Telaprevir ( LY-570310; VX950; MP-424)

genotype 1 HCV NS3-4A protease (Ki = 7 nM)

Telaprevir inhibits the NS3-4A serine protease of the hepatitis C virus, which stops the virus's ability to process polyproteins. This, in turn, causes a time- and dose-dependent decrease in viral RNA replication, total HCV RNA levels, and protein levels in Con1 (genotype 1b) subgenomic HCV replicon cells. Telaprevir exhibits a noteworthy increase in inhibitory effect on HCV RNA replication over time, with IC50 values for 24, 48, 72, and 120 hours of incubation, respectively, of 0.574 μM, 0.488 μM, 0.210 μM, and 0.139 μM. Three separate experiments using a 48-hour incubation period show that telaprevir has an average IC50 of 0.354 μM and an average IC90 of 0.830 μM, respectively. Telaprevir has no appreciable cytotoxicity against HepG2, parental Huh-7, or HCV replicon cells after 48 hours of incubation. After incubating for 13 days without any rebound, replicon cells are completely free of HCV RNA when telaprevir (17.5 μM) is withdrawn. In comparison to treatment with each agent alone, telaprevir and IFN-α exhibit an additive to moderate synergistic effect on suppression of resistance mutations and reduction of HCV RNA replication without a discernible increase in cytotoxicity.[1]

In the mice model, oral telaprevir administration decreases HCV protease-dependent cleavage and subsequent liver-secreted SEAP into the blood to 18.7% and 18.4% at dosages of 10 and 25 mg/kg, respectively. [/2] In HCV-infected human hepatocyte chimeric mice with genotype 1b, administration of Telaprevir at 200 mg/kg for one week causes a 1.9 log reduction in HCV RNA; when treatment is combined with MK-0608 (50 mg/kg) for four weeks, viruses are eradicated from the mice.[3]

Stable Huh-7 cells containing the self-replicating, subgenomic HCV replicon, which is identical in sequence to the I377neo/NS3-3'/wt replicon are used for anti-HCV assays. Telaprevir serially diluted in DMEM plus 2% FBS and 0.5% dimethyl sulfoxide (DMSO) is incubated with replica cells at 37 °C for the specified amount of time. Using an RNeasy-96 kit, total cellular RNA is extracted, and a quantitative real-time polymerase chain reaction (QRT-PCR) assay is used to determine the copy number of HCV RNA in order to assess the 50% inhibitory concentration (IC50).

Evaluating Telaprevir (VX-950) or IFN-α in HCV replicon cells involves determining its IC50, IC90, and CC50. To sum up, 96-well plates are plated with 1×10⁴ replicon cells each well. Using antiviral agents serially diluted in DMEM plus 2% FBS and 0.5% DMSO, replicon cells are incubated at 37°C for the specified amount of time on the following day. With an RNeasy-96 kit, total cellular RNA is extracted, and a quantitative RT-PCR (QRT-PCR) assay is used to calculate the copy number of HCV RNA. The mean of five replicates in cell culture is represented by each datum point. A tetrazolium (MTS)-based cell viability assay is used to measure the cytotoxicity of Telaprevir under the same experimental conditions. One million parental Huh-7 cells or four million HepG2 cells per well are used in the cytotoxicity assay using human hepatocyte cell lines. In order to assess the cytotoxicity of Telaprevir against resting peripheral blood monoclonal cells, 1×10⁵ cells per well are cultured with Telaprevir in RPMI-1640 medium (without serum) for 48 hours, after which the MTS-based assay is used to determine the cell viability. Precoated with anti-human CD3 antibody, a 96-well plate is filled with 1×10⁵ cells per well in RPMI-1640 medium to test the cytotoxicity of VX-950 against proliferating PBMC. The cells are cultured for 72 hours at 37°C with Telaprevir and anti-human CD28 antibody. The [³H]thymidine update is used to measure the cell growth between the 48th and 72nd hours[1].

