Viral Hepatitis: The Search for a Cure · PDF file“Viral Hepatitis: ... • Viral...

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2017 Drug Design and Delivery Symposium

“Viral Hepatitis: The Search for a Cure”

The 2017 DDDS is co-produced with ACS Division of Medicinal Chemistry and the AAPS

Michael Sofia Co-founder and Chief Scientific

Officer, Arbutus Biopharma

Stephen Mason Executive Director, Discovery,

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Michael J. Sofia, Chief Scientific Officer

Arbutus Biopharma, Inc.

17

Viral Hepatitis:

The Search for a Cure

Forms of Viral Hepatitis

Five forms of viral hepatitis: Hepatitis A, B, C, D, E

• Hepatitis A – Acute self-limiting infection

– Contracted by eating contaminated foods

– Rarely leads to permanent liver damage

• Hepatitis B – Acute infection can lead to chronic infection

– Contracted by vertical infection or from contaminated blood sources

– Lead to liver damage and HCC

• Hepatitis C – Acute infection can lead to chronic infection

– Contracted from contaminated blood sources

– Lead to liver damage and HCC

• Hepatitis D – Occurs only in conjunction with HBV

– Leads to a more sever form of HBV-related liver disease

• Hepatitis E – Typically only an acute self-limiting infection – problem in immune compromised

individuals

– Fecal to oral transmission route

18

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Chronic Viral Hepatitis: HBV & HCV

• Every third person on the planet shows evidence of infection with viral hepatitis

• 500 million people are chronically infected with hepatitis B or C

• 1 million die every year: 1 every 30 seconds

• Globally 57% of cirrhosis and 78% of primary liver cancer are due to these 2 diseases

• 80-90% of liver transplants associated with HBV & HCV infection

• The majority of those chronically infected are undiagnosed – hepatitis B and C are often asymptomatic for years

• The sheer size of the problem is intimidating - as many people are chronically infected with viral hepatitis in 2 African countries as there are people living with HIV/AIDS in the whole world

19

Summary of Epidemiology and Natural

History of Chronic Viral Hepatitis

• HCV – 170-200 Million infected – 20% lifetime risk of cirrhosis – 4% lifetime risk of HCC – Leading cause of liver transplant in North America

and Europe – No vaccine available

• HBV – 2 Billion ever infected – ~400 Million infected now – 1 Million die each year of HCC or cirrhosis – 25% life time risk for each HBsAg+ patient of HCC

or cirrhosis – Second most common carcinogen (liver cancer)

after cigarettes – Preventive vaccine available

• Linked to the co-existence of multiple co-morbidities

20

-30

yea

rs

Normal

Cirrhotic

HCC

20

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Challenge Question

ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT

• HIV

• HCV

• HBV

• None of the above

Which chronic viral disease has the highest worldwide prevalence rate?

21

HBV vs HIV vs HCV HIV HCV HBV

U.S. Prevalence 1 million 3 million 1.3– 3 million

Worldwide Prevalence 35 million 160 million 350 million

Percent Diagnosed in U.S. 80% 50% 30%

Percent Diagnosed Who Are

Treated in U.S.

70% 33% 6-10%

Nature RNA retrovirus RNA virus DNA virus

Virions Produced per Day 1010 1012 1013

Enzyme Targets for Therapy Multiple Multiple One

Curable? Unclear; lifelong suppression

with HAART therapy

Yes Unclear; lifelong suppression

with Nuc therapy

Why Easy / Difficult? Proviral DNA integrated into host

genome, difficult to eliminate

RNA virus existing in the host

cytoplasm; can eradicate with

cocktail of small molecules

DAAs

cccDNA inside the nucleus, also

integrated into host genome,

difficult to eliminate

Need Immune Component in

Therapeutic Regimen for

Cure?

Maybe No Maybe

Transmission Infected blood/needles, sex Infected blood/needles, sex Infected blood/needles, sex

Vertical Transmission Yes No Yes

Vaccine No No Yes

2115 U.S. Sales $9.3 billion $13.3 billion $700 million 22

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Can it become a disease of the past?

