Table 2.6.3.2: Primary Pharmacodynamics
Test Article: Asunaprevir (BMS-650032) In Vitro
Type of Study Test System Noteworthy Findings Testing
Facility Report No. Location in Dossier Anti-HCV NS3
protease activity Measured activity against purified recombinant HCV NS3/4A protease by monitoring the cleavage of a FRET-labeled peptide substrate loosely based on a cleavage site of authentic polyprotein
ASV exhibited IC50 values of 0.3 to 320 nM against purified NS3/4A protease enzyme complexes generated from 9 strains representative of each of the 6 major genotypes. In a survey of 3 strains representative of GT-1, the compound exhibited IC50 values ranging from 0.3 to 1.8 nM. GT-2 and GT-3 representatives were least susceptible (IC50 values ranging from 15 to 320 nM). GT-4, GT-5, and GT-6 representatives exhibited IC50 values ranging from 0.9 to 1.7 nM.
BMS 930023123 CTD 4.2.1.1.1
Selectivity for HCV
NS3 protease Measured activity of respective purified recombinant proteases by monitoring the cleavage of their respective substrates
ASV exerted minimal to no activity against the closely related viral serine protease, GBV-B NS3 protease, and a panel of 10 human serine and cysteine proteases.
BMS 930023123 CTD 4.2.1.1.1
Anti-HCV activity in
cell culture Measured luciferase or FRET signals in HuH-7 cells either constitutively or transiently expressing HCV (hybrid) replicons
ASV exhibited EC50 values ranging from 1 to 230 nM against HCV replicons representing GT-1a, GT-1b, and GT-2a. Against GT-2b and GT-3a NS3 protease replicon hybrids, ASV exhibited EC50 values of 0.48 μM and 1.2 μM, respectively. Against genotype 4 NS3 protease replicon hybrids, ASV exhibited EC50 values ranging from 1.8 to 7.6 nM.
BMS 930023123
930057191 CTD 4.2.1.1.1 CTD 4.2.1.1.6
Human serum effect on
anti-HCV potency Measured HCV RNA replication in HuH-7 cells constitutively expressing subgenomic HCV replicons
ASV activity was moderately attenuated in the presence of 40% human serum (6.5-fold increase in EC50 value against a replicon representing genotype subtype 1b).
BMS 930023123 CTD 4.2.1.1.1
Cytotoxicity Proliferation assays against a panel
of cell lines CC50 values ranging from 11 to 38 μM were obtained against 6 human cell lines derived from T-cells, liver, lung, cervix, and kidney cells.
BMS 930023123 CTD 4.2.1.1.1
Anti-HCV viral
activity in cell culture Measured HCV RNA replication in HuH-7 cells infected with a full length infectious virus construct
ASV exhibited an EC50 value of 39 nM against HCV infectious virus representing a genotype 2a strain.
BMS 930023123 CTD 4.2.1.1.1
Table 2.6.3.2: Primary Pharmacodynamics
Test Article: Asunaprevir (BMS-650032) In Vitro
Type of Study Test System Noteworthy Findings Testing
Facility Report No. Location in Dossier Selectivity for HCV in
cell culture Antiviral assays performed using 4
different RNA viruses ASV is a selective inhibitor of HCV genotype 1 RNA replication (EC50 1.2 to 4 nM);
showed no significant activity against BVDV (EC50 = 12 μM), HIV (EC50 = 14 μM), HRV (EC50 > 100 μM) or HCoV (EC50 > 100 μM).
BMS 930023123 CTD 4.2.1.1.1
Mode of action against
the HCV NS3 protease Characterized activity using purified recombinant HCV NS3/4A protease complex and monitoring the cleavage of a FRET-labeled peptide substrate
ASV competitively inhibited the binding of substrate to NS3/4A protease complex in a continuous FRET assay. ASV bound directly and reversibly to the protease, Ki 0.24 to 1.0 nM, depending on the genotype strain employed.
BMS 930023124 CTD 4.2.1.1.2
Resistance Cell culture passage of HCV replicons in the presence of ASV;
luciferase activity monitored in HuH-7 cells either constitutively or transiently expressing HCV replicon variants
HCV GT-1a and GT-1b replicons with reduced susceptibility to ASV were selected.
