HCV NS3/4A protease(IC50: 3.5~11.3 nM)
Hepatitis C virus (HCV) NS3/4A protease (IC₅₀ = 3.5 to 11.3 nM; EC₅₀ = 0.21 to 4.6 nM for HCV replicons of genotypes 1-6; median EC₅₀ = 0.30 nM for replicons from 40 patient samples of genotypes 1-5) [1]
Hepatitis C virus (HCV) NS3/4A protease (formerly ABT-493) [2]

Glecaprevir (formerly ABT-493) is a novel hepatitis C virus (HCV) NS3/4A protease inhibitor (PI) with pangenotypic activity. It inhibited the enzymatic activity of purified NS3/4A proteases from HCV genotypes 1 to 6 in vitro (half-maximal [50%] inhibitory concentration = 3.5 to 11.3 nM) and the replication of stable HCV subgenomic replicons containing proteases from genotypes 1 to 6 (50% effective concentration [EC50] = 0.21 to 4.6 nM). Glecaprevir had a median EC50 of 0.30 nM (range, 0.05 to 3.8 nM) for HCV replicons containing proteases from 40 samples from patients infected with HCV genotypes 1 to 5. Importantly, glecaprevir was active against the protease from genotype 3, the most-difficult-to-treat HCV genotype, in both enzymatic and replicon assays demonstrating comparable activity against the other HCV genotypes. In drug-resistant colony selection studies, glecaprevir generally selected substitutions at NS3 amino acid position A156 in replicons containing proteases from genotypes 1a, 1b, 2a, 2b, 3a, and 4a and substitutions at position D/Q168 in replicons containing proteases from genotypes 3a, 5a, and 6a. Although the substitutions A156T and A156V in NS3 of genotype 1 reduced susceptibility to glecaprevir, replicons with these substitutions demonstrated a low replication efficiency in vitro Glecaprevir is active against HCV with most of the common NS3 amino acid substitutions that are associated with reduced susceptibility to other currently approved HCV PIs, including those at positions 155 and 168. Combination of glecaprevir with HCV inhibitors with other mechanisms of action resulted in additive or synergistic antiviral activity. In summary, glecaprevir is a next-generation HCV PI with potent pangenotypic activity and a high barrier to the development of resistance.

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Glecaprevir inhibited the enzymatic activity of purified HCV NS3/4A proteases from genotypes 1 to 6 with IC₅₀ values ranging from 3.5 to 11.3 nM. It also suppressed the replication of stable HCV subgenomic replicons containing proteases from genotypes 1 to 6, with EC₅₀ values of 0.21 to 4.6 nM. For replicons from 40 patient samples infected with HCV genotypes 1 to 5, the median EC₅₀ was 0.30 nM (range: 0.05 to 3.8 nM), showing potent activity against genotype 3 (a difficult-to-treat genotype) comparable to other genotypes. In drug-resistant colony selection studies, it selected substitutions at NS3 position A156 in replicons of genotypes 1a, 1b, 2a, 2b, 3a, and 4a, and substitutions at D/Q168 in genotypes 3a, 5a, and 6a. Substitutions A156T and A156V in genotype 1 reduced susceptibility but impaired replicon replication efficiency. It was active against HCV with common NS3 substitutions (e.g., positions 155 and 168) associated with resistance to other HCV protease inhibitors. Combination with other HCV inhibitors showed additive or synergistic antiviral activity [1]

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In an open-label dose-ranging trial, treatment-naive adults with HCV genotype 1 infection (with or without compensated cirrhosis) received 3-day monotherapy with glecaprevir (doses: 100 mg, 200 mg, 300 mg, 400 mg, 700 mg). The mean maximal decrease in HCV RNA levels from baseline was approximately 4 log₁₀ IU/ml across all doses. Viral load declines were similar in patients with and without compensated cirrhosis. Fifty percent of baseline samples had NS3/4A protease inhibitor resistance-associated variants (RAVs), but viral load reductions remained consistent in these patients [2]

Glecaprevir inhibited the enzymatic activity of HCV genotype 1 to 6 NS3/4A proteases, with the half-maximal (50%) inhibitory concentration (IC50) values ranging from 3.5 to 11.3 nM in a biochemical assay. When glecaprevir was tested against six human serine proteases (chymase, chymotrypsin type II, chymotrypsin type VII, elastase, kallikrein, and urokinase) and one human cysteine protease (cathepsin B), no inhibition was observed at concentrations up to 200,000 nM. These results indicate that glecaprevir demonstrates a high level of selectivity for the HCV NS3/4A protease over the human proteases tested.