23

HCV: Prevalence, Total Infected, Genotype

24 Razavi H, Gower E, Estes C, Hindman S. Global HCV Genotypes. AASLD 2013; 2013 Nov 1-5; Washington, DC, United States.

North America Europe, Western Europe, Central Europe, Eastern Asia, Central Asia Pacific, High Income Asia, South

Asia, Southeast

Asia, East

Australasia

Sub-Saharan Africa, West Sub-S Africa, Southern Sub-S Africa, Central North Africa/Middle East Latin America, Andean Latin America, Southern

Latin America, Tropical

Latin America, Central

Caribbean

Total Infected (Viremic)

200K-650K

650K-1.9M

0-200K

1.9M-3.5M

3.5M-9.2M

Prevalence (Viremic)

0.0%-0.6%

0.6%-0.8%

0.8%-1.3%

1.3%-2.9%

2.9%-7.8%

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Growing Burden of Mortality Associated with

Viral Hepatitis in the US (1999-2007)

• National multiple-cause mortality data 1999-2007

• 73 % of HCV and 59 % of HBV-related deaths in persons aged 45-64

• Co-morbidities associated with increased odds ratio of mortality

– Chronic Liver Disease (32.1;HCV and 34.4;HBV)

– co-infection with other hepatitis virus (29.9;HCV and 31.5;HBV)

– Alcohol related (4.6;HCV and 3.7;HBV)

– HIV co-infection (1.8;HCV and 4.0;HBV)

• Mortality rates of HBV, HCV, and HIV; United States 1999-2007

Holmberg SD , et al. 62nd AASLD; San Francisco, CA; November 4-8, 2011. Abst. 243 25

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Hepatitis C Virus:

Morphology and Characteristics

• Nucleic Acid: 9.6 kb ssRNA(+)

• Classification: Flaviviridae, Hepacivirus

• Genotypes: 1 to 6

• Enveloped

• No known viral reservoir

• Does not integrate into host genome

27

Clotting factor treatment prior to 1987

Injection drug use

Injection treatments prior to universal precautions

Long-term hemodialysis

CDC, MMWR 1998; 47:4

High Risk of Infection

170 Million Infected

Worldwide

1 in 30

Baby Boomers

Infected

28

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The Hepatitis C Virus

HCV Lifecycle HCV Genome

Lindenbach, B and Rice, C., Nature, 2005, 436,933

• Error-prone RNA-dependent, RNA polymerase

- poor proofreading function

- high replication rate in vivo

~9.6 kb genome: 0.1-1 error per RNA synthesized

Replication Rates

HCV HIV

1012 1010-1011

29

Key Target Areas of Drug

Discovery Focus and Key Drugs

Adapted from : Liver International pages 69-78, 23 DEC 2013 DOI: 10.1111/liv.12423 http://onlinelibrary.wiley.com/doi/10.1111/liv.12423/full#liv12423-fig-0001

Protease Inhibitors Telaprevir

Boceprevir

Asunaprevir

Grazoprevir

Simeprevir

Paritaprevir

NS5A Inhibitors Daclatasvir

Ledipasvir

Velpatasvir

Ombitasvir

Elbasvir

Polymerase Inhibitors Nucleosid(t)e

Sofosbuvir

Non-nucleoside

Dasabuvir

Beclabuvir

30

http://onlinelibrary.wiley.com/doi/10.1111/liv.12423/full#liv12423-fig-0001






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Challenge Question

ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT

• Harvoni® (sofosbuvir + ledipasvir)

• Viekira Pak® (ombitasvir + paritaprevir + dasabuvir +ritonavir)

• Zepatier® (grazoprevir + elbasvir)

• Sovaldi® (sofosbuvir) + RBV

What was the first IFN-free HCV cure therapy to be approved by the US FDA?

31

FDA Approved IFN-Free

HCV Cure Drug Combinations

32

2013 2014 2015 2016 2017

Year and Order of Approval

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Time

SVR

(G

eno

typ

e 1

)

Tre

atm

en

t D

ura

tio

n (

Wee

ks)

48

24

16

08

0

1989 1998 3x Weekly Weekly Weekly Weekly Daily

~33-36%

~42-46%

90%

~94 - 99%

2001 2011 2013 2014

100%

60%

80%

40%

20%

10%

0% 3x Weekly

~10-11%

RBV

+ Harvoni

RBV

+

Protease

+ RBV

+ RBV

+

Sovaldi

+

IFN IFN PEG IFN PEG IFN PEG IFN

~67- 75%

The History of HCV Therapy Development

33

HCV Curative Therapy Today

• IFN-Free curative therapies are a reality

• Simple oral fixed-dose and short duration therapies

• >95% cure rates across multiple genotypes

• High cure rates in difficult to treat patient populations

• Patient access is the issue • HCV can become a rare disease in the future

34 34

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Is there a path to a cure?

