, with Ki values of 14 nM for each subtype.
... Boceprevir is a ketoamide protease inhibitor that binds reversibly to the HCV nonstructural NS3 protease active site inhibiting intracellular viral replication. Phase III clinical studies have demonstrated that, in combination with the current standard of care, boceprevir significantly increases the a sustained virological response rate in both treatment-naive and previously treated patients with genotype 1 CHC. ...

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.)