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CRV431: An Optimized Cyclophilin Inhibitor with Multiple Anti-HBV Activities, High Selectivity Index, and Synergy with CMX157

Robert Foster1, John Kulp2, Daren Ure1, Daniel Trepanier1, Philippe Gallay3 1Contravir Pharmaceuticals, Edison, NJ 2Baruch S. Blumberg Institute, Doylestown, PA 3Scripps Research Institute, La Jolla, CA

CRV431 CRV431, a novel cyclophilin inhibitor (“CPI”), designed to be layered

on top of HBV therapeutic drugs including NUC backbone

CMX157 CMX157, a nucleotide (“NUC”) analogue of tenofovir, designed to

reduce viral load and serve as backbone HBV therapy

IDEAL THERAPEUTIC DRUG COMBINATION Additive to synergistic, with wide in vitro Selective Index (“SI”) to

optimally position combination for wide Therapeutic Index (“TI”) in clinic, while reducing exposures compared with monotherapy

Targets multiple stages of the HBV life cycle

INTRODUCTION

CRV431 has less cytotoxicity than other cyclophilin inhibitors

Each curve = one experiment in one cell type

Cells plated in triplicate at moderate density and treated for 3 days with compounds or DMSO vehicle. Viability assessed with resazurin-based (metabolic) assay and normalized to DMSO.

Primary Human Cells

Renal epithelial Bronchial smooth muscle Dermal fibroblast Umbilical vein endothelial

Human Cell Lines

HepaRG hepatocyte Jurkat lymphocyte

•In vitro anti-HBV IC50 ≈ 0.03 - 0.7 µM •Anticipated plasma Cmax and Ctrough ≈ 2 µM and 0.7 µM (based on alisporivir clinical efficacy)

COMPARISONS

Other studies show that NTCP inhibition by cyclosporin analogs occurs through a cyclophilin-independent mechanism.

Uptake of HBVpreS peptide-FITC by NTCP-Huh7 cells (generous gift from Dr. Urban).

CRV431 IC50 ≈ 1 µM

NTCP-Huh7 (infection assays) and Huh7 (transfection assays). CRV431 treatment begun prior to infection and transfection. Measure extracellular HBsAg by ELISA on Day 6 post-infection or Day 3 post-transfection.

7.9

2.5

0.79

0.25

0.079

-40%

-20%

0%

20%

40%

60%

80%

100%

0.16

0.5

1.6

516

50

80%-100%

60%-80%

40%-60%

20%-40%

0%-20%

-20%-0%

-40%--20%

SELECTIVE INDEX MODEL

CRV431 is a highly potent cyclophilin inhibitor

Cyclophilin A inhibition was assessed with the chymotrypsin-coupled isomerase assay using 10 nM recombinant cyclophilin A and succinyl-AAPF-pNA peptide substrate.

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0 .0 0 0

0 .0 0 5

0 .0 1 0

0 .0 1 5

0 .0 2 0

0 .0 2 5

0 .0 3 0

C s A

A lis p o r iv ir

C R V 4 3 1

D ru g n M

Ca

taly

tic

K (

s-1

)

IC 5 0

1 .8 n M

2 .8 n M

1 6 .8 n M

C R V 4 3 1

% V

iab

ilit

y

1 1 0 1 0 0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

IC 5 0 = 2 3 .6 M

C sA M

% V

iab

ilit

y

1 1 0 1 0 0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

IC 5 0 = 1 2 .6 M

A lis p o riv ir M

% V

iab

ilit

y

1 1 0 1 0 0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

IC 5 0 = 1 5 .2 M

A . B . C .

ANTI-HBV ACTIVITIES

CRV431 blocks HBV DNA replication in AD38 cells

CRV431 + CMX157 COMBINATION

CONCLUSIONS

CRV431 inhibits NTCP-mediated HBV entry

D ru g c o n c e n tra t io n ( M )

HB

V p

ep

tid

e u

pta

ke

via

NT

CP

(flu

ore

sc

en

ce

un

its

)

0.0

3125

0.0

625

0.1

25

0.2

50.5 1 2 4 8 1

6

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

A lis p o r iv ir

C R V 4 3 1

D ru g c o n c e n tra t io n ( M )