35

Hepatitis B Virus (HBV)

• Hepadnaviridae member that primarily infects liver cells • DNA virus • 100 times more infective than HIV

• Found in blood and body fluids – Able to survive in dried blood for longer than 1 week

• Viral reservoir: cccDNA in nucleus of hepatocytes • Small segments of viral DNA do integrate but do not code for viral

proteins

Ott et al. J Pediatr Health Care. 1999;13(5):211-216.

Ribeiro, et al. Microbes and Infection. 2002;4:829-835.

MMWR. 2003;52:1-33. 36

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Chronic Hepatitis B: By The Numbers

1 WHO. Available at: www.who.int/csr/disease/hepatitis/en/. 2 Ferlay et al. Globocan 2002, Cancer incidence, mortality and prevalence worldwide, IARC Press, Lyon 2004. 3 Records of the thematic press conference of the Ministry of Health of the PRC at April 21, 2008, from the website of the Ministry of Health of the People's Republic of China; 4 Ulmer, T et al. (2007). European orientation towards the better management of hepatitis B in Europe; 5. CDC. Hepatitis B FAQs for Health Professionals. Available at http://www.cdc.gov/hepatitis/HBV/HBVfaq.htm#overview.

•7 HBV Genotypes (based on complete HBV genome): A-G

– A – World-wide

– B & C – Asia

– D – Southern Europe, Middle East

– E – Africa

– F – South America, Polynesia

– G – USA and Europe

More than 350 million or 1 in 20 people worldwide have chronic hepatitis B infection1

(Compared with the 33 million living with HIV2)

14 million in Europe3,4

1.46-2 million people in

the United States are

chronically infected5

112 million in Asia-Pacific

(93 million people in China)1,2

7 HBV Genotypes (based on complete HBV genome): A-G A – World-wide B & C – Asia D – Southern Europe, Middle East E – Africa F – South America, Polynesia G – USA and Europe

1 Million die each year of HCC or cirrhosis 25% life time risk for each HBsAg+ patient of HCC or cirrhosis Second most common carcinogen (liver cancer) after cigarettes

37

The Hepatitis B Virus

Glebe, D., et al, Sem. Liver Dis, 33, 2013, 103

Genome Structure of HBV

• 4 Promoter elements

• 2 enhancer elements

• 10 start sites

5 mRNAs:

• Pregenomic/core/pol (3.5 kb)

• Precore (3.5 kb)

• PreS1 (2.4 kb)

• PreS2/S (2.1 kb)

• X (0.7 kb)

Source: Gerlich, W. 2013. Virology Journal,

10:239 38

http://www.who.int/csr/disease/hepatitis/en/





http://www.cdc.gov/hepatitis/HBV/HBVfaq.htm









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Transmission of HBV

6% infected after age 5 years become chronically infected

90% infected infants become chronically infected

Horizontal Transmission

Host Recipient

Child-to-Child

Contaminated Needles

Sexual

Health Care Worker

Transfusion

Vertical Transmission

Mother

Infant

Perinatal

No clear risk factors in 20-30% of patients

CDC Fact Sheet. http://www.cdc.gov/ncidod/diseases/hepatitis/b/. Accessed: October 2, 2004.

Lee. N Engl J Med. 1997;337(24):1733-1745.

Lavanchy. J Viral Hepat. 2004;11(2):97-107. 39

Three Phases of Chronic HBV Infection

Source: Gerlich, W. 2013. Virology Journal, 10:239

40

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REVEAL-HBV:

Clearance of HBV DNA Reduces Risk of HCC

• REVEAL-HBV study cohort (N = 2946; aged 30-65 yrs)

– Pts recruited 1991-1992, serum markers evaluated every 6-12 mos until June 30,

2004; HCC rates followed until December 31, 2008

• HBV DNA suppression independently associated with significantly reduced

risk of HCC

– Pts with HBeAg suppression (n = 185) still had high HBV DNA levels and still at

high risk of HCC

– HBsAg suppression not associated with reduced incidence of HCC, but study not

powered to detect difference

• Greatest reduction in HCC incidence observed among pts with high baseline

HBV DNA (≥ 100,000 copies/mL) who cleared HBV DNA during follow-up

– HCC incidence highest in pts HBeAg seropositive throughout follow-up

41

HBV Approved Therapies

Nucleosides/Nucleotides

Tenofovir

Alafenamide VEMLIDY® Gilead Sciences 2016

Tenofovir VIREAD® Gilead Sciences 2006

Telbivudine TYZEKA™ Idenix/Novartis 2006

Entecavir BARACLUDE™ Bristol-Myers Squibb 2005

Adefovir Dipivoxil HEPSERA™ Gilead Sciences 2002

Lamivudine EPIVIR-HBV® GlaxoSmithKline 1998

Interferons

Peginterferon

alfa-2a PEGASYS® Roche Laboratories 2005

Interferon alfa-2b

recombinant INTRON® A Schering/Merck 1992

Preferred Therapies – AASLD Guidelines

42

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Relative Efficacy of Approved HBV Therapies