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Purified HCV NS3/4A proteases from genotypes 1 to 6 were prepared, and the enzymatic inhibition activity of glecaprevir was measured. The assay was conducted to determine the half-maximal inhibitory concentration (IC₅₀) by assessing the ability of the drug to block protease activity, with results ranging from 3.5 to 11.3 nM for different genotypes [1]

HCV subgenomic replicon cells containing the protease from genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a, or 6a were passaged in the presence of glecaprevir at concentrations 10- or 100-fold its EC50s for the respective replicon cell lines to select for colonies with substitutions that conferred resistance to glecaprevir. Glecaprevir generally selected a low number of colonies containing resistance-conferring substitutions in replicon cells across the different HCV genotypes. Higher colony counts were observed with selections using replicons with the genotype 2a JFH-1 backbone (i.e., the genotype 2a and 2b replicons in this study), regardless of the selection concentrations. This was likely due to the exceptionally high replication rates for replicons with this backbone.

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Stable HCV subgenomic replicon cell lines containing proteases from genotypes 1 to 6 were established. These cells were treated with various concentrations of glecaprevir, and the 50% effective concentration (EC₅₀) for inhibiting viral replication was determined, with values ranging from 0.21 to 4.6 nM. Additionally, replicon cells from 40 patient samples (genotypes 1-5) were used to evaluate the drug’s activity, yielding a median EC₅₀ of 0.30 nM. Drug-resistant colony selection was performed by treating genotype 1a and 1b replicon cells with glecaprevir (10-fold EC₅₀) plus G418 (400 μg/ml) for approximately 3 weeks; surviving colonies were fixed, stained with crystal violet, and counted [1]

Absorption, Distribution and Excretion
In healthy subjects, the time to peak plasma concentration (Tmax) is approximately 5 hours. The mean peak plasma concentration (Cmax) in non-cirrhotic HCV-infected individuals is 597 ng/mL. Food intake increases gucarrevir absorption by 83-163% compared to a fasting state. The primary route of excretion is the bile-fecal route, with 92.1% of the administered drug excreted in feces and 0.7% in urine. Metabolism/Metabolites Gucarrevir undergoes limited secondary metabolism primarily via CYP3A in vitro. Biological Half-Life The elimination half-life (t1/2) is approximately 6 hours.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Gcaprevir has not been studied in breastfeeding women receiving treatment for hepatitis C. Because it binds to maternal plasma proteins at a rate as high as 97.5%, its concentration in breast milk is likely to be very low.
Hepatitis C is not transmitted through breast milk, and breast milk has been shown to inactivate the hepatitis C virus (HCV). However, the U.S. Centers for Disease Control and Prevention (CDC) recommends that mothers infected with hepatitis C should consider discontinuing breastfeeding if they experience nipple fissures or bleeding. It is unclear whether this warning applies to mothers receiving treatment for hepatitis C.
Infants born to mothers infected with hepatitis C should be tested for hepatitis C; nucleic acid testing is recommended because maternal antibodies are present in the infant for the first 18 months after birth and until the infant develops an immune response.
◉ Impact on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
Pirentavir binds to human plasma proteins in 97.5%. The plasma-to-serum ratio is approximately 0.57.

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In a 3-day monotherapy clinical trial, no serious or grade 3 (severe) adverse events were observed in patients treated with gliclazorvir at doses ranging from 100 mg to 700 mg. No clinically significant laboratory abnormalities were reported [2].

[1]. Antimicrob Agents Chemother.2017;62(1). pii: e01620-17.

[2]. Antimicrob Agents Chemother.2015;60(3):1546-55.

[3]. Signal Transduct Target Ther. 2021 May 29;6(1):212.

Glecaprevir is a direct-acting antiviral drug and an inhibitor of the hepatitis C virus (HCV) NS3/4A protease, targeting viral RNA replication. When used in combination with [DB13878], lecaprevir is effective in treating patients who have failed other NS3/4A protease inhibitors. It has a high genetic barrier against viral resistance mutations. In cell culture, amino acid substitutions at the NS3 resistance-related sites A156 or D/Q168 in HCV genotype 1a, 2a, or 3a replicons lead to decreased sensitivity to lecaprevir. Combinations of amino acid substitutions at the NS3 site Y65H and D/Q168 also result in further decreased lecaprevir sensitivity, and the NS3 Q80R mutation in genotype 3a patients also leads to lecaprevir resistance. Glecaprevir and [DB13878] are marketed as an oral combination under the brand name Mavyret. This fixed-dose combination therapy was approved by the FDA in August 2017 for the treatment of adult patients with chronic hepatitis C virus (HCV) genotypes 1-6 infection without cirrhosis (liver disease) or with mild cirrhosis, including patients with moderate to severe renal disease and those undergoing dialysis. Mavyret is also indicated for patients with HCV genotype 1 infection who have previously received a regimen containing an NS5A inhibitor or an NS3/4A protease inhibitor (but not simultaneously). Hepatitis C virus infection often leads to decreased liver function and eventually liver failure, severely impacting patients' quality of life. The ultimate goal of this combination therapy is to achieve sustained virological response (SVR) and cure hepatitis C virus infection. In clinical trials, this combination therapy achieved an SVR12 rate of ≥93% in genotypes 1a, 2a, 3a, 4, 5, and 6, meaning undetectable hepatitis C virus for 12 weeks or longer after the end of treatment.
Gelacaprevir is a hepatitis C virus NS3/4A protease inhibitor. Glecaprevir's mechanism of action is as an HCV NS3/4A protease inhibitor, P-glycoprotein inhibitor, breast cancer resistance protein inhibitor, organic anion transporter 1B1 inhibitor, organic anion transporter 1B3 inhibitor, cytochrome P450 3A inhibitor, cytochrome P450 1A2 inhibitor, and UGT1A1 inhibitor. Indications: It is indicated for the treatment of adult patients with chronic hepatitis C virus (HCV) genotype 1, 2, 3, 4, 5, or 6 who have no cirrhosis or compensated cirrhosis (Child-Pugh A). MAVYRET is also indicated for the treatment of adult patients with HCV genotype 1 infection who have previously received a treatment regimen containing an HCV NS5A inhibitor or an NS3/4A protease inhibitor (PI) (but not concurrently with either inhibitor).
FDA Label
Maviret is indicated for the treatment of chronic hepatitis C virus (HCV) infection in adults aged 3 years and older and children. Maviret coated granules are indicated for the treatment of chronic hepatitis C virus (HCV) infection in children aged 3 years and older.