In genotype 1a replicons, the following substitutions were observed in the NS3 protease domain: T40A, Q41E, V51A, R62K, D79E, T95A, I114V, R123G, R155K, D168G, I170T, N174Y, L175P, G176E. NS3 protease substitution R155K, when introduced into recombinant HCV replicons, conferred resistance to ASV in a cell-based assay. The level of resistance was moderate for this substitution (21-fold loss in EC50 value compared with parent GT-1a replicon). In GT-1b replicons, the following substitutions were observed in the NS3 protease domain:
Q41R, Q80R, Q86R, P89L, Y105C, D168A/G/H/V/Y, E173G, and E176G. NS3 protease substitution D168V, when introduced into recombinant HCV replicons, conferred resistance to ASV in a cell-based assay. The level of resistance was high for this substitution (280-fold loss in EC50 value compared with parent GT-1b replicon).
BMS 930023124
930035268 CTD 4.2.1.1.2 CTD 4.2.1.1.3
Table 2.6.3.2: Primary Pharmacodynamics
Test Article: Asunaprevir (BMS-650032) In Vitro
Type of Study Test System Noteworthy Findings Testing
Facility Report No. Location in Dossier Cross-resistance HCV replicon transient luciferase
cell-based assays to monitor activity against NS3 protease clinical resistance variants
HCV replicons conferring resistance to inhibitors such as boceprevir and telaprevir were examined for their susceptibility to ASV inhibition. Against GT-1a replicons carrying NS3 protease substitutions at amino acid positions V36 (V36A/L/M), T54 (T54A/S), R155 (R155K), and V36-R155 (V36M-R155K), ASV exhibited no/minimal to moderate cross-resistance with EC50 values ranging from 0.33 to 42 nM (parent GT-1a replicon EC50 = 0.76 nM). Against GT-1b replicons carrying NS3 protease substitutions at amino acid positions A156 (A156S/T/V), demonstrated low to moderate resistance with EC50 values of 5.4 to 17 nM (parent GT-1b replicon EC50 = 0.86 nM).
BMS 930057191 CTD 4.2.1.1.6
Combination studies HCV replicon cell-based assay in HuH-7 cells to monitor effects of combining HCV inhibitors of different mechanisms
Combinations of ASV with other classes of HCV inhibitors including interferons alfa and lambda, ribavirin, NS5A,
showed additive to synergistic antiviral activity in vitro, with no antagonistic antiviral activity.
No apparent enhancement of cytotoxicity was seen. Treatment of HCV GT-1a replicons and HCV GT-1a replicons carrying the NS3 substitution R155K with a combination of ASV, daclatasvir, and interferon-alfa similarly suppressed HCV RNA replication.
BMS 930023123
930029019 930033004 930057191
CTD 4.2.1.1.1 CTD 4.2.1.1.4 CTD 4.2.1.1.5 CTD 4.2.1.1.6
Abbreviations: ASV = asunaprevir or BMS-650032, BVDV = bovine viral diarrhea virus, CC50 = concentration of drug to kill 50% of cells, EC50 = 50% effective concentration in reducing viral replication, FRET = fluorescence resonance energy transfer, GBV-B = GB virus B, GT = genotype, HIV = human immunodeficiency virus, HRV = human rhinovirus, HCoV = human coronavirus, HCV = hepatitis C virus, IC50 = 50% inhibitory concentration, Ki = inhibition constant, μM = micromolar, nM = nanomolar, NS3 = nonstructural protein 3, NS3/4A = nonstructural protein 3/nonstructural protein 4A, RNA = ribonucleic acid
3 副次的薬理試験
Table 2.6.3.3: Secondary Pharmacodynamics
Test Article: Asunaprevir (BMS-650032)
In Vitro Test System Noteworthy Findings Testing
Facility Report No. Location in Dossier Type of Study
Mode of action on the
IFNβ response Cardif proteolytic cleavage assay and IFNβ luciferase reporter assay in 293T cells.
