Zadaxin

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This APASL 2006 breakfast session is sponsored by
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Thymosin a1 In The Treatment of Chronic
Hepatitis B
Rong-Nan Chien M.D. Liver Research Unit Chang
Gung Memorial Hospital and University Keelung,
Taiwan
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Hepatitis B Virus Infection
, perinatally
. Decompensation, cirrhosis, HCC
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A dynamic state of interactions
HBV-DNA








1400
HBeAg
Anti-HBe
1200
1000
ALT (U/L)
800
600
400
200
0
0
4
8
12
16
20
24
28
32
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Months of Follow-up
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chronic HBV infection
30-40
60-70
HBeAg ()
HBV-DNA ()
inactive carrier state
active hepatitis
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0.1
26
24
cirrhosis
decompensation
death / transplantation
/year
HCC
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Goals of Therapy
Hepatitis virus elimination/suppression
HBeAg, HBV-DNA, HBsAg loss Reduction of
hepatic necroinflammation hepatitis
resolution prevent decompensation
Prevention of disease progression
hepatitis flares/decompensation
cirrhosis and/or HCC Improvement in survival
Initial/maintained
Sustained/durable
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?N (n36)

N1.5xN (n72)

1.52.5xN (n77)

2.55xN (n70)

510xN (n85)

1025xN (n105)

gt25xN (n105)
gt 5x ULN
Spontaneous HBeAg Clearance
2.5- 5x ULN
Liaw JGH 1997
Higher ALT more vigorous CTL response higher
clearance
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Treatment Options for Chronic HBV
Immunomodulating

TH

TS

-

TC
-

Immunosuppressives
NK

IFNs
-
-
Antivirals
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Advantages and Limitations of Current Therapies
for CHB

• More economical than IFN (if given for 1 year
  only)
• Well tolerated
• Durability of response is lower than IFN
• Long-term therapy associated with increasing risk
  of drug-resistant mutants

• Active against lamivudine-resistant mutants
• Low resistance
• More costly than lamivudine
• Durability of response, long-term safety and risk
  of drug-resistance unclear

• Finite duration of treatment
• More durable response (HBsAg loss/seroconversion
  )
• Lack of drug-resistant mutants
• Peak-related unfavourable safety profile

• Finite duration of treatment
• More durable response (HBsAg loss/seroconversion
  )
• Lack of drug-resistant mutants
• Improved pharmacological profile allows
  once-weekly dosing
• Less favourable safety profile compared with NAs

Lamivudine
Adefovir
IFN?
PEG IFN?2a
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Treatment Options for Chronic HBV
X
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Biological Activities of Thymosina1
Cytokines ?interferon a and interferon ?
?interleukin 2 (IL-2) and IL-2 receptor
affinity Cell of the Immune System
?differentiation/ production/ activity of
?natural killer cells ?lymphocytes (CD3,
CD4, CD8)
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Thymosin-?1 Dual Mechanism of Action
No significant adverse effects reported
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HBV Monotherapy Regimen
T0
Thymosin
T6
1.6 mg sc biw
Thymosin
T12
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20
21
22
23
24
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
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Month (s)
Chien et al Hepatology 1998
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Taiwan trial patient demographics
Chien et al. (1998) Hepatology 27(5)
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Cumulative and Delayed Response to Thymosin
Untreated Control
6 Month
12 Month
Chien et al Hepatology 1998271385
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PBMC Proliferation and Cytokine Response to rHBcAg
Cytokine Response
Stimulation Index
Peak ALT (U/L)
HBV
Outcome
Acute 1 1345 7.0 Th1 SR 2 1706 8.0
Th1 SR 3 1434 5.4 Th1 SR T?1-treated 1
800 7.2 Th1 CR 2 677 7.0 Th1 CR 3
550 5.5 Th1 CR 4 89 4.0 Th1 CR 5
573 16.0 Th1 CR 6 292 3.6 Th1Th2 NR IFN
-treated 1 2280 5.9 Th1 CR 2 1424 5.0
Th1 CR 3 926 7.7 Th1 CR 4 108
3.0 Th1Th2 NR
Tsai et al 2003
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Thymalfasin monotherapy in chronic hepatitis B
HBeAg()
HBeAg (-)
Thymalfasin 1.6 biw x 6m
IFN-a 5Mu tiw x 6m
No treatment
30
50
25
40
20
30
15
HBV DNA / HBeAg negative
HBV DNA negative/ ALT normal
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10
10
5
0
0
6 mo after end
at end
at end
6 mo after end
Andreone et al. (1996) Hepatology 24 774
Mutchnick et al. (1999) J Viral Hep 6 397
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HBV Thymalfasin monotherapy
Patients
Methods Opened labeled. Randomized 317 patients
enrolled (49 sites) 1.6mg b.i.w vs. 0.8mg
b.i.w 6 months therapy 12 months follow-up
283 patients completed Tx F U.
139 patients had 1.6mg Ta1 b i w
144 patients had 0.8mg Ta1 b i w


