CYP450 Induction in vitro

Tested in human hepatocytes.

Low potential for drug-drug interactions.

Dimenthyl Sulfoxide

CMX157

CMX157

CMX157

Omeprazole

Phenobarbital

Rifampin

1.00 ± 0.19

1.03 ± 0.24

1.05 ± 0.16

1.08 ± 0.20

44.9 ± 24.5

2.60 ± 0.59

2.04 ± 0.27

0.1% (v/v)

1 M

10 M

100 M

100 M

750 M

10 M

Phenacetin O-dealkylation

(CYP1A2)Treatment Concentration

Bupropionhydroxylation

(CYP2B6)

Testosterone 6 -hydroxylation

(CYP3A4/5)

1.00 ± 0.26

0.954 ± 0.117

0.974 ± 0.098

0.849 ± 0.356

12.8 ± 9.0

20.1 ± 7.8

10.3 ± 4.5

1.00 ± 0.09

1.15 ± 0.09

1.10 ± 0.09

1.40 ± 0.09

2.56 ± 0.42

7.02 ± 0.66

6.32 ± 0.33

Fold Increase

• CMX157 is a novel, lipid conjugate prodrug of tenofovir. It is highly liver targeted, with rapid uptake and e�cient conversion to TFV-PP, the active moiety. CMX157 demonstrated potent anti-HBV activity and was at least as active as TAF in vitro.

• CMX157 showed no signi�cant potential for cytotoxicity, mitochondrial toxicity, CYP450 or drug transporter interactions.

• These results support ongoing clinical studies to determine the safety, pharmacokinetics and antiviral activity of CMX157 in healthy subjects and subjects with chronic HBV infection.

Conclusions

P H A R M A C E U T I C A L S

Preclinical Characterization of CMX157,A Novel Tenofovir Prodrug for the Treatment

of Chronic Hepatitis B InfectionContraVir Pharmaceuticals, Inc. 399 Thornall Street, 1st Floor Edison, NJ 08837

JSullivan-Bolyai@ContraVir.com

Background

Novel lipid conjugated prodrug of tenofovir designed to utilize lipid uptake pathways in order to:• Increase bioavailability• Enhance target tissue penetration for increased antiviral potency• Decrease renal and bone toxicity by reducing circulating TFV

Method

Results

AD-38 cells: Three independent experiments, run on di�erent days,in triplicate.

CMX157 is at least as active as TAF.

1x10-11

100

90

80

70

60

50

40

30

20

10

0

1x10-10 1x10-9

HBV DNA Inhibition

1x10-8

Inhibitor Concentration (M)

Perc

ent I

nhib

ition

1x10-7 1x10-6 1x10-5

CMX-157

TAF

TDF

Test Article EC50 (nM)Mean ± SD

EC90 (nM)Mean ± SD

CMX-157

TDF

TAF

9.3 ± 3.6

52.7 ± 16.8

32.4 ± 17.1

186 ± 53

776 ± 427

474 ± 261

Antiviral Activity in vitro

AD-38 cells, two separate experiments, triplicate wells.

CMX157 delays viral rebound longer than TAF and other antivirals.Results are consistent across two di�erent experimental conditions.

Viral Rebound in vitro

CMX IC90

110.0%

90.0%

70.0%

50.0%

30.0%

10.0%

-10.0%TAF IC90

HBV

DN

A C

opy

Num

ber

TFV IC90

All Antivirals Tested at IC90

TDF IC90 DMSO 3TC

Day 6

Day 11

Day 15

DMSO

1E+7

1E+6

1E+5

1E+4

1E+3

1E+2

1E+1

1E+0

TDFTFV

TAF

CMX-157 Tet3TC

HBV

DN

A C

opy

Num

ber

Day 6

Day 9

Day 13

All Antivirals Tested at 5 nM

Metabolism in PrimaryHuman Hepatocytes

Rapid, e�cient conversion to TFV-PP.Hepatocytes fully viable.Conversion is time and concentration dependent.

Metabolite Production over Time

0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

20 40 60 80

Time (hours)

TFV_10 uM CMX-157

TFV_50 uM CMX-157

TFV-PP_10 uM CMX-157

TFV-PP_50 uM CMX-157

Time Point

TFV, nM

0 00

65.191.3238275395

00

70.784.3135143188

46.050.394.9152392460860

00

76101185175361

148

244872

TFV-PP, nM TFV-PP, nMTFV, nM

Systemic, Portal Vein and HepaticPharmacokinetics in Rats

CMX157 20 mg/kg, single oral dose in Sprague-Dawley rats.