ASV inhibited proteolytic cleavage of Cardif by HCV NS3/4A protease, as visualized by Western immunoblotting. Cleavage of Cardif by the presence of NS3/4A protease subsequently leads to suppression of transcription of IFN genes; addition of ASV restores transcription of IFN genes in a dose-dependent manner.
BMS 930023124 CTD 4.2.1.1.2
Abbreviations: ASV = asunaprevir or BMS-650032, Cardif = caspase recruitment domain adaptor inducing IFNβ, HCV = hepatitis C virus, IFN = interferon, IFNβ = interferon-beta, NS3/4A = nonstructural protein 3/nonstructural protein 4A.
4 安全性薬理試験
Table 2.6.3.4: Safety Pharmacology
Test Article: Asunaprevir (BMS-650032) Organ Systems
Evaluated Species/
Strain Method of
Administration Doses (mg/kg)
Gender and No.
per Group Noteworthy Findings GLP
Compliance
Study No./
Document Control Receptors, No.
enzymes, ion channels, and transporters
Human, rat, guinea
pig In vitro 10 μM
(7.5 μg/mL) NA BMS-650032 did not exhibit clear selectivity for any of the 37 targets tested (biochemical, enzyme activity, and ligand binding assays).
nonGLP DT07078/
930022950
Cardiovascular,
Electrophysiology Human/HEK 293 cells and hERG channel
In vitro 10, 30 μM
(7.5, 22.5 μg/mL)
NA ≥ 10 μM: Mild-moderate inhibition of hERG potassium currents (8.2-20.6% vs pretest); IC50 > 30 μM
nonGLP DT07089/
930023617
Human/HEK 293 cells and sodium channel (SCN5A)
In vitro 10, 30 μM
(7.5, 22.5 μg/mL)
NA ≥ 10 μM: Mild-moderate inhibition of sodium currents (26.6-65.9% vs pretest at 1 Hz stimulation frequency and 29.8-71.6%
vs pretest at 4 Hz)
nonGLP DT07089/
930023617
Human/HEK 293 cells and L-type calcium channel
In vitro 30 μM
(22.5 μg/mL) NA 30 μM: Mild-moderate inhibition of L-type
calcium (Cav 1.2) (18.3% vs pretest). nonGLP DT07089/
930023617
Rabbit/cardiac
Purkinje fiber In vitro 3, 10, 30 μM (2.2, 7.5, 22.5 μg/mL)
NA ≤ 30 μM: No BMS-650032-related findings. nonGLP DT07089/
930023617
Dog/Beagle
(telemetered) Oral (capsule) 0, 100
(single dose) 3 M, 3 F 100 mg/kg: Unformed and/or liquid stool in 2 M and 2 F 1 day post dose, bloody stool on Day 6 and unformed/mucoid stool on Days 6-9 in 1 M.
Maximum BMS-650032 plasma
concentration (4 h post dose): 43.68 μg/mL (M); 70.36 μg/mL (F).
nonGLP DS07001/
930021202
Table 2.6.3.4: Safety Pharmacology
Test Article: Asunaprevir (BMS-650032) Organ Systems
Evaluated Species/
Strain Method of
Administration Doses (mg/kg)
Gender and No.
per Group Noteworthy Findings GLP
Compliance
Study No./
Document Control Cardiovascular, No.
Electrophysiology Rabbit/
isolated heart In vitro 0, 10 μM
(0, 7.5 μg/mL) 2 or 3 F 10 μM: No BMS-650032-related findings. nonGLP DT07013/
930023505
Rabbit/NZW IV infusion 0, 3, 10, 30 3 M ≥ 3 mg/kg: Minimal dose-dependent increases (10-24% vs pretreatment) in blood pressure.
Plasma BMS-650032 concentrations 15-180 μg/mL.
nonGLP DT06116/
930023510
Respiratory and
Neurologic Mouse/
Crl:CD1 (ICR) Oral (gavage) 0, 200, 600, 2000 (single dose)
5 M, 5 F ≤ 2000 mg/kg: No respiratory or CNS
effects. GLP DM07027/
930024006
Rat/Crl:CD (SD) Oral (gavage) 0, 200, 600, 2000 (single dose)
5 M, 5 F ≤ 2000 mg/kg: No respiratory or CNS
effects. GLP DM07028/
930023864
Rat/Crl:CD (SD) Oral (gavage) 0, 30, 100, 600
(1 month) 15 M, 15 F ≤ 600 mg/kg: No noteworthy findings.
