Research Article

The cyclophilin inhibitor SCY-635 suppresses viral replicationand induces endogenous interferons in patients with chronic

HCV genotype 1 infectionq

Sam Hopkins1,⇑, Betty DiMassimo1, Pamela Rusnak1, Douglas Heuman2, Jacob Lalezari3,Ann Sluder1, Bernard Scorneaux1, Sarah Mosier1, Paul Kowalczyk1, Yves Ribeill1,

James Baugh4, Philippe Gallay4

1Scynexis, Inc., Durham, NC, United States; 2McGuire Veterans Affairs Medical Center, Richmond, VA, United States; 3Quest Clinical Research,San Francisco, CA, United States; 4Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, CA, United States

Background & Aims: SCY-635 is a non-immunosuppressive ana- may represent a major mechanism through which cyclophilin

log of cyclosporin A that inhibits cyclophilins A and B and hepa-titis C virus (HCV) replication in vitro. In a phase 1b multi-doseescalation study, we evaluated the safety, plasma pharmacoki-netics, and antiviral activity of 15 days of monotherapy withSCY-635 in adults with chronic genotype 1 HCV infection.Methods: Twenty adults with chronic HCV genotype 1 were ran-domized to SCY-635 oral doses of 100, 200, or 300 mg three timesdaily for 15 days.Results: No dose-limiting clinical or laboratory toxicities wereidentified. On day 15, the mean decline in plasma viremiawas 2.24 ± 1.74 log10 IU/ml with SCY-635 900 mg/d. Individualantiviral responses correlated with host IL28B genotype. Posthoc analyses indicated treatment with SCY-635 increasedplasma protein concentrations of interferon a (IFNa), IFNs k1

and k3, and 2050 oligoadenylate synthetase 1 (2050OAS-1), withthe greatest increases in IL28B CC and CT subjects. Changesin plasma concentrations for all markers were coincident withchanges in the plasma concentration of SCY-635. Peaks of IFNsa, k1, and k3 and 2050OAS-1 were observed within 2 h afterdrug administration. In replicon cells, SCY-635 enhanced secre-tion of type I and type III IFNs and increased the expression ofIFN-stimulated genes (ISG).Conclusions: These studies establish clinical proof of concept forSCY-635 as a novel antiviral agent and suggest that restoration ofthe host innate immune response to chronic hepatitis C infection

Journal of Hepatology 20

Keywords: SCY-635; Cyclophilin inhibitor; Chronic hepatitis C; Interferon a;Interferon k; 2050OAS-1; IL28B.Received 4 April 2011; received in revised form 18 January 2012; accepted 13February 2012; available online 13 March 2012q ClinicalTrials.gov identifier: NCT01290965.⇑Corresponding author. Address: Autoimmune Technologies, 1010 CommonStreet, Suite 1705, New Orleans, LA 70112, United States. Tel.: +1 919 522 6490.E-mail address: S.Hopkins@autoimmune.com (S. Hopkins).Abbreviations: HCV, hepatitis C virus; CsA, cyclosporin A; EC50, 50% effective co-ncentration; HIV-1, Human Immunodeficiency Virus Type-1; PPIase, peptidylprolyl isomerase; TEAE, treatment-emergent adverse event; CTCAE, CommonTerminology Criteria for Adverse Events; IFNa, interferon alpha; IFNb, interferonbeta; IFNk1, interferon lambda 1; IFNk3, interferon lambda 3; 2050OAS-1, 2050oli-goadenylate synthetase 1; CPK, creatinine phosphokinase; ALT, alanine amino-transferase; AST, aspartate aminotransferase; ISG, IFN-stimulated gene.

inhibitors exert clinical antiviral activity.� 2012 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.

Introduction

For patients with chronic infection with hepatitis C virus (HCV),response to interferon (IFN)-based regimens is dependent in parton host IL28B genotype [1], suggesting host innate immune fac-tors are critical to the likelihood of cure. In vitro, efficient replica-tion of sub-genomic and full length HCV RNA genomes dependsupon expression of the host protein cyclophilin A (cyclophilin18) [2–4]. SCY-635 is an orally bioavailable, novel analog of cyclo-sporin A (CsA) [5] that is a reversible, competitive, active site-directed inhibitor of cyclophilin A and inhibits peptidyl prolylisomerase (PPIase) catalytic activity at low nanomolar (nM) con-centrations. The 50% effective concentration (EC50) of SCY-635 forinhibiting HCV-specific RNA replication against a subgenomicCon1-derived replicon is 132 ng/ml (100 nM), with a selectivityindex of approximately 130. In vitro, SCY-635 exhibits additiveto synergistic antiviral activity when tested in combination withribavirin, IFNa-2b, nucleoside analogs, and protease inhibitors[6].