Mice: Recombinant adenovirus Ad-WT-HCVpro-SEAP, with 10⁹ IFU per mouse, is injected via the tail vein into five groups of six-week-old SCID mice (six animals per group). Two oral doses of Telaprevir (VX-950) at a dose of 10, 25, 75, 150, or 300 mg/kg are administered to each group of mice. First dose of Telaprevir is administered two hours prior to adenovirus injection; second dose is administered ten hours following injection. A second set of ten mice is given the vehicle on its own. Serum samples are taken 24 hours after injection, and the SEAP activity in each group administered with Telaprevir is contrasted with the vehicle group's. Rat and Canine Rats and dogs are used to assess the oral and intravenous pharmacokinetics of telaprevir (VX-950). One intravenous bolus dose of 0.95 mg/kg Telaprevir is given intravenously to three male Sprague-Dawley rats weighing 250–300 g. Heparinized tubes are used to collect serial blood samples prior to dosage administration and at intervals of 0.083, 0.167, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours following the dose. Telaprevir in 10% ethanol, 40% polyethylene glycol 400, and 50% D5W is given intravenously as a bolus dose to three male beagle dogs (8–12 kg). Heparinized tubes are used to collect serial blood samples prior to dosage administration as well as at 0.083, 0.167, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 hours later. Telaprevir is formulated in polyvinylpyrrolidone (PVP) K-30 plus 2% sodium lauryl sulfate and dosed as an oral gavage for oral studies in rats and dogs. Oral dosages of 40 mg/kg VX-950 are given to three male Sprague-Dawley rats (250–300 g) and 9.6 mg/kg VX-950 are given to four male Beagle dogs (10.9–12.0 kg). Blood samples are obtained before dosage administration and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours following dose administration in both oral studies. Plasma samples are obtained by centrifugation and kept at -70°C until analysis in both intravenous and oral studies. Samples from the oral studies are analyzed using an achiral LC/MS/MS method, while samples from the intravenous studies are analyzed using a chiral liquid chromatography followed by tandem mass spectrometry (LC/MS/MS) method.

[1]. Antimicrob Agents Chemother . 2006 May;50(5):1813-22.

[2]. Antimicrob Agents Chemother . 2006 Mar;50(3):899-909.

[3]. J Hepatol . 2011 May;54(5):872-8.

C36H53N7O6

679.85

679.41

C, 63.60; H, 7.86; N, 14.42; O, 14.12

402957-28-2

Telaprevir-d4

solid powder

CCC[C@@H](C(=O)C(=O)NC1CC1)NC(=O)[C@@H]2[C@H]3CCC[C@H]3CN2C(=O)[C@H](C(C)(C)C)NC(=O)[C@H](C4CCCCC4)NC(=O)C5=NC=CN=C5

BBAWEDCPNXPBQM-GDEBMMAJSA-N

InChI=1S/C36H53N7O6/c1-5-10-25(29(44)34(48)39-23-15-16-23)40-33(47)28-24-14-9-13-22(24)20-43(28)35(49)30(36(2,3)4)42-32(46)27(21-11-7-6-8-12-21)41-31(45)26-19-37-17-18-38-26/h17-19,21-25,27-28,30H,5-16,20H2,1-4H3,(H,39,48)(H,40,47)(H,41,45)(H,42,46)/t22-,24-,25-,27-,28-,30+/m0/s1

(3S,3aS,6aR)-2-[(2S)-2-[[(2S)-2-cyclohexyl-2-(pyrazine-2-carbonylamino)acetyl]amino]-3,3-dimethylbutanoyl]-N-[(3S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl]-3,3a,4,5,6,6a-hexahydro-1H-cyclopenta[c]pyrrole-3-carboxamide

VX-950; LY-570310; MP-424; VX950; LY570310; MP424; VX 950; LY 570310; MP 424; trade names: Incivek; Incivo

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)