Cmax values at 30, 100, and 600 mg/kg were up to 0.306, 13.7, and 43.4 μg/mL.
GLP DM07024/
930024094
Rat/Crl:CD (SD) Oral (gavage) 0, 40, 80, 200
(6 months) 25 M, 25 F ≤ 200 mg/kg: No noteworthy findings.
Cmax values at 40, 80, and 200 mg/kg were up to 2,650; 22,700; and 124,000 μg/mL.
GLP DM08025/
930039270
Rat/Crl: CD (SD) Oral (gavage) BMS-650032:
0, 30, 60 BMS-790052:
0, 10, 60 (1 month)
10 M, 10 F BMS-650032/BMS-790052 at
≤ 60/60 mg/kg: No drug-related CNS, or respiratory effects
BMS-650032: Cmax ≤ 15.2 μg/mL BMS-790052: Cmax ≤ 5.7 μg/mL
GLP DS08126/
930035562
Cardiovascular, Neurologic, and Respiratory
Dog/Beagle Oral (capsule) 0, 20, 60, 300
(1 month) 3 M, 3 F ≤ 300 mg/kg: No noteworthy findings.
Cmax values at 20, 60, and 300 mg/kg were up to 6.12, 28.9, and 114 μg/mL.
GLP DM07020/
930024088
Table 2.6.3.4: Safety Pharmacology
Test Article: Asunaprevir (BMS-650032) Organ Systems
Evaluated Species/
Strain Method of
Administration Doses (mg/kg)
Gender and No.
per Group Noteworthy Findings GLP
Compliance
Study No./
Document Control Cardiovascular, No.
Neurologic, and Respiratory
Dog/Beagle Oral (capsule) 0, 15, 50, 100
(9 months) 4 M, 4 F ≤ 100 mg/kg: No noteworthy findings.
Cmax values at 15, 50, and 100 mg/kg were up to 6.29, 26.8, and 59.7 μg/mL.
GLP DM08026/
930038894
Rat/Crl: CD (SD) Oral (gavage) BMS-650032:
0, 30, 60 BMS-790052:
0, 10, 60 (1 month)
10 M, 10 F BMS-650032/BMS-790052 at
≤ 60/60 mg/kg: No drug-related CNS, or respiratory effects
BMS-650032: Cmax ≤ 15.2 μg/mL BMS-790052: Cmax ≤ 5.7 μg/mL
GLP DS08126/
930035562
Monkey/
Cynomolgus Oral (gavage) BMS-650032:
0, 72, 129.5 BMS-790052:
0, 15, 50 (1 month)
4 M, 4 F BMS-650032/BMS-790052 at
≤ 129.5/50 mg/kg: No drug-related CV, CNS, or respiratory effects.
BMS-650032: Cmax ≤ 18.1 μg/mL BMS-790052: Cmax ≤ 4.65 μg/mL
GLP DS08143/
930035546
Monkey/
Cynomolgus Oral (gavage) BMS-650032:
0, 45, 80 BMS-790052:
0, 15, 50 (3 months)
4 M, 4 F BMS-650032/BMS-790052 at
≤ 80/50 mg/kg: No drug-related CV, CNS, or respiratory effects.
BMS-650032: Cmax ≤ 9.00 μg/mL BMS-790052: Cmax ≤ 3.75 μg/mL
GLP DM09008/
930039780
Abbreviations: Cmax = Mean maximum plasma drug concentration; CNS = Central nervous system; CV = Cardiovascular; HEK = Human embryonic kidney cells; hERG = Human ether-a-go-go-related gene; Hz = Hertz; IV = Intravenous; L-type = Long acting-type; NZW = New Zealand white; PEG = Polyethylene glycol; Peg IFN = Pegylated interferon α-2b;
qod = Every other day; RBV = Ribavirin; SD = Sprague Dawley; TPGS = d-α-tocopheryl polyethylene glycol 1000 succinate.