Entecavir1,2 Tenofovir3 PEG-IFN α-2a4,5

HBeAg positive n = 354 n = 176 n = 271

HBV DNA undetectable 67% 76% 25%a

HBeAg seroconversion 21% 21% 27%

ALT normalisation 68% 68% 39%

HBsAg loss 2% 3.2% 2.9%b

HBeAg negative n = 325 n = 250 n = 177

HBV DNA undetectable 90% 93% 63%a

ALT normalisation 78% 76% 38%

HBsAg loss 0.3% 0% 0.6%b

Results at 48 weeks a HBV DNA < 400 copies/mL; b At 72 weeks

1. Chang T-T, et al. N Engl J Med 2006;354:1001–10. 2. Lai C-L, et al. N Engl J Med 2006;354:1011–20. 3. Marcellin P, et al. N Engl J Med 2008;359:2442–55.

4. Lau GKK, et al. N Engl J Med 2005;352:2682–95. 5. Marcellin P, et al. N Engl J Med 2004;351:1206–17.

43

Therapy

HB

V D

NA

ch

an

ge f

rom

ba

seli

ne

(lo

g 1

0 c

/mL

)

0.0

-1.0

-2.0

-3.0

-4.0

+1.0

Time

Long-term Therapy is Required

to Maintain Viral Suppression

HBsAg

HBVDNA

cccDNA

Werle et al, Gastroenterology 2004 44

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What Does a Cure Look Like?

Functional Cure Absolute Cure

Clinical Scenario As if recovery after

acute HBV infection

As if never infected

HBsAg Negative Negative

Anti-HBsAg Positive Positive

Serum HBV DNA Not Detected Not Detected

HBV cccDNA Detected, but not

transcriptionally

active

Not Detected

Hepatic integrated

HBV DNA

Detected Not Detected

Current Status Achievable in a few

patients

Not yet achievable

Jiang, et al., DDW, 2016 45

HBV Chronic Infection

CD4+ T cell

CD8+ T cell Antigen presenting cell

Nucleus Cytoplasm Hepatocyte

Vesicular transport to cell membrane

Endoplasmic reticulum

Core particle plus strand

synthesis

HBV

Core particle (capsid)

Repair

Transcription

Translation

Core particle minus strand synthesis

Core assembly and RNA packaging

46

• 1013 virons produced per day

• Infection is not cytopathic

• Outcome of infection and

severity of associated liver

disease are determined by

nature and magnitude of host

immune response

HBV Viral Life Cycle

46

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HBV and the Host Immune Response

• Inhibition of innate immune signaling

• Inhibition of HBV specific T cell responses

• Inhibition of antibody responses to HBV

• Outcome: Immune tolerance, chronicity 47

How to Achieve a Cure?

• Control viral replication

– Cripple the virus

• Reactivate the host immune response

– Release immune tolerance

• Clear cccDNA

Fu

ncti

on

al

Cu

re

Ab

so

lute

Cu

re

48

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HBV Cure: Potential DAA Drug Targets

49

HBV Cure: Emerging Strategies

Viral Attachment Inhibition

• Preclinical and Clinical POC

• Clinical results modest and variable

• Effects in HDV also

50

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Entry Inhibition: Myrcludex B

1. Infection of PHH-transplanted uPA/SCID mice

with HBV for 3 weeks

2. Treatment of infected mice with Myrcludex B

for another 3 weeks blocks HBV spread

Myrcludex B monotherapy in chronically infected patients

Myrcludex reduces HBV DNA by 0.8log (w12) 2016 AASLD/EASL HBV Workshop Sept. 8th 2016

51

Nucleoside Prodrugs

CMX157

Phase II Clinical Development Launched as Vemlidy®

Liver Targeted Tenofovir Prodrugs

Advantages

• Increase drug levels in liver

• Reduce renal and bone toxicity associated with Tenofovir

52

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• Hepatitis B virus replication is strictly

dependent upon capsid assembly

around pregenomic RNA (pgRNA) prior

to rcDNA synthesis and subsequent

cccDNA synthesis.

