The Hepatitis B Antivirals: Mechanism to Drug Development

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The Hepatitis B Antivirals Mechanism to Drug
Development

• Savita Srivastava, MD

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Outline

• Mechanisms for Therapeutic Approaches
• Replication Cycle
• Immune Mediated Injury
• FDA approved antivirals
• Alpha interferon (pegylated)
• Lamivudine
• Adefovir
• Entecavir
• Telbivudine
• Up and Coming Drug Therapies in the Pipeline
• This is NOT an exhaustive discussion on the
  outcomes data pertaining to the efficacy and
  durability of anti-virals!

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Replication Cycle
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Virology 101 Understanding Hepatitis B at its
Core

• Belongs to the family of hepadnaviruses which
  include duck, woodchuck, ground squirrel
  hepatitis viruses
• 42 nm in diameter
• Complete virion includes
• Envelope proteins and lipids
• Core nucleocapsid protein, viral genome,
  polymerase protein

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The Genome

• Genome consists of circular partially double
  stranded DNA 3200 base pairs in length consisting
  of 4 partially overlapping open reading frames
  (ORF)
• envelope (pre-S/S)
• Core (precore/core)
• Polymerase
• X proteins

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Genotypes A-H
DNA polymerase lacks proofreading activity
accounting for genetic differences
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HBV Replication Cycle Targeting Replication

• cccDNA conversion
• mRNA transcripts translate into the following
• 3.5 kd -gt HBeAg, DNA pol, nucleocapsid
• 2.4kd and 2.1 kd -gt HBsAg proteins for envelope
• minor mRNA -gt Xproteins

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DNA polymerase The target of current nucleoside
analogues
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First approved DNA polymerase inhibitor developed
at Yale!Doong S et al. PNAS 1991
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Immune-Mediated Injury
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Dissecting Apart the Immunologic Network Targets
for Therapy
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Cytolytic and Non-cytolytic action by
CTLsChisari F. Hepatology 1995

• Fas ligand (Fas-L) and peforin mediated apoptosis
  of infected hepatocytes
• CTLs secrete IFN gamma and TNF alpha that
  abolishes HBV gene expression and replication

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Host Immune Response Implicated in the
Development of Chronic HBV Infection

• Self Limited HBV infection vigorous,
  polyclonal, and multispecific cytotoxic and
  helper T cell response
• cytokine mediated inhibition of HBV replication
• CTL directed killing via perforin Fas ligand and
  TNF-alpha mediated death pathways
• Persistent HBV infection too weak to eliminate
  all HBV infected hepatoclytes but strong enough
  to destroy some and induce non-specific
  inflammatory response that progressive injures
  the liver and may lead to fibrosis/cirrhosis

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Possible Causes of Persistent Infection

• Viral
• High viral load
• High replication rate
• Viral inhibition of antigen presentation
• Viral mutations that antagonize antigen
  recognition
• Immunosuppressive effects of virus
• Host
• Immunologic tolerance
• Exhausted T cell response
• Insufficient co-stimulation of virus specific T
  cells
• Inefficient viral presenting cells
• Alteration of Th1 and Th2 balance

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Mechanism of Interferon alphaPeters M.
Hepatology 1996

• Increases expression of anti-viral genes that are
  not well understood in mechanism oligoadenylate
  synthetase, protein kinase, Mx protein homologue,
  beta 2 microglobulin

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FDA Approved Drugs for HBV Treatment
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But Wait!Who Should We Treat?
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Pegylated Interferon alpha-2a
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Pegylated Interferon alfa-2a

• Pegasys by Roche is the only pegylated interferon
  approved by the FDA for HBV treatment
• Structure covalent conjugate of recombinant
  alfa-2a interferon with single branched
  bis-monomethoxy polyethylene glycol chain
• Produced using DNA technology in which a cloned
  human leukocyte interferon gene is inserted and
  expressed in E. coli
• Mechanism of Action antiviral, immunomodulatory,
  antiproliferative

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Pegylated Interferon alfa-2a

• Dose 180 mcg/week SQ for 48 weeks (injectable
  solution in vials or in pre-filled syringes)
• Hepatic impairment ALT gt5x ULN consider
  decreasing to 135mcg/week or discontinuing until
  after flare subsides
• Renal dosing in ESRD and hemodialysis, use 135
  mg/ week
• Pharmacokinetics Maximal serum concentrations
  occur between 72-96 hours post dose ½ life
  50-120 hours

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Pegylated Interferon alfa-2a

• Contraindications hypersensitivity, autoimmune
  liver disease, cirrhosis with decompensation
• Adverse effects
• Neuropsychiatric Depression, suicidal ideations,
  psychosis
• Infections severe bacterial infections
• Bone Marrow toxicity
• Increased ALTgt10x ULN in 12-18
• Other flu-like symptoms, fatigue, N/V, headache,
  loss of appetite, irritability, fatigue
• Pregnancy category C

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Pegylated Interferon alfa-2a Drug Interactions
inhibit P450 CYP1A2