We evaluated the safety, pharmacokinetics, and antiviralactivity of SCY-635 in adults with chronic HCV. In part 1 ofthe study, SCY-635 or placebo was administered once dailyfor 15 days to 37 patients. Total daily doses of SCY-635 were30 mg (Cohort 1), 100 mg (Cohort 2), or 300 mg (Cohort 3).SCY-635 was safe and well tolerated; however, antiviral activ-ity was not observed (data on file at Scynexis, Inc.). Therefore,in part 2, which is described in this report, the total daily doseand the dosing frequency were increased. Total daily doses ofSCY-635 equaling 300 mg (Cohort 4), 600 mg (Cohort 5), or900 mg (Cohort 6) were given on a divided schedule (threetimes daily; t.i.d.) for 15 consecutive days. SCY-635 antiviralactivity and effects on markers of host immune response wereevaluated.

12 vol. 57 j 47–54

Research Article

Patients and methods

Study design for SCY-635-104

Part 2 of this randomized, double-blind, placebo-controlled, dose-escalationstudy was conducted at the McGuire Research Institute, Richmond, Virginia. Priorto study initiation, the protocol and informed consent form were reviewed andapproved by the site’s institutional review board. After completion of the treat-ment period, subjects reconsented to IL28B genotyping and analysis of markersof innate immune activation.

Between December 2007 and October 2008, three cohorts of subjects chron-ically infected with HCV genotype 1 were sequentially enrolled. In each cohort,subjects were randomly assigned to 15 consecutive days of orally administeredSCY-635 or placebo in a 6:1 (active:placebo) ratio. The randomization code waskept under locked conditions by an unblinded study drug dispenser, who didnot participate in evaluating study subjects.

Subjects were housed in the clinical research unit from day �1 until day 4.Days 4 through 13 were conducted on an outpatient basis. Subjects returned tothe research unit on day 13 for pharmacokinetic assessments and were dis-charged on day 15. A follow-up visit occurred on day 22 (7 ± 3 days after theend of treatment). Subjects in Cohort 4 received 300 mg SCY-635 per day(100 mg t.i.d.) or matching placebo capsules; those in Cohort 5 received 600 mgSCY-635 per day (200 mg t.i.d.) or placebo; and those in Cohort 6 received900 mg SCY-635 per day (300 mg t.i.d.) or placebo.

Subjects

Males and females were eligible for inclusion if they were between 18 and65 years of age, exhibited quantifiable levels of HCV-specific RNA in excess of100,000 IU/ml, and demonstrated a negative urine assay for amphetamines, bar-biturates, cocaine, opiates, and phencyclidine. Participants were excluded if theywere infected with any HCV genotype other than genotype 1; exhibited positiveantibody tests for HIV-1, HIV-2, or hepatitis B virus; received an investigationalagent in the prior 3 months; received any FDA-approved anti-HCV therapy inthe prior 3 months; exhibited evidence of decompensated liver disease (bilirubin>4 mg/dl, albumin <3 g/dl, prothrombin time >2 s prolonged, or history of bleed-ing esophageal varices, ascites, or hepatic encephalopathy); received an organtransplant; exhibited ALT values >2.5 times the upper limit of normal at screen-ing; exhibited evidence of hepatocellular carcinoma; or exhibited evidence ofongoing alcohol or substance abuse.

Antiviral assessments

Antiviral activity was evaluated by measuring plasma HCV RNA levels (IU/ml)at baseline; on days 1, 2, 3, 5, 8, 11, and 15; and at 7 (±3) days post-treatment.The baseline blood sample was collected immediately prior to initiation oftreatment on day 1. The day 15 sample was collected prior to the morningdose of study drug. Plasma HCV RNA levels were determined by polymerasechain reaction (Roche COBAS TaqMan Assay, Roche Diagnostics)performed by Esoterix, Inc. (Cranford, NJ) with a lower limit of quantitationof 15 IU/ml.

IL28B genotyping

IL28B genotyping was performed using real time PCR with allele-specific Taqmanprobes to detect a single nucleotide polymorphism rs12979860 C/T on chromo-some 18q13 [1].