% I

nh

ibit

ion

HB

V D

NA

0 .0 1 0 .1 1 1 0 1 0 0

0

2 0

4 0

6 0

8 0

1 0 0

0 0 .02

C R V 4 3 1 E C 5 0 = 2 9 n M , E C 9 0 = 6 8 n M

A lis p o r iv ir E C 5 0 = 1 - 5 M

E m a x = 4 0 - 5 0 %

E m a x = 9 0 - 1 0 0 %

CRV431 blocks HBeAg and HBsAg production and/or secretion in infected and transfected cells, unlike entecavir

H B e A g

C R V 4 3 1 M

HB

eA

g n

g/m

l

0 .1 1 1 0

0

1

2

3

P o s t - in f e c t io n D a y 6

P o s t - t r a n s fe c t io n D a y 3

E n te c a v ir C o n tro l (H B e A g )

HB

eA

g n

g/m

l

D M S O E n te c a v ir D M S O E n te c a v ir

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

3 .0

3 .5

IN F E C T IO N T R A N S F E C T IO N

p 0 .0 1

H B s A g

C R V 4 3 1 M

HB

sA

g p

g/m

l

0 .1 1 1 0

0

1 0 0

2 0 0

3 0 0

4 0 0P o s t - t r a n s fe c t io n D a y 3

P o s t - in f e c t io n D a y 6

E n te c a v ir C o n tro l (H B s A g )

HB

sA

g p

g/m

l

D M S O E n te c a v ir D M S O E n te c a v ir

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

IN F E C T IO N T R A N S F E C T IO N

p 0 .0 1

CRV431 NON-VIRAL EFFECTS

Novel drug combinations targeting multiple HBV activities are needed to eliminate HBV.

Optimized selective index of CRV431 may provide for enhanced clinical utility.

Possible utility may be further enhanced by combination therapy with CMX157.

CRV431 has a wide SI, as defined by the ratio of CC50 to IC50 in vitro

The SI of CRV431 is the widest of any known CPI, potentially offering a wide TI in patients

Thus far, CRV431 addresses many of the identified endpoints relevant to HBV drug therapy including:

Reduction of HBV DNA

Suppression of HBeAg and HBsAg

Inhibition of viral entry via NTCP

CRV431, in combination with CMX157, is synergistic (reduction of HBV DNA)

CRV431 has potential beneficial effects on progression of liver fibrosis

CRV431 inhibits HBV synergystically with CMX157

AD38 cells treated with multiple combinations of CRV431 and CMX157 for 5 days. Measurement of intracellular HBV DNA.

% In

hib

itio

n

Cyclophilins are implicated in fibrotic mechanisms, such as collagen maturation, degradation, and hepatic stellate cell activation.

CRV431 IN VIVO

CRV431 is suitable for oral dosing

Single oral dose of CRV431 at 10 mg/kg in 6 male and 6 female rats. Male and female rats showed similar responses.

H o u r

Co

nc

en

tra

tio

n i

n b

loo

d (

M)

0 4 8 1 2 1 6 2 0 2 4

0 .0

0 .5

1 .0

1 .5

C s A

C R V 4 3 1

M e a n s S D , n = 6 /g ro u p

AU

C0

-24

(

mo

lh

rm

l-1

)

C s A C R V 4 3 1

0

2

4

6

8

1 0

1 2

1 4

1 6

CRV431 additionally reduces liver fibrosis through mechanisms independent of viral inhibition

Vehicle 5 m/k/d CRV431 20 m/k/d CRV431

NASH Model of Liver Fibrosis in Mice (Stelic Research, Japan)

CRV431 TREATMENT

V e h ic le 5 m g / k g / d 2 0 m g / k g / d

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Sir

ius

re

d-p

os

itiv

e f

ibro

sis

(%o

f s

am

ple

d a

re

a)

C R V 4 3 1 C R V 4 3 1

n s

p < 0 .0 1

C o n c e n t r a t io n ( M )

Fra

cti

on

of

ma

xim

al

re

sp

on

se

0 .1 1 1 0 1 0 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

CRV431 effect

ALV toxicity

ALV effect

CRV431 toxicity

= CC50/IC50

= 23,600 nM / 29 nM (AD38)

= 814

CRV431 Selective Index in AD38 cells (HBV DNA)

Selective index depends on cell type and marker (e.g. HBV DNA, HBeAg, HBsAg)