• Decrease metabolism
• Theophylline
• Warfarin
• Flurouracil
• Zidovudine

• Increase metabolism
• Melphalan

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DNA polymerase Inhibitors
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• Lamivudine
• Epivir by Glaxo-Smith Kline
• Structure Synthetic nucleoside dideoxy analogue
  of cytadine (-) enantiomer of the 2,3-dideoxy
  3thiacytidine
• Adefovir
• Hepsera by Gilead Sciences
• Structure Acyclic nucleotide analog of adenosine
  monophosphate

• Entecavir
• Baraclude by Bristol-Myers Squibb
• Structure cyclopentyl guanosine nucleotide
  analog
• Telbivudine
• Tyzeka by Idenix Pharmaceuticals and Novartis
• Structure synthetic thymidine nucleoside
  analogue

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Mechanism of Action

• Mechanism inhibits DNA polymerase (reverse
  transcriptase) via DNA chain termination after
  incorporation of the nucleotide or nucleoside
  analog into the viral DNA chain

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Lamivudine

• Dose 100 mg/daily for unclear duration lower
  doses in renal insufficiency no dose adjustment
  in hepatic impairment
• Treatment endpoints
• HBeAg positive One year of rx HBeAg
  seroconversion additional six months
  consolidation rx
• HBeAg negative unknown
• Emergence of lamivudine resistant virus
• Pharmacokinetics Cmax 0.9 hours (fasting) and
  3.2 hours (fed state) half life 5-7 hours

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Adefovir

• Dose 10mg/day for unclear duration modified
  dosing for renal impairment
• Treatment endpoints
• HBeAg positive One year of rx HBeAg
  seroconversion additional six months
  consolidation rx
• HBeAg negative Continue indefinitely if HBV DNA
  suppressed
• Emergence of adefovir resistant virus
• Pharmacokinetics Cmax is 0.58 4 hours ½ life
  is 7.5 hours

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Entecavir

• Dose 0.5 mg/daily in treatment naïve for unclear
  duration 1 mg/daily in patients with prior
  lamivudine resistance dose adjustment in renal
  insufficiency
• Treatment endpoints
• Continue rx indefinitely if virus is suppressed
  (no long term data yet)
• emergence of entecavir resistant virus
• Pharmacokinetics Cmax occurs between 0.5 and 1.5
  hours ½ life is 5-7 days

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Telbivudine

• Dose 600 mg/daily dose adjustment in renal
  insufficiency no dose change in hepatis
  insufficiency
• Treatment endpoints
• continue rx indefinitely if virus is suppressed
  (no long term data)
• emergence of entecavir resistant virus
• Pharmacokinetics Cmax 1-4 hours ½ life 40-49
  hours

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Adverse Events
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Drug-Drug Interactions Class Effects

• No cytochrome P450 interactions
• Excretion in the urine
• Pregnancy category B or C (see below)

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Drug-Drug InteractionsEach Drug

• Lamivudine
• Bactrim decreased clearance of lamivudine with
  concominant use
• Zalcitabine competative inhibitor
• Zidovudine increased level of drug by 39
• Adefovir Increased nephrotoxicity with
  concominant administration of
• Aminoglycosides
• NSAIDs
• Cyclosporine
• Vancomycin
• Entecavir and Telvibudine report no additional
  interactions

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Treatment Strategies HBeAgLok A and McMahon B.
Hepatology 2007
Anti-Viral Entecavir gt Telbivudine gt Lamivudine
gt Peg-IFN gt Adefovir Seroconversion PegIFN gt
DNA polymerase inhibitors
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Treatment Strategies HBeAg-Lok A and McMahon B.
Hepatology 2007
Anti-Viral Entecavir gt Telbivudine gt Lamivudine
gt Peg-IFN gt Adefovir
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Drug Resistance Hinders Current Rx
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Rate of Developing Resistance
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Cross Resistance

• Lamivudine and Entecavir in LAM-R
  mutants(rtL180M and rtM204V/I) cell based assays
  show 8-30 fold reduction in entecavir
  susceptibility
• Lamivudine and Telbivudine in LAM-R
  mutants(rtL180M and rtM204V/I) cell based assays
  show 1000 fold reduction in telbivudine
  susceptibility
• Adefovir and Telbivudine ADV-R mutant (rtA181v)
  showed 3-5 fold reduced susceptibility to
  telvibudine in cell based assays
• BOTTOM LINE Lamivudine and Entecavir
  resistant mutants are fully susceptible to
  Adefovir

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Evolution of the Multi-drug Resistant HBVYim HJ
et al. Hepatology 2006

• Multi-drug resistant HBV has been reported in
  patients who receive sequential anti-viral
  therapy secondary to initial lamivudine
  resistance
• Purpose To determine whether mutations which
  confer resistance to multiple agents co-locate on
  the same genome in vivo

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85 of isolated clones had mutations conferring
resistance to multiple drugs on the same
genome Clonal analysis of patient sera over a
period of time showed evolution of all clones
from 1) LAM-R clones only 2) LAM-R clones
multi-drug resistant clones 3) All clones are
multi-drug resistant
Conclusion Therefore, combination therapy will
NOT adequately suppress the multi-resistant
virus Future Consideration De novo combination
therapy?
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Combination Therapy The Next Frontier?
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One Combination May Prove BeneficialLamivudine
Adefovir in LAM-R virusFung SK et al. J
Hepatol 2006.