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Mechanism of Action
Gaviret is an inhibitor of the HCV NS3/4A protease, a viral enzyme responsible for hydrolyzing the HCV-encoded polyprotein into mature NS3, NS4A, NS4B, NS5A, and NS5B proteins. These multifunctional proteins, including NS3, are essential for viral replication. The N-terminus of the NS3 protein confers its serine protease activity, while its C-terminus encodes a DExH/D-box RNA helicase that hydrolyzes NTPs for energy, unwinding double-stranded RNA along the 3′ to 5′ direction during viral genome RNA replication. NS4A is a cofactor of NS3, guiding NS3 localization and regulating its enzymatic activity. Gcaprevir interferes with intracellular processes of the viral life cycle by inhibiting NS3/4A protease activity, blocking the cleavage of downstream HCV peptide linkages and the proteolytic processing of mature structural proteins.

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Gcaprevir (formerly known as ABT-493) is a new generation HCV NS3/4A protease inhibitor with pangenotypic antiviral activity[1][2]
Gcaprevir has a high resistance barrier due to its activity against common drug-resistant variants and its inhibition of the replication efficiency of drug-resistant HCV replicons[1]
This drug has been used in a phase II clinical trial in combination with other direct-acting antiviral drugs to treat patients with chronic HCV infection with or without compensated cirrhosis[2]

C38H46F4N6O9S

838.87

838.298

C, 54.41; H, 5.53; F, 9.06; N, 10.02; O, 17.16; S, 3.82

1365970-03-1

1365970-03-1 (free);1838572-01-2 (hydrate);

66828839

White to off-white solid powder

1.5±0.1 g/cm3

1.610

1.71

3

15

7

58

1760

7

S(C1(C)CC1)(NC([C@]1(C[C@H]1C(F)F)NC([C@@H]1C[C@@H]2CN1C([C@H](C(C)(C)C)NC(=O)O[C@@H]1CCC[C@H]1OCC=CC(C1C(=NC3C=CC=CC=3N=1)O2)(F)F)=O)=O)=O)(=O)=O |c:41|

MLSQGNCUYAMAHD-ITNVBOSISA-N

InChI=1S/C38H46F4N6O9S/c1-35(2,3)28-32(50)48-19-20(17-24(48)30(49)46-37(18-21(37)29(39)40)33(51)47-58(53,54)36(4)14-15-36)56-31-27(43-22-9-5-6-10-23(22)44-31)38(41,42)13-8-16-55-25-11-7-12-26(25)57-34(52)45-28/h5-6,8-10,13,20-21,24-26,28-29H,7,11-12,14-19H2,1-4H3,(H,45,52)(H,46,49)(H,47,51)/b13-8+/t20-,21+,24+,25-,26-,28-,37-/m1/s1

(3aR,7S,10S,12R,21E,24aR)-7-tert-butyl-N-{(1R,2R)-2(difluoromethyl)-1-[(1-methylcyclopropane-1-sulfonyl)carbamoyl]cyclopropyl}-20,20-difluoro5,8-dioxo-2,3,3a,5,6,7,8,11,12,20,23,24a-dodecahydro-1H,10H-9,12methanocyclopenta[18,19][1,10,17,3,6]trioxadiazacyclononadecino[11,12-b]quinoxaline-10carboxamide

ABT-493; ABT493; ABT 493; A-1282576; A 1282576; A1282576; A-1282576.0; Glecaprevir; MAVYRET.

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 : 83.3 ~100 mg/mL ( 99.30 ~119.2 mM )
Ethanol : ~100 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.48 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 20.8 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: ≥ 2.08 mg/mL (2.48 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.08 mg/mL (2.48 mM)

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