Research ArticleDrug Discovery

Repurposing of the antihistamine chlorcyclizine and related compounds for treatment of hepatitis C virus infection

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Science Translational Medicine  08 Apr 2015:
Vol. 7, Issue 282, pp. 282ra49
DOI: 10.1126/scitranslmed.3010286
  • Fig. 1. Activities of NPC compounds in qHTS and confirmation of CCZ analogs.

    (A) Three-axis plot of the activities of NPC compounds in the qHTS. Activity (%) was calculated by normalizing luciferase signals to the mean signal from the dimethyl sulfoxide (DMSO) control wells. Compounds were sorted according to curve classes. Red, active compounds in curve classes 1 and 2; green, weakly active compounds in curve class 3; blue, inactive compounds in curve class 4. (B and C) Chemical structures (B) and in vitro dose-response curves (C) of CCZ analogs identified in the NPC screen. The percent inhibition of the compound in the HCV-Luc assay is shown in blue triangles, and the cytotoxicity effect in the ATPlite assay of host cells is shown in red circles. EC50 and CC50 values are indicated. Data are means ± SEM (n ≥ 3 replicates). Curves are representative results from at least three independent experiments.

  • Fig. 2. Anti-HCV activities of CCZ.

    (A) Human hepatocytes (Huh7.5.1 cells) were infected with wild-type HCVcc in the presence of the compounds at 10 μM overnight followed by incubation with compound treatment for an additional 48 hours. Viral RNA was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). (B) Primary human hepatocytes were infected with wild-type HCVcc in the presence of (S)-CCZ titration overnight followed by incubation with (S)-CCZ titration for an additional 48 hours. Intracellular viral RNA levels were evaluated by qRT-PCR. (C) In the presence of compound treatment, Huh7.5.1 cells were passaged every 3 days for seven passages and were plated on 96-well plates 3 days before ATPlite assay to measure cell viability. (D) HCV replication cycle assays were carried out with (S)-CCZ at 10 μM. Cyclosporin A (10 μM) was a control in HCV single-cycle infection (HCVsc), transient replicon genotype 1a, and replicon genotype 1b and 2a assays. Bafilomycin A1 (10 nM) was used as a control in HCV pseudoparticle (HCVpp) genotype 1a and 1b, vesicular stomatitis virus G pseudoparticle (VSV-Gpp), and murine leukemia virus pseudoparticle (MLVpp) assays. Results were normalized to DMSO. RLU, relative luminescence units. GT, genotype. (E) At t = −2 hours, HCV-Luc was incubated with Huh7.5.1 cells at 4°C for 2 hours for attachment. At t = 0 hours, the unbound virus was removed, and the plates were moved to 37°C to allow synchronous infection and incubated for 48 hours before virus load measurement. (S)-CCZ (10 μM), bafilomycin A1 (10 nM), and sofosbuvir (10 μM) were added either continuously or at the indicated time points and incubated for 2 hours. (F) Results from (E) were normalized to DMSO continuous treatment. Data are means of replicates ± SEM (n ≥ 3). *P < 0.05, **P < 0.005, ***P < 0.0001, versus DMSO (Student’s t test).

  • Fig. 3. In vitro and in vivo ADME and pharmacokinetics of CCZ.

    (A) The microsomal stability of CCZ and nor-CCZ was measured in vitro by incubation with human or mouse microsomes. Intrinsic clearance (Clint) and T1/2 were calculated. (B) The protein binding adjusted EC50 and CC50 values of (S)-CCZ were measured in 40% of human serum (HS) with HCV-Luc and ATPlite assays. (C and D) Plasma, brain, and liver concentrations of the drug were measured over time after a single intraperitoneal dose of (S)-CCZ (50 mg/kg) or (S)-nor-CCZ (10 mg/kg). Results are means of replicates ± SEM (n = 3). Asterisks indicate statistical significance of liver concentration compared with plasma concentration by Student’s t test (*P < 0.05, **P < 0.005, ***P < 0.0001) (C). T1/2, the highest concentration after administration of compounds (Cmax), and time to reach Cmax (Tmax) are provided in (D).

  • Fig. 4. In vivo efficacy and pharmacokinetics of CCZ in mice infected with HCV genotype 1b or 2a.

    Alb-uPA/SCID mice were engrafted with primary human hepatocytes and then infected with HCV serum samples of genotype 1b or 2a. The mice were monitored for serum HCV RNA and human albumin for 4 to 6 weeks before treatment. Pretreatment HCV RNA values were determined by averaging HCV RNA levels at weeks −2, −1, and 0. (A) Changes in the genotype 1b and 2a HCV titers from pretreatment baseline over time. For 1b, over a period of 4 weeks during (S)-CCZ treatment and 4 weeks of follow-up with or without treatment. For 2a, over a period of 6 weeks of (S)-CCZ treatment and 4 weeks of follow-up without treatment in both groups. The result at each last dosing time point (4 and 6 weeks for 1b and 2a, respectively) was compared to the corresponding pretreatment level using the Mann-Whitney test after the Shapiro-Wilk normality test. Significant difference in HCV RNA was obtained with all the treatment conditions (P < 0.05). (B) The concentrations of (S)-CCZ and (S)-nor-CCZ were measured in plasma samples collected at weeks 1, 2, 3, and 4 in genotype 1b–infected mice and at weeks 1, 2, 3, 4, 5, and 6 in genotype 2a–infected animals. The weekly concentrations were averaged for each dosing group and shown. (C) The concentrations of (S)-CCZ and (S)-nor-CCZ in plasma, liver, and brain samples collected at day 28 in genotype 1b–infected mice were measured by liquid chromatography–mass spectrometry (LC-MS). Data are means of mice in each group ± SEM (for genotype 1b, n = 5 in the 50 mg/kg group, n = 4 in the 10 mg/kg group, and n = 5 in the 2 mg/kg group; for genotype 2a, n = 8 in the 50 mg/kg group and n = 5 in the 10 mg/kg group).