Determination of IFNs a, b, and k, and 2050OAS-1 concentrations in human plasma andfrom cell culture medium

The concentrations of IFNs a, b, k1, and k3, and 2050OAS-1 were detected andquantified from subject plasma pharmacokinetic samples and from cell culturemedium using commercially available ELISA-based assays. The ELISA kitsincluded PBL multi-subtype kits for IFNa and IFNb, USCN kits for IL29 (IFNk1)and 2050OAS-1, and Kamiya Biomedical Company kit for IFNk3. All samples weredivided into multiple aliquots and stored at �80 �C to avoid multiple freeze/thawcycles.

48 Journal of Hepatology 20

Pharmacokinetic assessments

Blood samples for determining SCY-635 concentrations in plasma were collectedon days 1, 2, 3, and 14. Samples were collected prior to dosing and at 0.5, 1, 2, 3, 4,6, and 8 h after the first daily dose. Trough PK samples were collected prior to thefirst daily dose on days 2, 3, 4, 5, 8, 11, 12, 14, and 15 and prior to the third dailydose on days 1, 2, 3, and 13. Plasma levels of SCY-635 were determined using avalidated HTLC-MS/MS method with an assay range of 4.76–952 ng/ml. PKparameters were calculated using non-compartmental methods in WinNonlin(Pharsight, Mountainview, CA).

Safety assessments

Safety assessments included physical examinations, vital signs, 12-lead electro-cardiograms, hematology and clinical chemistry assessments, urinalysis, andreporting of spontaneous treatment-emergent adverse events. Severity of adverseevents was graded on the basis of Common Terminology Criteria for AdverseEvents (CTCAE) version 3.0.

Statistical analyses

Analyses included all randomized subjects who received at least one dose ofstudy medication. Descriptive statistics were reported for baseline demographic,clinical laboratory, and vital signs data. Baseline adjusted log10 IU/ml HCV RNAvalues were summarized using descriptive statistics.

Correlation of concentration versus time data for SCY-635 and markers of innateimmune function

Two methods, one graphical and one statistical, were used to assess the relation-ship between the plasma concentration of SCY-635 and the plasma concentra-tions of IFN a, b, and k, and 2050OAS-1. For the graphical analysis, the measuredconcentrations of both SCY-635 and the various analytes were rescaled ontothe range (0,1). The rescale function in ggplot2 (http://had.co.nz/ggplot2/) wasused for data transformation. For a given data set, range scaling transformedthe measured data to map the minimum concentration to 0.0 and the maximumconcentration to +1.0. A simple linear interpolation scheme was used to mapintermediate concentration values. To quantify the relation between the SCY-635 and a specific analyte concentration time course, we calculated the pairwisePearson correlation coefficients between the plasma concentrations of SCY-635and the various analytes. These correlations measure the linear dependencebetween the plasma concentrations of SCY-635 and the various analytes. Correla-tions approaching +1.0 show that the analyte plasma concentration is positivelycorrelated with the SCY-635 plasma concentration; correlations near 0.0 show norelationship; and correlations approaching �1.0 show that the analyte plasmaconcentration is negatively correlated with the SCY-635 plasma concentration.

Replicon assays

Parental Huh-7 cells were maintained at 37 �C, 5% CO2 in DMEM complete growthmedia (10% heat inactivated fetal bovine serum, 10 mM HEPES, NEAA, 2 mM L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin). Huh-7-Con1-Lucsubgenomic replicon cells were maintained in the same growth media supple-mented with neomycin (300 lg/ml). For all replicon studies, neomycin wasremoved from complete growth media 2 days prior to treatment. Cells in lineargrowth were seeded in 6-well plates at variable cell densities to obtain 90% con-fluence at each time point of collection. Parental or Huh-7 cells containing theCon1 subgenomic replicon encoding a luciferase reporter gene (called hereHuh-7-Con1-luciferase cells) [7] were treated in triplicate with either DMSO con-trol (0.01% final concentration) or the cyclophilin A inhibitors SCY-635, Alisporivirand CsA (Sigma) at a final concentration of 2 lM beginning 24 h after seeding thecells for 0, 1, 2, 3, and 4 days. To harvest, plates were centrifuged at 4000 rpm for4 min at 4 �C. Supernatants were transferred to sterile plates and stored at �80 �Cuntil ELISA analysis for IFNs and 2050OAS-1. Cell monolayers were washed twicewith ice cold PBS and stored in 300 ll sterile water at �80 �C for protein analysis.Cells were collected and sonicated, and total protein amounts were determinedusing a BCA protein assay (Fisher Scientific). To account for the effects of cellulargrowth, all results for 2050OAS-1 and IFN a and k1 were normalized to the concen-tration of total cellular protein.