Japan phase 3 trial
Iino, May 2005, Journal of Viral Hepatitis
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Demographics
Iino, May 2005, Journal of Viral Hepatitis
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HBV Thymalfasin monotherapyEnd of Treatment
Japan phase 3 trial
Patients serological status at 6 months (end of
Tx)
Iino, May 2005, Journal of Viral Hepatitis
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HBV Thymalfasin monotherapyEnd of 12 month
Follow-up
Japan phase 3 trial
Patients serological status at 18 months (end of
study)
Iino, May 2005, Journal of Viral Hepatitis
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HBV Genotypes
Kao et al. J Gastroent Hepatol 2002
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Demographic and Clinical Characteristics of
Patients With or Without Thymosin a1 Treatment
a,P0.034 b,P0.027 c,P0.007 d,P0.019
e,P0.003 f,P0.059 g,P0.006
Chien RN et al. J Viral Hepat 2006 in press
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Comparison of Patients With HBV Genotype B and C
Chien RN et al. J Viral Hepat 2006 in press
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Stepwise Logistic Regression Analysis on Clinical
Features and Complete Response
Chien RN et al. J Viral Hepat 2006 in press
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Baseline Characteristics of Patients With and
Without Response to Thymosin a1 Therapy
Chien RN et al. J Viral Hepat 2006 in press
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Thymosin-?1 therapy in patients with chronic
hepatitis B
Ta1 1.6 mg 2x/w x 26 wk is effective
Arresting HBV replication Reducing lobular
activity Delayed complete response
increase gradually after end of therapy Safe
and effective alternative HBV therapy
Combination with direct antivirals reasonable
approach!
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Summary
? HBV genotype, precore stop codon mutation
and Ta-1 therapy major determinants to
complete response. ? Patients with genotype B
and presence of precore stop codon mutation
showed significantly better response to Ta-1
therapy.
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Thank You for Your Attention
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Combination therapy with interferon and thymosin
alpha 1 SciClone Breakfast Symposium APASL
Manila 2006 A/Prof Lim Seng Gee, FRACP, FRCP,
FAMS, MD Chief, Dept of Gastroenterology
Hepatology Principal Investigator National
University Hospital Immunovirology Group National
University of Singapore Centre for Molecular
Medicine Singapore ASTAR, Singapore
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Therapy for Chronic Hepatitis B entering exciting
phase

• Licensed treatments are increasing
• Lamivudine
• Adefovir
• Entecavir
• Interferon peg-interferon
• Thymosin (Asia)
• Potential to optimise therapy based on
  combination therapy

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Combination studies to date for HBeAg CHB

• combination monotherapy
• Immunomodulator
• nucleoside
• IFN-lamivudine 33 15 lamivudine
• Peg-IFN-lam 27 32 Pegasys
• 19 lamivudine
• Dual nucleos(t)ides
• Lamivudine-ADV 19 20 lamivudine
• Lamivudine-LdT no difference to LdT alone

Barbaro, J Hepatol 2001
Sung, EASL 2003
Lau, NEJM 2005
Lai, Gastroenterol 2005
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Combination immunomodulator therapy - rationale

• HBeAg seroconversion is an immunological event
• Nucleos(t)ides have not been able to increase
  HBeAg seroconversion rate gt 25 despite potency
• A more intense immunological approach may
  optimise response

