CONCLUSIONS RESULTS ACKNOWLEDGEMENTS REFERENCES ...

CRV431 and CMX157 (TXL; tenofovir exalidex): Anti-HBV combination effects in vitro between a cyclophilin inhibitor and a nucleotide prodrug R. FOSTER1, D. TREPANIER1, D. URE1 , J. GREYTOK1, J.L. KULP2, P. GALLAY3 1 Contravir Pharmaceuticals Inc, Edison, New Jersey, USA and Edmonton, Alberta, Canada 2 Baruch S. Blumberg Institute, Doylestown, Pennsylvania, USA 3 The Scripps Research Institute, La Jolla, California, USA

INTRODUCTION

RESULTS

CONCLUSIONS

It is expected that a cure for HBV will require drug

replication and propagation. Our lead drug, tenofovir

CRV431 and TXL independently inhibit HBV replication in HepAD38, DE19, and DES19 cells

concentrations of TFV, while minimizing off-target effects

allows for suppression of HBV DNA, HBsAg, HBeAg, inhibition of viral

6000

H B V D N A c o p ie s /w e ll

phosphonate designed to deliver high intrahepatic

T X L a lo n e 5000

C R V 4 3 1 a lo n e 4000

SYNERGY PLOT

C R V431 IC 5 0 = 3 3 n M

2000

D E 1 9 C e lls

1000 6000 0 4

8

16

32

64

128

256

512

1024

D r u g c o n c e n t r a tio n ( n M )

earlier-stage molecule, is a host targeting antiviral that inhibits cyclophilins, namely cyclophilin A (cypA), a peptidyl prolyl isomerase. As a cypA inhibitor, CRV431

T X L a lo n e C R V 4 3 1 a lo n e 4000

reduces HBV DNA, suppresses HBsAg, inhibits viral

3000

METHOD The current study measured inhibition of intracellular HBV

Shipman MacSynergy. Additionally, CRV431 cytotoxicity was examined in a number of primary human cells and

cyclosporine, alisporivir, and sanglifehrin A, to confirm cell viability in the assays.


-6 - -39

extend to drugs with other modes of activity including, for example, core inhibitors.

ACKNOWLEDGEMENTS

1000 0 4

8

16

32

64

128

256

512

1024

We greatly thank Drs. Christoph Seeger and Ju-Tao Guo for the


4000

HepAD38, HepDE19 and HepDES19 cells.

TXL IC 5 0 = 2 1 5 n M

3000

C R V431 IC 5 0 = 3 6 n M

SYNERGY PLOT

2000 1000

8

16

32

64

128

256

512

1024

SYNERGY

136.98

95% confidence interval

222 - 52

ANTAGONISM

-13.88


0 - 27

SYNERGISTIC


HepAD38, HepDE19 (HBeAg-deficient) and HepDES19 (HBeAg- and HBsAgdeficient) cells were incubated for two weeks with tetracycline to suppress HBV replication. Tetracycline was then removed and cells treated with increasing concentrations of the indicated drugs. After 3 days, medium was removed and replaced with fresh medium and drugs. After 3 days (6 days of drug exposure), intracellular HBV DNA was purified and quantified by qPCR. HBV DNA data are expressed as number of intracellular HBV DNA copies per well in triplicate.

Phillips S et al. Alisporivir inhibition of hepatocyte cyclophilins reduces HBV replication and hepatitis B surface antigen production. Gastroenterology 2015; 148:403-414. Shimura S et al. Cyclosporin derivatives inhibit hepatitis B virus entry without interfering with NTCP transporter activity. J Hepatol 2017; 66:685-692

SYNERGY PLOT SYNERGY

175.8


270-82

ANTAGONISM

-3


ns

SYNERGISTIC DMSO

( flu o r e s c e n c e u n its )

Tian X et al. Hepatitis B virus (HBV) surface antigen interacts with and promotes cyclophilin A secretion: Possible link to pathogenesis. J Virol 2010; 84:3373-3381 Prichard-Shipman MacSynergyII – manual and spreadsheet https://www.uab.edu/medicine/peds/macsynergy

90000

V ia b le c e lls

REFERENCES Gallay PA et al. The novel cyclophilin inhibitor CPI-431-32 concurrently blocks HCV and HIV-1 infections via a similar mechanism of action. PLOS One 2015; 10(8):e0134707

CRV431 and other cyclosporins are much less cytotoxic than sanglifehrin A (alternate class of cyclophilin inhibitor)

C y c lo s p o r in A A lis p o riv ir

60000

C R V431 S a n g life h r in A 30000

S a p o n in 0 .5 % ( p o s itiv e c o n tr o l)

SYNERGISM/ANTAGONISM 0-25 = additivity (log volume < 2)

25-50 = mild synergism (log volume = 2-5)

50-100 = moderate synergism (log volume = 5-9)

>100 = strong synergy (log volume > 9)

0 0.0625 0.125

0.25

0.5

1

2

4

8

D r u g c o n c e n t r a t io n ( µ M )

cell lines, utilizing DMSO and 0.5% saponin as negative and positive controls, respectively, comparing


C R V 4 3 1 a lo n e

640 nM. Both drugs were tested in vitro in AD38, DE19,

concentration versus effect was evaluated using Prichard-

-22.86

5000

alone, and in combination with CMX157 ranging from 0-

triplicate wells, using DMSO as control. Drug

ANTAGONISM

2000

DNA at concentrations of CRV431 ranging from 0-320 nM

and DES19 cells. Studies were each conducted twice, in

324 - 140

T X L a lo n e

AIM measuring HBV DNA levels.


complementary actions of CRV431 with CMX157 may reasonably

C R V431 IC 5 0 = 3 5 n M

6000

4

combination anti-HBV effects of CMX157 and CRV431 by

232.15

TXL IC 5 0 = 2 1 7 n M

0

The aim of the current study was to investigate the

SYNERGY

SYNERGISTIC

5000

D E S 1 9 C e lls

impede HBx-cypA binding.

entry, and blocking of cypclophilin A binding to HBx. The

TXL IC 5 0 = 2 1 2 n M

3000

caused by high levels of circulating TFV. CRV431, our

uptake via NTCP and, more recently, has been shown to

therapeutic drug strategy towards the cure of HBV. This strategy

A D 3 8 C e lls

exalidex (TXL; formerly CMX157), a tenofovir (TFV) prodrug, is a novel lipid acyclic nucleoside (NUC)

Combination treatment with CRV431 and TXL inhibits HBV synergistically (Prichard-Shipman MacSynergyII)

exploits the complementary modes of action of the two drugs, which


combinations that interact at more than one stage of viral

CRV431 and CMX157, tested in combination, represents a viable

HepG2 cells in 96-well plates were treated with the indicated compounds for 3 days. Cell viability was then assessed with the Live/Dead Assay involving flow cytometric measurement of calcein-AM and the DNA-binding ethidium homodimer.

HepAD38, HepDE19 and HepDES19 cells (triplicate) were treated with increasing concentrations of the two indicated drugs. Antagonistic, additive and synergistic effects were analyzed by quantification of intracellular HBV DNA by qPCR and analysis by the MacSynergyII program. This program is based on the Bliss independence model that is defined by the following equation: Exy = Ex + Ey − (Ex × Ey), where Exy is the additive effect of drugs x and y as predicted by their individual effects, Ex and Ey.

CONTACT INFORMATION Dr. Robert Foster Chief Scientific Officer, Contravir Pharmaceuticals Inc. 399 Thornall Street, 1st Floor, Edison, New Jersey 08837 [email protected] 780-909-5041