12 vol. 57 j 47–54

Table 1. Baseline demographic and disease characteristics.

SCY-635Placebo(n = 3)

300 mg/d(n = 6)

600 mg/d(n = 5)

900 mg/d(n = 6)

Gender, No. (%)

Male 3 (100) 6 (100) 5 (100) 6 (100)

Race, No. (%)

African American 2 (67) 5 (83) 2 (40) 6 (100)

Caucasian

Pacific Islander

1 (33) 0 2 (40) 0

Native Hawaiian/ 0 1 (17) 1 (20) 0

Age, yr

Median 52 56 52 54

Range 50-56 52-59 28-58 46-59

HCV RNA, log10 IU/ml

Mean (SD) 6.92 (0.40) 6.45 (0.55) 6.78 (0.30) 6.61 (0.54)

ALT, IU/L

Median 53 48 48 66

Range 36-61 33-131 37-63 20-111

AST, IU/L

Median 54 40 35 65

Range 26-70 35-107 26-56 26-114

Prior hepatitis C treatment, No. (%)

2 (67) 4 (67) 2 (40) 0

Table 2. Number of subjects (%) with most frequently reporteda treatment-emergent adverse events.

SCY-635

Placebo(n = 3)

300 mg/d(n = 6)

600 mg/d(n = 5)

900 mg/d(n = 6)

Elevated CPK 0 4 (66.7) 0 0

Headache 0 2 (33.3) 1 (20.0) 1 (16.7)

Hypokalemia 0 0 1 (20.0) 2 (33.3)

Jaw pain 1 (33.3) 1 (16.7) 0 0

Liver function test elevated

0 1 (16.7) 0 2 (33.3)

Nausea 0 0 0 2 (33.3)

a Events occurring in two or more subjects.CPK, creatine phosphokinase.

JOURNAL OF HEPATOLOGY

Results

Subject enrollment

The study population for part 2 comprised 20 subjects. In Cohort5 (600 mg SCY-635 per day), six rather than seven subjects wereenrolled. Because initial subjects who received 600 mg/d did notshow appreciable changes in viral load, enrollment into Cohort 6(900 mg/d) began early. All subjects were male and 15 (75%)were African American (Table 1).

Safety

Thirty-one treatment-emergent adverse events (TEAEs) werereported in 16 (80%) subjects: 2 (67%) receiving placebo, 6 (100%)receiving 300 mg/d SCY-635, 3 (60%) receiving 600 mg/d SCY-635, and 5 (83%) receiving 900 mg/d SCY-635. There were no seri-ous AEs. No subjects discontinued treatment because of an AE. AllTEAEs resolved. The most frequently reported TEAEs with SCY-635were elevated serum creatinine phosphokinase (CPK), headache,hypokalemia, elevated liver function tests, and nausea (Table 2).All four elevations in serum CPK occurred in patients who received300 mg/d. All elevations in CPK were Grade 1 and occurred duringoutpatient treatment on days 8 or 11 and returned to below base-line on day 15. Grade 1 hypokalemia was reported in one subjectwho received 600 mg/d and in two subjects who received900 mg/d. Prior to enrollment, all three subjects were taking apotassium wasting diuretic for hypertension. Hypokalemiaresolved within 24 h of onset for two subjects. The third subjectexhibited hypokalemia at the follow-up visit.

Three subjects demonstrated increased values for alanineaminotransferase (ALT) or aspartate aminotransferase (AST).

Journal of Hepatology 20

One subject who received 300 mg/d exhibited baseline valuesof 40 IU/ml for ALT and 37 IU/ml for AST. On day 5, these valuesincreased to 71 IU/ml for ALT and 65 IU/ml for AST (Grade 1). ALTand AST values returned to within normal limits on day 8 and day15, respectively. Two subjects who received 900 mg/d had ele-vated transaminases. One subject exhibited baseline values of59 IU/ml for ALT and 47 IU/ml for AST. On day 5, ALT and ASTincreased to 114 IU/ml (Grade 1). ALT and AST values returnedto near baseline on day 15 and to within normal limits at fol-low-up. The second subject exhibited AST values of 26 IU/ml atbaseline and 118 IU/ml (Grade 2) on day 2. AST returned towithin normal limits on day 15. All elevations in liver enzymesin all three subjects were asymptomatic, were not associatedwith changes in bilirubin or alkaline phosphatase, and resolvedwithout interruption of study medication.