• Assembly of HBV nucleocapsid is

dependent on ordered folding of the

viral capsid protein.

• Interfering with HBV capsid assembly

with small molecule inhibitors has been

shown to translate into antiviral activity

in vitro and in vivo and constitutes a

novel mechanism that is distinct from

the nucleos(t)ide analogues currently

available for clinical use. HBV capsid assembly pathway and examples of inhibitors [core protein allosteric modulators (CpAM)]

Inhibition of HBV Capsid Assembly

and pgRNA Encapsidation

Class II Class I

53

First Clinical POC of Capsid Inhibitors (NVR-3-778)

IFN alone

IFN + NVR

NVR 3-778

d28 HBV DNA d28 HBV RNA

(log10 from BL) (log10 from BL)

NVR 3-778 -1.72 -0.82

PegIFNa-2a -1 -0.73

NVR3 -778 + PegIFN -1.97 -1.51

Treatment

(NVR 3-778 @ 600 mg BID; Peg-IFN 180 ug/wk)

Summary Table

Yuen et al EASL 2016 Oral Presentation

NVR 3-778 + PEG IFNalpha-2a Combo in treatment naïve HBeAg positive patients

NH

Ar S

O OO

NR

R'R"

54

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Capsid Assembly Inhibitor AB-423

In vitro AB-423 showed:

additive/synergistic activity in combination with Nucs and RNAi

agents

potent activity against HBV NucR variants and pan-genotypic

activity

no significant activity against unrelated viruses

AB-423 inhibited cccDNA synthesis during de novo HBV

infection of C3AhNTCP cells

Data suggests AB-423 has a dual mode of inhibition:

Inhibits encapsidation of pgRNA during ongoing infection

Inhibits cccDNA synthesis presumably via inhibition of the

capsid uncoating step

0.0

1.0

2.0

3.0

4.0

Infected MyrcludexB

IFNα AB-423

cop

y n

um

ber

/cel

l

cccDNA

0.0

0.5

1.0

1.5

Infected Myrcludex IFN-α AB-423

pgR

NA

re

lati

ve le

vel

pgRNA

H B V $ e n t e r s $ c e l l $

N T C P $ ( r e c e p t o r ) $

I n t r a c e l l u l a r $ a m p l i f i c a : o n $

V e s i c u l a r $ t r a n s p o r t $ t o $ G o l g i $ a n d $ c e l l $ $

m e m b r a n e $

D N A $ s y n t h e s i s $

C o r e $ a s s e m b l y $ R N A $ p a c k a g i n g $

p g R N A $ C o r e $ P o l $

T r a n s l a : o n $

C y t o p l a s m $

E n d o p l a s m i c $ r e : c u l u m $

T r a n s c r i p : o n $

R e p a i r $ c c c D N A $

P r o t e i n F f r e e $ v i r a l $ D N A $

C a p s i d $ d i s a s s e m b l y $

H B V $ $ v i r i o n $

M u l : v e s i c u l a r $ $ $ b o d y $

I n t e r m e d i a t e $ C o m p a r t m e n t $

S u r f a c e $ a n : g e n $

N u c l e u s $

S u b v i r a l $ p a r : c l e s $

x

x

D u a l m o d e o f i n h i b i ti o n b y c a p s i d a s s e m b l y i n h i b i to r s

0 7 1 4 2 1 2 8

0 .1

1

1 0

1 0 0

D a y

Se

rum

HB

V D

NA

(%

Pre

-Do

se

)

V e h ic le

A B -4 2 3 , 1 0 0 m g / k g B ID

A B -4 2 3 + E T V

A B -4 2 3 + P e g a s y s

E T V

55

Capsid (Core Protein) Assembly Inhibitors

Class I Class II

Heteroaryldihydropyrimidine (HAP) Propenamides

Sulfonylbenzamides

56

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29

Capsid Assembly Inhibitor Patent Landscape

Novira

NH

Ar SN

O OO

n

OH

NH

ArD

BS

OE A

O

NR

R'

Janssen

NH

Ar XS

N

OO

O

NH

R

X

R '

ArN

O

N

OR'

R

NH

Ar

O

O

N

O R'

R

N

R4

NH

Ar S

O OO

NR

R'

NH

O

NH

Ar SN

O OO

n

OH

NH

S

NH

O

HN R

Assembly

R" R"

R"

O

N

O

NH

Ar

XY

R

Roche

N

N

O

NH

Ar

N

R

NH

N

R

O

O

R'

R"

ArNH

N

N

O

MeO F

F

Cl

F

BAY-41-4109

NH

N

O

EtO

Br

F

N

O

S

N

GLS-4

Bayer

Sunshine Lake Pharm

57

Crystal Structure of Bound

Capsid Assembly Inhibitor

- Proc. Natl. Acad. Sci. 2015, 112, 15196.