Rapid, e�cient extraction and conversion to high liver levels of the active antiviral, TFV-PP after single dose in rat • 86% �rst pass extraction by the liver • Low systemic levels of TFV

CMX157

Drug Concentration in Liver (± SD)

100,000

10,000

1,000

100

100 6 12 18 24

Hours Post Dosing

Conc

entr

atio

n [n

g/g]

TFV

TFV-PP

CMX157 - Systemic Plasma

CMX157 - Portal Plasma

TFV - Portal Plasma

TFV - Systemic Plasma

0

10,000

1,000

100

106 12 18 24

Conc

entr

atio

n [n

g/m

L]

Hours Post Dosing

Drug Concentration in Hepatic Portal Veinvs. Systemic Circulation (± SD)

Whole Body Tissue Distribution in Rats

Single dose, whole body autoradiography in L-E rats.

Highly liver targeted.No substantial accumulation or retention in the heart.

Time after Dose Administration (h) Time after Dose Administration (h)

Tissue Concentrations of Radioactivity After OralAdministration of 20 mg/kg 14C-CMX157 to Rats

Tissue Concentrations of Radioactivity After IVAdministration of 2 mg/kg 14C-CMX157 to Rats

00.010

0.100

1.000

10.000

100.000

1000.000

10 20 30 40 50 00.010

0.100

1.000

10.000

100.000

10 20 30 40 50

Small intestine

Liver

Kidney (cortex)

Kidney (medulla)

Plasma

Heart

Tested in standard tissue culture and vesicle-based assays.

Low potential fordrug-drug interactions.

Compound00

< 10

00

11

< 10< 10

< 100000

0 0

93*32

< 10< 1011

< 10< 10

BSEPMRP2BCRP

OATP1B1OATP1B3

OAT1OAT3OCT2MATE1MATE2-K

P-gp* > 26,000 fold above protein-adjusted Cmax at 400 mg SD

% Inhibition

Cellular Transporter Inhibition in vitro

Tested in human liver microsomes.

Low potential for drug-drug interactions.

CYP450 Inhibition in vitro

Enzyme Zero-Minute Preincubation 30-Minute Preincubation

CYP1A2CYP2B6CYP2C8CYP2C9CYP2C19CYP2D9CYP3A4/5CYP3A4/5CYP3A4/5

3918

4.4*113127122418

3215

3.3*152826121916

Direct Inhibition Time-DependentInhibition

*Value for CYP2C8 is > 3,500 fold above the protein-adjusted Cmax at 400 mg SD

HepG2 cells in a standard assay.

Low potential for mitochondrial toxicity.

Mitochondrial Toxicity in vitro

Viable Cells (% vehicle)

MediumVehicleChloramphenicol

Glucose Galactose Glucose Galactose

CMX 157

TDF

TFV

0.11

10100

0.11

10100

0.11

10100

30

SDH-A: COX-I Ratio

117.3100.0

20.7

83.288.370.4

76.069.362.054.7

* Insu�cient cells for analysis

68.261.757.059.8

2.12.32.02.3

1.71.82.01.8

106.583.265.4

1.92.22.2

2.12.01.9

****

********

72.261.5

94.429.0

2.42.3

1.92.1

14.0 10.1 6.8

102.8100.0

2.32.0

2.21.9

Tested in a large panel of proliferating or non-proliferating cell lines for up to 14 days. Tissues represented include liver, brain, heart, kidney, lymph, muscle, cervix, blood, bone marrow and gut.

Low potential for cytotoxicity.

Cell Line Tissue Origin

3 D

ay In

cuba

tion

6 D

ay In

cuba

tion

12 o

r 13

Day

Incu

bati

on14

Day

Incu

bati

on

HepG2 LiverKidneyBrain

ColorectalPBMC

Muscle

LymphCervicalCervical

LiverLiver

Blood

Blood

Blood

Brain

Liver

Blood

Heart

>100.0>100.0>100.0

>100.0>100.0>100.0

40.216.046.6

61.6

>10.0>10.0>10.0>10.0

8.8>10.0

7.4

7.4

>10.0

>10.0

>10.0

12.5>100.0>100.0>100.0>100.0>100.0

>100.0>100.0>100.0>100.0>100.0>100.0

>100.0

>100.0

>100.0

>100.0

>100.0

28.086.057.7

Proliferating>10.0>10.0>10.0>10.0>10.0>10.0

Non-proliferating

Proliferating

Non-proliferating

>10.0

>10.0

>10.0

>10.0

>10.0

Non-proliferating>100.0 >100.0

Caki-1SNB-78COLO-205CCRF-CEMSJCRH30

CEM-SSHeLaME 180Huh-7HepG2PBMC (stimulated)

PBMC (unstimulated)MonocyteMacrophageDendritic

Hepatocyte

PBMC (unstimulated)

Colorectal 12.9>100.036.0COLO-205Muscle 9.1>100.017.4SJCRH30

Heart 12.8>100.0>100.0Cardiomyocyte

BoneMarrow

1.894.722.46GM-CFU0.53.484.62E-BFU

Cardiomyocyte

Proliferating

Cytotoxicity in vitro

R Rush1, J Greytok2, T Matkovits2, JZ Sullivan-Bólyai2, and D Standring3 1Allon Preclinical Consulting LLC, USA; 2ContraVir Pharmaceuticals Inc., USA;

and 3David Standring Consulting, USA.