One subject receiving 600 mg/d exhibited a single Grade 4 ele-vation in triglycerides that was considered severe and possiblytreatment-related. The subject entered the study with a Grade1 baseline triglyceride level of 298 mg/dl, which increased to1827 mg/dl (Grade 4) on day 5. This subject reported that he con-sumed several large, high-fat meals. The subject modified hisdiet, and blood triglycerides fell below baseline without interrup-tion of study medication.

Pharmacokinetics of SCY-635

SCY-635 plasma exposures (Cmax and AUC) increased in a greaterthan proportional manner. Mean values for Cmax, AUC0–8, and C8

increased from days 1 to 3 for all three cohorts.SCY-635 administered as 300 mg/d generated C8 plasma con-

centrations that were below the replicon-derived EC50 value of132 ng/ml (100 nM) [5] (Fig. 1). SCY-635 600 mg/d produced C8

plasma concentrations that were approximately equal to theEC50 value. SCY-635 900 mg/d resulted in significant distributionof SCY-635 into the plasma compartment. C8 plasma concentra-tions for this cohort remained at or slightly below the EC90 con-centration of 463 ng/ml (350 nM) from day 3 to day 15.

Response to SCY-635 monotherapy

Treatment with placebo or SCY-635 300 or 600 mg/d was associ-ated with minimal changes in viral load (Fig. 2A). One subject(0065) in Cohort 5 (SCY-635 600 mg/d) exhibited a virologicalresponse with a nadir of �0.89 log10 IU/ml below baseline on

12 vol. 57 j 47–54 49

10

100

1000

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336

SCY-

635

troug

h pl

asm

aco

ncen

tratio

n (n

g/m

l)

Time (hour)

SCY-635 300 mg/d (n = 6)SCY-635 600 mg/d (n = 5)SCY-635 900 mg/d (n = 6)

EC50 (132 ng/ml)

EC90 (463 ng/ml)

Fig. 1. Mean trough plasma concentrations of SCY-635.

0.5

0.0

-0.5

-1.0

-1.5

-2.00 4 8 12 16

Med

ian

log 10

cha

nge

in H

CV

RN

A fro

m b

asel

ine

(IU/m

l)

Study day0 4 8 12 16

Study day

A B

1.0

0.0

-1.0

-2.0

-3.0

-4.0

-5.0

-6.0Log 10

cha

nge

in H

CV

RN

A fro

m b

asel

ine

(IU/m

l)

Placebo

SCY-635 300 mg/d (n = 6)SCY-635 600 mg/d (n = 5)SCY-635 900 mg/d (n = 6)

Fig. 2. Effects of treatment with SCY-635 on the plasma concentration ofHCV-specific RNA over time. (A) Median plasma HCV RNA during treatment and(B) individual plasma HCV RNA with 900 mg/d SCY-635.

Research Article

day 11. In contrast, all six subjects who received 900 mg/d exhib-ited decreases in plasma viremia, with individual maximumchanges in viral load ranging from �0.84 to �5.47 log10 IU/ml(Fig. 2B). On day 15, the median viral load reduction for all sub-jects who received 900 mg/d SCY-635 was �1.90 log10 IU/ml; thegroup mean (±SD) viral load reduction was �2.24(±1.74) log10 IU/ml. On day 15, one subject exhibited plasmaHCV RNA that was equal to the limit of quantitation (15 IU/ml).

All subjects who received 900 mg/d SCY-635 experiencedincreases in plasma HCV RNA after cessation of treatment onday 15; however, all values measured at the day 22 follow-upvisit remained below baseline (data not shown). At the day 22follow-up visit, the mean (±SD) log10 IU/ml change from baselinewas �1.38 (±1.73) log10 IU/ml.

Influence of IL28B genotype on antiviral responses to SCY-635monotherapy

Subjects with IL28B genotype CC had the greatest reductions inviral load with SCY-635 treatment (Table 3). The correlationwas most apparent with SCY-635 900 mg/d. All subjects receiving900 mg/d were treatment-naive African American men 46 to59 years old, and five of the six were infected with genotype 1avirus. Among subjects receiving 900 mg/d, Cmax plasma concen-trations and AUC0–8 values differed by 2.6- and 2.9-fold, respec-tively; however, the maximum virological response differed byapproximately 43,000-fold.