HAP (Class I)

58

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30


Inhibit HBsAg Production or Secretion

RNAi Approach Nucleic Acid Polymers

Sequestration Small Molecules

• Clinical and preclinical

POC

• Potency & safety

• Clinical POC?

• MOA?

• Preclinical in vitro

POC

LNP Delivery

Triple Trigger

Chol-siRNA

GalNAc-siRNA

~40 mer

?

59

Controlling S-Antigen Production

via RNAi (ARC-520)

• Preclinical study in 9 HBV-

infected chimpanzees (9-37y)

• NUC pretreatment 8-24w,

repeated injections of ARC-

520

• Dose escalation study (single dose i.v.):

• 32 HBeAg-, 8 HBeAg+ CHB patients

under ENT

• Well tolerated up to 4 mg/kg under

pretreatment with oral antihistamine

• Reduction of serum HBsAg up to

0.5 log (HBeAg- patients)

0.7 log (HBeAg+ patients)

• Reduction of HBeAg up to 2 log

Program Terminated do to tox signal

60

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Controlling S-Antigen Production

via RNAi (ARB-1467)

0

20

40

60

80

100

120

0 7 14 21 28 35 42

Seru

m H

BsA

g as

%

Bas

elin

e

Day

Serum HBsAg

Untreated TKM-HBV 0.3 mg/kg

Treatments

Liver cccDNA Liver Serum

HBV DNA

n=5, mean ± SEM

0

25

50

75

100

125

Day 42 (terminal analysis)

% U

ntr

eate

d a

t D

ay 4

2

PXB Mouse Study

Human Clinical Study • Single-dose results show

significant reductions in serum

HBsAg levels

• Multi-dose results show a step-

wise, additive reduction in

serum HBsAg Reductions of ≥ 1.0 log10 in 3/5 patients (after 3 monthly doses at 0.4 mg/kg)

• LNP Delivery Technology

• Triple trigger RNAi

Streinu-Cercel, A., et al., EASL 2017, Abst # SAT-155 61

Controlling sAg via

Nucleic Acid Polymers (NAPS)

Al-Mahtab M, Bazinet M, Vaillant A (2016) Safety and Efficacy of Nucleic Acid Polymers in Monotherapy and Combined with Immunotherapy in Treatment-Naive Bangladeshi Patients with HBeAg+ Chronic Hepatitis B Infection. PLoS ONE 11(6): e0156667. doi:10.1371/

Mono therapy with REP 2139-Ca

62

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Reducing or Eliminating cccDNA

Long term nuc treatment results in multi-log

reduction of cccDNA pool

IFN treatment inhibits transcription and capsid stability, reduces cccDNA pool

pgRNA to rcDNA conversion

rcDNA to cccDNA

Conversion cccDNA

decay?

Virus

entry

Can inhibition

contribute to reduction

of cccDNA pool?

63

cccDNA Formation and Stability

Capsid conformational

shift

rcDNA deproteinization

--TDP2 implicated but

unconfirmed

Completion of (+) strand

and removal of RNA primer

---PolK implicated

DNA ligation of both

strands over gap

--Factors? DNA ligase 1+ 3 implicated

Chromatization

--Complexed with H3 and H4 histones,

acetylation regulates HBV replication

Regulation of expression

--HBX destabilizes SMC5/6

episomal silencing complex

--HBX itself is likely transcribed very

early, active at low levels

Modulation of cccDNA copy number

in non-dividing cells?

--IFN/TNF/LTß upregulation of

APOBEC3A

What We Know and What We Don’t Know

64

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33

LOW-REPLICATIVE STATE HIGH-REPLICATIVE STATE

Regulating cccDNA Transcription

Histones

PCAF p300 PCAF

p300 Sirt1

Sirt1 HDAC1 HDAC1

Histones

Pollicino et al. Gastroenteroplogy 2006

Levrero et al. J Hepatol, 2009

Lucifora et al, J Hepatol 2012

Belloni et al, PNAS 20O9

Belloni et al, J Clin Invest 2012

Epigenetic regulation:

- Histones acetylases, deacetylases,

methyltransferases

- Transcription factors

- Binding of viral proteins: HBc & HBx

Silencing

Interferon alpha,

Capsid inhibitors,

Epigenome modifyers

Epigenetic Control of cccDNA

65

Gene Editing

NHEJ – Non-Homologous End-Joining; results in short mutations, insertions and deletions (indels)