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immunomodulators

• Interferon
• Acts through upregulation of MxA protein, 2-5
  Oligosynthetase, a protease dependent pathway,
  activation of cytokines
• Meta-analysis shows significant effect over
  placebo with HBeAg loss 33

• Thymosin
• Improves T cell function and maturation, enhanced
  T cell cytotoxicity, NK cell function and
  increased B cell function
• Thymosin shows significant benefit over placebo
  with HBeAg seroconversion of 25-40.9

Wong, Ann Int Med 1993
Chien, Hepatol 1998
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Combination Low-Dose Lymphoblastoid Interferon
and Thymosin alpha 1 (thymalfasin) in the
Treatment of Chronic Hepatitis B

• Rasi G et al
• J Viral Hep 1996 3191-6

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Combination Low-Dose Lymphoblastoid Interferon
and Thymosin alpha 1 (thymalfasin) in the
Treatment of Chronic Hepatitis B

• STUDY DESIGN
• Phase II Open Label
• Six months treatment
• Six months observation
• Additional six months observation
• 15 patients, 11 previous IFN non-responders
• Rasi G. et al. J Viral Hep 1996

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Combination Low-Dose Lymphoblastoid Interferon
and Thymosin alpha 1 (thymalfasin) in the
Treatment of Chronic Hepatitis B

• DOSAGE
• Loading dose Ta1 1 mg SC for 4 days
• IFN 3 mu IM day 4
• Maintenance dose (week 2-26)
• Ta1 1mg SC biw
• IFN 3 mu IM biw

Week 0
Week 26
Month 12
Month 18

• END POINTS
• ALT Normalization
• HBV-DNA clearance

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Combination Low-Dose Lymphoblastoid Interferon
and Thymosin alpha 1 (thymalfasin)in the
Treatment of Chronic Hepatitis B

• RESPONSE -Normal ALT
• HBV-DNA -ive (Abbott assay)
• No. of patients Total treated IFN Failures
• Responding____N15________N11__________
• 6 mo. 6 (40) 4 (36)
• 12 mo. 8 (53) 5 (46 )
• 18 mo. 9 (60) 6 (55)
• HBsAg loss 6 (40)

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Combination Low-Dose Lymphoblastoid Interferon
and Thymosin alpha 1 (thymalfasin) in the
Treatment of Chronic Hepatitis

• CONCLUSIONS
• The combination of Ta1 IFN results in greater
  response rates than would have been expected with
  IFN alone.
• The combination of Ta1 and IFN appears to be
  extremely promising therapy esp with a high
  remission rate, and surprising high HBsAg loss
• No additional toxicity due to Ta1 (thymalfasin)
  was seen.

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Long-term Outcomes of Thymosin alpha-1 and
Interferon alpha-2b Combination Therapy in HBeAg
Negative Chronic Hepatitis BSaruc M et al,
Journal of Pharmaceutical Sciences, 2003, 9,
1386-1395
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Study design

• 52 pts with anti-HBe CHB
• Non-randomised clinical trial
• ThymosinIFN (gp 1)
• IFN alone (gp 2)
• IFNLAM (gp 3)

• Entry criteria
• ALTgtULN
• DNAgt2.5pg/ml (Abbot)
• Exclusion criteria
• Cirrhosis
• HIV, HCV, HDV, AICAH

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• Study Design

G1 n27
G2 n10
G3 n15
Week
0
26
52
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LBx
LBx
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Definitions of Response

• Primary endpoint
• SVR loss of HBV DNA normalisation of ALT 6m
  post-treatment
• Virological response loss of HBV at wk 52 (End
  of Treatment)
• Biochemical response normalisation of ALT at wk
  52 (End of Treatment)
• Secondary endpoint
• 2 point improvement in Knodell histology score

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Baseline characteristics
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Long Term Response Rates By Treatment Group



SVR
(TThymalfasin, IFN Interferon, LLamivudine)
plt0.05
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Conclusions

• Thymalfasin in HBeAg -ive disease
• Significant inhibition of HBV DNA maintained for
  12-18 months post-therapy with combination
  therapy
• Possible role of Ta1 in maintaining long-term off
  treatment remission