50 Journal of Hepatology 20

Effects of nominal dose and drug concentration on the plasmaprotein concentrations of endogenous IFNs and 2050OAS-1

SCY-635 concentration versus time plots was integrated with theabsolute plasma protein concentrations of IFNa and IFNk1 andthe fold-change from baseline for the plasma protein concentra-tion of 2050OAS-1. Subjects receiving SCY-635 300 or 600 mg/ddid not have consistent changes in plasma protein concentrationsof IFNs a, k1, and 2050OAS-1 from baseline throughout the 8-hdosing interval (data not shown). For subjects receiving SCY-635 900 mg/d, IFNs a and k1 and 2050OAS-1 increased above base-line, and the increases were sustained throughout the 8-h dosinginterval and coincided with SCY-635 peak plasma concentration(data not shown). Treatment with SCY-635 (900 mg/d) led todecreased IFNb plasma levels but increased plasma levels ofIFNk3. Maximum plasma concentrations of SCY-635 coincidedwith minimum observed plasma concentrations of IFNb. Maxi-mum plasma protein concentrations of IFNk3 coincided withplasma Cmax values for SCY-635.

Correlation between the plasma concentration of SCY-635 andchanges in endogenous IFNs and 2050OAS-1

In all subjects who received SCY-635 900 mg/d, there was con-cordance between the absorption and disposition of SCY-635and changes in the plasma protein concentrations of endoge-nous IFNs and 2050OAS-1 (Table 4 and Fig. 3). The majority ofthe Pearson correlation coefficients that were calculatedbetween SCY-635 and IFNs a, k1, and k3 and 2050OAS-1 fallwithin the range of +0.5 to +1.0, indicating a strong positivecorrelation for these covariates. All correlation coefficients cal-culated between SCY-635 and IFNb fall within the range of�0.5 to �1.0, indicating a strong negative correlation betweenthese two covariates.

In vitro confirmation of the SCY-635-mediated production ofendogenous IFNs and activation of IFN stimulated genes

Huh-7-Con1-luciferase, but not parental cells, expressed highluciferase activity in cell lysates (data not shown). No measurableamounts of IFNs a or k1 or 2050OAS-1 were detected in the cellsupernatants of Huh-7-Con1-luciferase cells treated with DMSO(Fig. 4A). Similarly, no measurable amounts of IFNs a, k1, or2050OAS-1 were detected in the supernatant of parental Huh-7cells treated with SCY-635, Alisporivir or CsA (Fig. 4A). In con-trast, significant amounts of IFNs a and k1 and 2050OAS-1 weredetected in the supernatant of Huh-7-Con1-luciferase cells trea-ted with SCY-635, Alisporivir or CsA (Fig. 4B).

Peak amounts of IFNs a and k1 and 2050OAS-1 were observed2 days after drug addition. The amounts of IFNs a and k1 and2050OAS-1 decreased after day 2 but remained detectable 3 and4 days after drug addition. CsA had a less dramatic effect thanSCY-635 and Alisporivir on IFNs a and k1 and 2050OAS-1.

Discussion

Oral administration of the non-immunosuppressive cyclophilininhibitor SCY-635 conferred dose-related, clinically relevantsuppression of plasma viremia in adults with chronic genotype 1

12 vol. 57 j 47–54

Table 3. Viral genotype, host IL28B genotype, racial/ethnic background, viral load response, and day 3 plasma pharmacokinetic parameters.

Dose(mg/d)

Subject number

Race IL28B genotype

HCV genotype

Max log10 decline in HCV RNA

Day 3 C8hr(ng/ml)

Day 3 AUC0-8 (hr*ng/ml)

300 0057 African American TT 1b 0.00* 26.3 482

300 0056 African American TT 1b 0.12 29.1 338

300 0053 Caucasian CT 1b 0.19 0** 0**

300 0055 African American CT 1a 0.22 27.6 245

300 0058 African American CT 1a 0.31 50.0 410

300 0059 African American CC 1a 0.46 38.5 644

300 0054 CC 1a 0.52 26.4 282

600 0063 African American CT 1a 0.11 0** 0**

600 0060 African American - 1a 0.17 38.1 1008

600 0064 TT 1a 0.40 76.3 1455

600 0062 Caucasian CT 1a 0.42 93.0 1177

600 0061 African American TT 1a 0.50 96.4 1269

600 0065 Caucasian CC 1a 0.89 127 2400

900 0068 African American CT 1b 0.40 0** 0**

900 0072 African American TT 1a 0.84 396 7090

900 0069 African American CT 1b 1.42 403 5353

900 0067 African American CT 1a 1.47 244 5307

900 0073 African American CC 1a 2.34 232 3776

900 0070 African American CT 1a 2.44 607 10,960

900 0071 African American CC 1a 5.47 518 10,590

Native Hawaiian/Pacific Islander

Native Hawaiian/Pacific Islander

⁄Subject showed a 0.06 log10 increase in viral load from baseline.⁄⁄Subject received placebo.