HR – Homologous Recombination; accompanied by donor DNA, capable to insert / replace sequence

Targeted DNA Cleavage Endpoints

Meganucleases/Homing endonucleases (HEs)

Zinc Finger Nucleases (ZFNs)

Tal-effector nucleases (TALENs)

CRISPR/Cas9

Engineered Endonucleases

cccDNA: A Target for Gene Editing

Nishimasu et al, Cell 2014 Stone et al, Curr Opin HIV/AIDS 2013

66

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34

cccDNA: A Target for Gene Editing

Gene Editing: Targeting HBV with CRISPR/Cas9

• Co-transfection of 1.3x WT HBV and sgRNA-Cas9-2A-mCherry plasmid by HDI in mice, followed by monitoring viral markers in mouse blood

• Total HBV DNA and cccDNA exhibit dramatic, increasing reductions over time

67

Immunmodulation:

Challenges on the Path to a Cure

1. Heterogeneous host immunity among HBV patients. -what is a clinical biomarker for host immune re-awakening?

2. Lack of understanding of the immunological function

of viral proteins.

-all inhibitory? or stimulatory?

HBV cure

68

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35

HBV Cure: Potential Immune

Modulatory Drug Targets

69


Bertoletti A, Gehring AJ (2013) Immune Therapeutic Strategies in Chronic Hepatitis B Virus Infection: Virus or Inflammation Control?. PLoS Pathog 9(12): e1003784. doi:10.1371/journal.ppat.1003784

Restoration of Antiviral Immunity

70

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36

TLR7 Agonist GS-9620

HBV DNA Levels

HBsAg Levels

GS-9620

Woodchuck Study

• 5 mg/kg QOD, 4-8 weeks

• Mean Max viral load decline of 6.1

2.9, and 5.8 observed

• sAg levels reduced to undetectable

in 100% of animals

• Reduced sAg levels were sustained

after cessation of therapy

Human Clinical Study

• Discontinued due to lack of

efficacy

• Dose limiting toxicity?

Menne, S, et al., J. Hepatology, 2015, 62, 1237-1245 71

RIG-I Agonist: SB9200

• Dinucleotide

• Reduction in

serum HBV DNA

• Reduction in sAg

levels

• Induction of ISGs

• Induction of type 1

IFN

Fletcher SP, Menne S (2015) PLOS Pathogens (Sept. 2015). 72

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37

STING Activation Controls HBV Replication

and Induces Cytokine Production

73

2’3’ cGAMP bisphosphorothioate Human/multi-species active

DMXAA Mouse STING agonist

Un

treate

d

mIF

N-a

DM

XA

A -

12.5

mg

/kg

cG

AM

P -

12.5

mg

/kg

cG

AM

P -

25 m

g/k

g

5

6

7

8

Se

ru

m H

BV

DN

A

Lo

g1

0(C

op

ies

/mL

)

Dose

2 3 1

Un

treate

d

mIF

N-

12.5

mg

/kg

12.5

mg

/kg

25 m

g/k

g

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

IL-6

(p

g/m

L)

B L O Q

D o s e

1 2 3

c G A M PD M X A A

Un

treate

d

mIF

N-

12.5

mg

/kg

12.5

mg

/kg

25 m

g/k

g

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

IP-1

0 (

pg

/mL

)

B L O Q

c G A M PD M X A A

• STING expressed in hepatocytes (low level), antigen presenting cells and T cells

• An innate immune adaptor that regulates responses to cytosolic/viral dsDNA

Thi, E. et al., ICAR, 2017 Abs # 135

HBV Cure: The Drug Discovery Landscape

Immune modulation • Toll-like receptors

agonists, Gilead, Roche

• Anti-PD-1 mAb, BMS, Merck, Roche, Medimmune, others

• Vaccine therapy Transgene, Gilead, Roche Innovio, others • STING: ABUS • Cyclophilin:

Contravir • RIG-I: Springbank,

Rigontec

Zoulim F, et al. Antiviral Res 2012;96(2):256–9; HBF Drug Watch, Available at: http://www.hepb.org/professionals/hbf_drug_watch.htm.

HBx

Endosome

rcDNA

cccDNA

Polymerase

pgRNA

Core

Surface

proteins

Entry inhibitors • Lipopeptides, e.g.