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A randomized placebo controlled trial of
Thymosin alpha 1 and lymphoblastoid interferon
for HBeAg Chronic Hepatitis B (AntiViral
Therapy-in press) Seng Gee Lim,1 Chun-Tao Wai,1
Yin Mei Lee,1 Yock Young Dan,1 Dede Selamat
Sutedja,1 Aileen Wee,2 Shirley Suresh,3 Ying Juan
Wu,3 David Machin,3 Chee Chian Lim,4 Kwong Ming
Fock,5 Evelyn Koay,6 Scott Bowden,7 Steven
Locarnini,7 and Shamsuddin Mohammed Ishaque8
1Department of Gastroenterology and Hepatology,
National University Hospital, Spore 2Department
of Pathology, National University Hospital,
Singapore 3Clinical Trials and Epidemiology
Research Unit, Singapore 4Department of
Medicine, Tan Tock Seng Hospital,
Singapore 5Department of Medicine, Changi General
Hospital, Singapore 6Molecular Diagnostic
Laboratory, National University Hospital,
Singapore 7Victoria Infectious Diseases Reference
Laboratory, Melbourne, Australia 8Department of
GI, Bangabandhu Sheikh Mujib Medical University,
Bangladesh
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Investigator initiated multicentre study-
acknowledgements

• National Medical Research Council
• Grant NMRC/0072/1995 NMRC/0427/2000
• SciClone Pharmaceuticals
• Supplying Thymosin placebo
• National University Hospital, Singapore
• Chun-Tao Wai, Yin Mei Lee, Yock Young Dan, Dede
  Selamat Sutedja, Aileen Wee, Evelyn Koay
• Tan Tock Seng Hospital, Singapore
• Chee Chian Lim, Kenneth Liew, Wei Lyn Yang

• Changi General Hospital, Singapore
• Kwong Ming Fock, Eng Kiong Teo
• Bangabandhu Sheikh Mujib Medical University,
  Bangladesh
• Shamsuddin Mohammed Ishaque
• Clinical Trials and Epidemiology Research Unit
  MoH Singapore
• Shirley Suresh, Ying Juan Wu, David Machin
• Victorian Infectious Disease Ref Lab, Melbourne,
  Australia
• Scott Bowden, Steven Locarnini

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Objectives

• To determine if combination therapy with
  interferon and thymosin was superior to
  interferon alone with regards to
• Primary endpoints
• HBeAg loss or
• HBeAg seroconversion
• Secondary endpoints
• Normalisation of ALT
• Reduction in HBV DNA
• Improvement in histology

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Randomised Placebo Controlled Trial

• Entry criteria
• HBsAggt6m
• ALT1.5lt10X ULN
• HBeAg
• HBV DNAgt106 cp/ml
• Absence of cirrhosis
• Treatment naïve

• Exclusion criteria
• HCV, HDV, HIV co-infection
• FBC biochemistry significantly outside normal
  range
• Absence of significant medical illnesses
• Decompensated liver disease

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Central Tel. Randomisation
Wellferon 5MIU 3x/w Thymosin 1.6µg 3x/w For 24
weeks
Wellferon 5MIU 3x/w Placebo 3x/w For 24 weeks
48 weeks Followup
48 weeks Followup
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Study size determination

• A pilot study (Rasi, J Viral Hep 1996) suggested
  that a difference of 30 between combination IFN
  Thymosin compared to IFN alone could be
  anticipated
• Based on two sided test size of 5 and power of
  85, estimated study size was 48 pts in each arm

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Results
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Baseline Characteristics of patients
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CONSORT Diagram
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ITT analysis
Digene assay
Pgt0.05
Pgt0.05

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ITT analysis
p0.067
p0.104

Digene assay
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Kaplan-Meier analysis of HBeAg loss over time
p0.169
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Mean HBV DNA response by treatment group
Treatment phase
HBV DNA measured by Bayer VERSANT HBV 3.0 Assay,
LLD2,000cp/ml
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Histological response

• HAI score
• All patients had decrease in HAI at the end of FU
  compared to baseline, from 10.50 5.35 to 8.82
  6.27 (mean reduction -1.99, 95 CI -3.28 to
  -0.69 p0.003)
• But no difference between combination and IFN
  monotherapy, mean difference 0.26, 95 CI -3.09
  to 2.57 p ns