Table 4. Pearson correlation coefficients.

Subject IFNα IFNβ IFNλ1 IFNλ3 2’5’OAS-1

72 0.8313 -0.7867 0.7332 0.9370 0.8728

69 0.4997 -0.5176 0.6591 0.6677 0.6421

67 0.7142 -0.6730 0.4361 0.7717 0.9342

73 0.6240 -0.7040 0.8549 0.3942 0.8405

70 0.8454 -0.6492 0.8532 0.1539 0.9495

71 0.8005 -0.7684 0.3981 0.8391 0.8580

JOURNAL OF HEPATOLOGY

HCV infection. Monotherapy with SCY-635 900 mg/d caused a meanreduction in HCV-specific RNA of �2.24 log10 IU/ml by day 15. Thiseffect is comparable to antiviral activity (�1.72 log10 IU/ml)observed following 15 days of monotherapy with the cyclophilininhibitor Alisporivir at a dose of 1000 mg/d [8].

In general, SCY-635 was well tolerated. Dose proportionalincreases in plasma pharmacokinetic parameters were observedbetween the 300 and 600 mg/d dose groups, and greater thanproportional increases were observed between the 600 and900 mg/d dose groups. Among subjects who received 900 mg/dof SCY-635, reduction in viral load correlated with IL28B geno-type, suggesting the antiviral activity of SCY-635 is linked toIFN response. All subjects who received the 900 mg total dailydose of SCY-635 had increases in the plasma concentrations ofIFNs a, k1, and k3 and 2050OAS-1 above pretreatment baseline val-ues. Therefore, SCY-635-mediated antiviral activity appears to be

Journal of Hepatology 20

driven, at least in part, by stimulation of IFN production and acti-vation of ISG.

SCY-635-mediated enhanced expression of components of theIFN response was only observed in the presence of the virus andwas dependent on drug concentration. Thus, inhibition of viral pro-tein synthesis and complete neutralization of host cyclophilinsrepresent critical parameters that contribute to the SCY-635-mediated activation of the IFN response. HCV has developed multi-ple strategies to counteract the innate response in HCV patients,suggesting that acute infection activates a robust innate immuneresponse, which must be overcome to establish chronic infection.Because HCV replicates via a double-stranded RNA (dsRNA)intermediate, it activates protein kinase R (PKR) [9], IFN regulatoryfactor 1 (IRF-1) [9,10], and IRF-3 [10,11] and downstream antiviralgenes that are activated by these factors [12,13]. Nevertheless,HCV appears to be resistant to these antiviral pathways [13,14].By demonstrating that SCY-635 both blocks HCV RNA replicationand reactivates the innate IFN response, this study providesevidence that HCV is capable of counteracting and controllingthe innate IFN response in the absence of antiviral therapy.

The effects exerted by SCY-635 on the plasma concentrationsof IFNs in patients with chronic HCV suggest that cyclophilins areinvolved in regulating the expression of type I and III IFNsthrough multiple independent pathways. Cyclophilins such ascyclophilin A or B could modulate production of type I and III IFNsby interacting directly or indirectly with components of theinnate response. It is also possible that NS5A binds directly tocyclophilin A or B and forms a stable ternary complex that pre-

12 vol. 57 j 47–54 51

1.00.80.60.40.20.0

0 2 4 6 8

Con

cent

ratio

nsc

aled

to 1

.0

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8Time (h) Time (h) Time (h) Time (h) Time (h)

Interferon α Interferon β Interferon λ1

Subject 0072

Interferon λ3 2’5’OAS-1

1.00.80.60.40.20.0

0 2 4 6 8

Con

cent

ratio

nsc

aled

to 1

.0

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8Time (h) Time (h) Time (h) Time (h) Time (h)

Subject 0069

1.00.80.60.40.20.0

0 2 4 6 8

Con

cent

ratio

nsc

aled

to 1

.0

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8Time (h) Time (h) Time (h) Time (h) Time (h)