• Method 43 patients rx with Adefovir for gt6
  months followed for mean 18 months to determine
  rate of resistance 79 of these patients with
  LAM-R prior to initiating adefovir therapy
• Result Cumulative probability of adefovir
  resistance 22 at 24 months
• Multivariate analysis Patients with adefovir
  resistance were more likely to have had prior
  lamivudine resistance
• 0 of the 16 LAM-R patients who received
  combination therapy had adefovir resistance
• 28 of the 18 LAM-R patients who received
  adefovir monotherapy had adefovir resistance
• Conclusion In LAM-R patients, combination
  therapy may prevent development of adefovir
  resistance

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Part III

• Emerging Therapies
• Thinking outside the BOX

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The phase III pipeline More DNA polymerase
Inhibitors

• Goal of DNA polymerase inhibitors in the
  pipeline achieving high rates of DNA suppression
  BUT similar rates of seroconversion
• Limitations Many of the newer agents confer
  similar mutations and are cross-resistant with
  lamivudine YMDD motif mutation
• The Drugs
• Emtricitabine
• Tenofovir
• Clevudine

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Novel Approaches to Treatment

• Antisense Molecules
• Prevent transcription of mRNA and translation to
  protein by utilization of antisense molecules or
  ribozymes that are complementary to DNA and RNA
  templates
• Theoretical decrease in mutation risk targeting
  multiple sites in the DNA and RNA
• Current impediments rapid degradation of
  molecules in vivo studies, lack of effective
  delivery system
• Feasibility studies underway

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Nuclease Resistant Ribozymes Directed Against HBV
RNAMorrissey et al. Journal of Viral Hepatitis
2002.

• Ribozymes targeted to HBV decrease in HBV DNA,
  HBsAg, and HBeAg in cell culture study using
  human hepatoblastoma cell lines transfected with
  HBV cDNA
• 14 day administration of ribozymes in transgenic
  mice which express HBV DNA and proteins show
  similar results

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Nuclease Resistant Ribozymes Directed Against HBV
RNAMorrissey et al. Journal of Viral Hepatitis
2002.

• HBsAg in human hepatoblastoma cells

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Nuclease Resistant Ribozymes Directed Against HBV
RNAMorrissey et al. Journal of Viral Hepatitis
2002.

• HBeAg in human hepatoblastoma cells

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Nuclease Resistant Ribozymes Directed Against HBV
RNAMorrissey et al. Journal of Viral Hepatitis
2002.

• HBV DNA in human hepatoblastoma cells

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Novel Approaches to Treatment

• Immunomodulatory Therapy Thymosin
• Thymosin alpha-1 is a bovine thymus extract
• Can stimulate T cell function and further induce
  IFN-gamma, TNF-alpha to mediate non-cytolytic
  viral clearance
• Approved in Asia for rx

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Meta-analysis of Thymosin trialsChan HL.
Aliment Pharmacol Ther 2001
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Novel Approaches to Treatment

• New Vaccines
• S and pre S antigen vaccines (GenHevac)
• (Couillin I et al. J Infect Dis 1999)
• Plasmid DNA vaccination that contain HBsAg gene
  can induce an anti-HBS response in transgenic
  mice (Mancini M et al. Proc Natl Acad Sci USA
  1996)
• T cell vaccine with HBcAg CTL epitope escalates
  T cell response but shows weak anti-viral effect
  in phase II CY-1899 T cell Vaccine Study Group
  (Heathcote J et al. Hepatology 1999)

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Novel Approaches to Treatment

• Bone Marrow Transplant via adoptive immunity
  transfer
• Donors who are anti-HBS and anti-HBC positive has
  been associated with clearance of HBV in
  recipients with chronic HBV infection (HBsAg
  positive gt 12 month)
• Study of BMT recipients suggests that specific T
  cells primed against HBcAg is the primary
  mechanism of immunity transfer (Lau GK et al.
  Gastroenterology 2002)

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Bone Marrow Transplant StudyLau GK et al.
Gastroenterology 2002.
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Novel Approaches to Treatment

• Alpha-glucosidase inhibitors
• Glucosidases are important cellular enzymes in
  glycoprotein biosynthesis and protein folding
  (cellular and viral)
• Inhibitors may have a role in controlling
  assembly of complete virion for secretion and
  escalation of infection

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Novel Approaches to Treatment

• Monoclonal antibodies
• Antibodies against different epitopes of HBsAg
  could decrease surface antigen and DNA levels

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Novel Approaches to Treatment

• Fibrosis inhibitors
• Interferon gamma is a cytokine secreted by T
  cells that can inhibit the proliferation of
  hepatic stellate cells and reduce collagen
  synthesis