  • Table 1. Anti-HCV activity, selectivity, and anti-histamine properties of CCZ analogs.

    Compounds were tested in the HCV-Luc infection assay in parallel with the ATPlite assay. HCV-Luc was used to infect Huh7.5.1 cells in the presence of compound titration. Viral infection and replication were measured by luciferase signal 48 hours after treatment, and cytotoxicity was evaluated by the adenosine 5′-triphosphate (ATP)-based cell viability assay. The concentration values that led to 50% and 90% viral inhibition (EC50 and EC90, respectively) and 50% cytotoxicity (CC50) were calculated with GraphPad Prism using a nonlinear regression equation. Cyclosporin A was a control. Data are means ± SEM from n ≥ 3 independent experiments. Antihistamine activity was obtained with the β-arrestin H1-histamine receptor assay. N.D., not determined.

    CompoundHCV-Luc EC50 (μM)HCV-Luc EC90 (μM)ATPlite CC50 (μM)SIAntihistamine activity at 10 nM (%)
    Racemic CCZ0.044 ± 0.0111.40 ± 0.4549.8 ± 17.299472.60
    (R)-CCZ0.020 ± 0.0051.09 ± 0.3737.5 ± 4.15187588.00
    (S)-CCZ0.024 ± 0.0091.44 ± 0.4333.4 ± 2.44139241.70
    (S)-Nor-CCZ0.034 ± 0.0120.578 ± 0.0999.31 ± 0.042742.24
    Cyclosporin A0.213 ± 0.0440.92 ± 0.20N.D.N.D.N.D.
  • Table 2. Synergistic antiviral effect of CCZ in combination with anti-HCV drugs.

    The level of synergy was defined in MacSynergy as follows: ++, moderate synergy (5 ≤ log volume < 9); +++, strong synergy (log volume ≥ 9). Combination indices (CI) are means ± SEM from combinations of the tested drug with (S)-CCZ at or near their EC50 values when tested alone (n ≥ 6). The level of synergy was defined by CalcuSyn as follows: ++, moderate synergy (0.7 ≤ CI < 0.85); +++, synergy (0.3 ≤ CI < 0.7).

    ProgramParameterRibavirinIFN-αTelaprevirBoceprevirSofosbuvirDaclatasvirCyclosporin A
    MacSynergyLog volume++++++++++++++++++++
    CalcuSynCI value0.630 ± 0.1060.609 ± 0.1280.426 ± 0.1380.691 ± 0.1140.362 ± 0.0750.427 ± 0.1420.727 ± 0.187
    Synergy volume+++++++++++++++++++++

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/282/282ra49/DC1

    Materials and Methods

    Fig. S1. CCZ does not affect the expression levels or cellular distribution of HCV entry factors.

    Fig. S2. Synergistic antiviral effects of CCZ in combination with anti-HCV drugs.

    Fig. S3. Antiviral activity of CCZ against dengue virus.

    Table S1. Structure, anti-HCV activity, and cytotoxicity of H1-antihistamine compounds from the NPC library.

    Table S2. Negligible amount of transformation of (S)-CCZ to (S)-nor-CCZ in vitro.

    Table S3. Antiviral activity of CCZ against HCV genotypes 1 to 7.

    Table S4. NIAID antiviral screen of CCZ against 13 viruses.

    References (4149)

  • Supplementary Material for:

    Repurposing of the antihistamine chlorcyclizine and related compounds for treatment of hepatitis C virus infection

    Shanshan He, Billy Lin, Virginia Chu, Zongyi Hu, Xin Hu, Jingbo Xiao, Amy Q. Wang, Cameron J. Schweitzer, Qisheng Li, Michio Imamura, Nobuhiko Hiraga, Noel Southall, Marc Ferrer, Wei Zheng, Kazuaki Chayama, Juan J. Marugan, T. Jake Liang*

    *Corresponding author. E-mail: jliang{at}nih.gov

    Published 8 April 2015, Sci. Transl. Med. 7, 282ra49 (2015)
    DOI: 10.1126/scitranslmed.3010286

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. CCZ does not affect the expression levels or cellular distribution of HCV entry factors.
    • Fig. S2. Synergistic antiviral effects of CCZ in combination with anti-HCV drugs.
    • Fig. S3. Antiviral activity of CCZ against dengue virus.
    • Table S1. Structure, anti-HCV activity, and cytotoxicity of H1-antihistamine compounds from the NPC library.
    • Table S2. Negligible amount of transformation of (S)-CCZ to (S)-nor-CCZ in vitro.
    • Table S3. Antiviral activity of CCZ against HCV genotypes 1 to 7.
    • Table S4. NIAID antiviral screen of CCZ against 13 viruses.
    • References (4149)

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