Subject 0067

1.00.80.60.40.20.0

0 2 4 6 8

Con

cent

ratio

nsc

aled

to 1

.0

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8Time (h) Time (h) Time (h) Time (h) Time (h)

Subject 0073

1.00.80.60.40.20.0

0 2 4 6 8

Con

cent

ratio

nsc

aled

to 1

.0

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8Time (h) Time (h) Time (h) Time (h) Time (h)

Subject 0070

1.00.80.60.40.20.0

0 2 4 6 8

Con

cent

ratio

nsc

aled

to 1

.0

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8

1.00.80.60.40.20.0

0 2 4 6 8Time (h) Time (h) Time (h) Time (h) Time (h)

Subject 0071

Fig. 3. Plasma SCY-635 concentration and endogenous IFNs and 2050OAS-1. The dotted line represents SCY-635 concentration and the continuous line the different IFNsor the 2050OAS-1 concentration.

Research Article

52 Journal of Hepatology 2012 vol. 57 j 47–54

0

5

10

15

20

25

30 Parental HuH7 cells

AlisporivirCsA

SCY-635DMSO

IFNα

A B

0

25

50

75

100

125 125

pg/m

g pr

otei

nµg

/mg

prot

ein

Parental HuH7 cells

IFNλ IFNλ

0255075

100125150175200

0 1 2 3 4 5

OAS-1Parental HuH7 cells

Days post-drug treatment

0 1 2 3 4 5 0

25

50

75

100

µg/m

g pr

otei

n

HCV Con1HuH7 cells

0255075

100125150175200

0 1 2 3 4 5

OAS-1HCV Con1HuH7 cells

Days post-drug treatment

0 1 2 3 4 5

0 1 2 3 4 5 0

5

10

15

20

25

30 HCV Con1HuH7 cells IFNα

pg/m

g pr

otei

n

pg/m

g pr

otei

n

pg/m

g pr

otei

n

0 1 2 3 4 5

AlisporivirCsA

SCY-635DMSO

Fig. 4. IFNa, IFNk, and OAS-1 production by subgenomic Con1 Huh-7 cells.(A) Parental Huh-7 cells and (B) HCV Con1 Huh-7 cells. CsA, cyclosporine A.

JOURNAL OF HEPATOLOGY

vents the contact of these cyclophilins with specific componentsof the innate response. In this model, exposure of cells to SCY-635causes the dissociation of NS5A from the ternary complex andrestores the ability of the cell to produce IFNs and ISG productsin response to chronic viral infection. This model is consistentwith published reports indicating that NS5A, expressed from aplasmid in uninfected Huh-7 cells, suppresses IFNa promoteractivity [15] and is consistent with our own reports indicatingthat SCY-635 causes the dissociation of cyclophilin A fromNS5A at nanomolar concentrations [16].

The specific suppression of IFNb, a potent antiviral cytokine,did not attenuate the clinical antiviral activity of SCY-635. Thetreatment-associated increases in the plasma protein concentra-tions of IFNs a, k1, and k3 imply that cyclophilins play opposingroles in regulating the production of IFNb in comparison to IFNsa, k1, and k3. Our data suggest that cyclophilins may exert tonicrepression on the production of IFNs a, k1, and k3 through a reg-ulatory protein that is common to type I and type III IFN produc-tion. This putative repressor mechanism may be similar to whathas been described for the regulation of interleukin-2 tyrosinekinase activity by cyclophilin A [17].

Journal of Hepatology 20

The data presented in this study demonstrate that SCY-635exhibits potent clinical anti-HCV activity and stimulates produc-tion of multiple species of endogenous IFNs. The kinetics and themagnitude of the antiviral effect together with the correlation ofresponse with IL28B genotype is consistent with treatment-asso-ciated secretion of endogenous IFNs as being the primary driverof clinical antiviral activity for SCY-635. These data imply thatcyclophilins play critical roles in regulating type I and type IIIIFN responses to chronic viral infection.

Financial support

All financial support for the conduct of this study was providedby Scynexis, Inc.

Conflict of interest

Sam Hopkins, Betty DiMassimo, Pamela Rusnak, Ann Sluder, Ber-nard Scorneaux, Sarah Mosier, Paul Kowalczyk and Yves Ribeillare current or former employees of Scynexis, Inc. No otherauthors received any form of compensation, payment, or salaryfrom Scynexis, Inc. for conducting this research project.

Acknowledgments

We thank Edie Gavis for her tireless efforts to recruit and recon-sent subjects who participated in this study and Jennifer King,Ph.D., for critical editing of the manuscript.

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