Myrcludex-B

Targeting cccDNA • ABUS, JNJ, Chromis, Enyo, Intellia, Gilead, Precision Bio

Inhibition of nucleocapsid assembly, JNJ, Assembly, Gilead, Janssen, Roche, ABUS, Enanta, Sunshine Lake

Polymerase inhibitors • Nucleoside :Gilead,

BMS, CoCrystal, Contavir

Targeting HBsAg ABUS, Replicor, Roche RNA interference,

ABUS, ARWR, ALNY, GSK/Ionis, Arcturus/JNJ

74

http://www.hepb.org/professionals/hbf_drug_watch.htm

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38

Current Pipeline of

Investigational Agents

Phase I Phase II Phase III Launch Preclincal

Tenofovir Alafenamide

Myrcludex B

REP 9AC’

GS-9620

JNJ-379

Assembly ABI-H0731

NVR3-778

75

ALN-HBV

Ionis-HBVrx SB-9200

AB-423 Enyo FXR

ARB-1740

Aicuris AIC 649

Chomis cccDNA

CoCrystal CRISPR

CMX157

Enanta Capsid

INtellia CRISPR

Precision Gene Editing

JnJ/Arcturus RNAi

GS-5801 Dicerna RNAi

RG-7834

Therapeutic Vax

Nucleotide

TLR Viral Entry Inhibitor

HBsAg Release Inhib.

RNAi

Capsid Inhibitor

Cyclophilin Inhibitor

RIG-I

Gene Editing

ARB-1467

75

Combination Therapy

• General belief that no single approach will be sufficient to deliver a cure

• As in HCV and HIV combinations of drugs with different MOA will be the solution

• Which combination will deliver the ultimate “cure” is yet to be determined

• How to assess combinations pre-clinically that may guide clinical studies is developing

76

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39

0 7 1 4 2 1 2 8 3 5 4 2 4 9 5 6 6 3 7 0

5

6

7

8

D a y

Se

rum

HB

V D

NA

(lo

g1

0c

op

ies

/mL

)

Combination Therapy

6 wk Treatment Phase 4 wk Follow-Up

0 7 1 4 2 1 2 8 3 5 4 2 4 9 5 6 6 3 7 0

0 .0 1

0 .1

1

1 0

1 0 0

D a y

Se

rum

HB

V D

NA

(%

Ba

se

lin

e)

V e h ic le fo r A B -4 2 3

A B -4 2 3

A B -4 2 3 + E T V

A B -4 2 3 + P e g IF N

P e g IF N

A R B -1 7 4 0

A B -4 2 3 + A R B -1 7 4 0

E T V

A B -4 2 3 + A R B -1 7 4 0 + P e g IF N

A B -4 2 3 + A R B -1 7 4 0 + E T V

LLOQ

Dosage Route Frequency

AB-423

(Capsid Assembly inhibitor)

100 mg/kg PO BID

ETV (Nuc) 1.2 µg/kg PO QD

PegIFN (Immune Stimulator)

30 µg/kg SQ 2×/wk

ARB-1740 (RNAi)

3 mg/kg IV biweekly 0 7 1 4 2 1 2 8 3 5 4 2 4 9 5 6 6 3 7 0

1

2

3

D a y

Se

rum

HB

sA

g(l

og

10 I

U/m

L)

6 wk Treatment Phase 4 wk Follow-Up Lee, A., et al., AASLD 2016, Abs # 232 77

Key Challenges in Finding an HBV Cure

• How to completely control viral replication?

• How to address the virus’ ability to control the host immune response?

• How to eradicate the viral reservoir, cccDNA?

• What is the best combination of MOA?

• Can significant reduction in the duration of therapy be achieved?

78

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40

HBV: Is There a Path to a Cure?

• Increased focus by both academic and industry labs well beyond historic levels

• Many new targets and strategies under investigation

• Increased efforts to understand the virus and how the host immune system responds to the virus

• Combination of drugs with different MOA have the potential to deliver major therapeutic advances

79

1966 1982 1983

HBV Discovered

HBV RT identified

1987

IFNα as OT

Lamivudine

2004 1992 2002

Adefovir

2003 2006

HBV cccDNA identified

1994

HEP2.2.15 cells identified

Non-cytolitic immune control

2008 2010 2011 2012 2013 2016

Capsid Inhibitor identified

Entecavir

Telbivudine

Tenofovir

A Cure Yet To Be Realized: HBV

?

CU

RE

1998

PegIFN

50 years without a cure

80 But light is at the end of the tunnel

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81








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2017 Drug Design and Delivery Symposium Save the Date for the next webinar!

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