• Fibrosis score
• Mean change in fibrosis score, at the end of FU
  compared to baseline, was 0.11 95 CI -0.46 to
  0.48, p ns
• No difference was found between combination and
  IFN monotherapy, mean difference, -0.31 95 CI
  -1.20 to 0.58 p-value ns

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Safety

• Combination IFN alone
• ALT flares(5x ULN)
• On treatment 25 28
• post-Rx 16 24
• SAEs 4 (8) 4 (8)
• Related to therapy retrobulbar grade 3 ALT
  flare neuritis
• AE 582 events 584 events
• (11.29/pt) (11.68/pt)
• No difference in top 10 AEs between the 2 groups

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Summary

• Combination therapy with IFN Thymosin compared
  to IFN alone, 48 weeks after therapy showed
• a difference of 17.8 with regards to HBeAg loss
  (nearing significance, p0.067) and
• a difference of 15.8 with regards to HBeAg
  seroconversion
• There was no difference between the two groups in
  
• normalisation of ALT,
• reduction in HBV DNA, or
• improvement in histology

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Comparison of combination trials
pegIFN vs LAM vs lam/pegIFN
LAM vs LAM/ADV
ADV vs LAM/ADV
LAM vs LAM/TELB
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30
seroconversion/resistance
25

20
15

10
5

0
-5
Log ?DNA
-10
LAM
LAMADV
ADV
LAMADV
LAM
LAMTELB
pegIFN
LAM
LAMpegIFN
Sung, EASL 2003
Peters, Gastro 2004
Lai, Gastro 2004
Lau, NEJM 2005
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Conclusions

• A trend towards HBeAg loss and seroconversion was
  seen in combination IFN Thymosin therapy
  compared to IFN monotherapy amounting to a
  difference of 17.8 which should be validated in
  larger studies
• Utilising a baseline rate of 28 from the study
  in the monotherapy arm, with test size 5 and
  power 85 as previously, and taking a
  conservative effect size of 15, a randomized
  control trial (11) would require 328 patients
• Combination therapy with two immunomodulators has
  potential to improve response to HBV
• The future of combination therapy is still to be
  fully explored and optimal combinations have yet
  to be discovered

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Immunological substudy

• Despite considerable research into immunology of
  HBV, there is surprising little information on
  the discriminants of HBeAg seroconversion
• Tsai et al (JCI 1992), found in 8/10 pts who
  seroconverted after spontaneous acute
  exacerbation that seroconverters were associated
  with increased T cell proliferation to HBeAg
  HBcAg
• There is little information on immunological
  responses associated with treatment of CHB

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Methods I

• As part of the IFN-Ta1 combination study, an
  immunological substudy was conducted
• All patients had T cells collected at baseline,
  end-of-treatment, and end-of-followup
• T cells were stored at -80C
• At the end of the study, T cells from the
  following groups were selected for analysis
• Responders HBeAg seroconversion
• Non-responders non-seroconverters

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Methods II

• Treatment IFN Monotherapy Combination
• Response RMResponder (9) RCResponder (8)
• NRMNon-responder (12) NRCNon-responder (7)
• The following analyses were performed
• T cell phenotyping by FACS CD4, CD8, CD56,
  CD25high, combinations of above
• T cell proliferation to HBcAg, HBeAg, HBsAg
• Cytokine secretion assay
• ELISPOT assay for IFN-?
• Controls were subjects who had previous exposure
  to HBV HBsAg-ive, anti-HBcive

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CD4 CD4/CD25high
RC
T Lymphocyte Subset-CD4
RM
80
NRC
70
NRM
60
Control
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p0.039
of T Lymphocytes
40

• At the end of FU there was significantly higher
• CD4 in RC vs RM
• CD4/CD25high in RM vs RC

30
20
10
0
pre-treatment
post-treatment
follow-up
Control
T Lymphocyte Subset-CD4 CD25
high
18
16
14
12
10
of CD4 Cells
8
p0.01
6
4
2
0
pre-treatment
post-treatment
follow-up
Control
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Cytotoxic T cells
T Lymphocyte Subset-CD56
RC
RM
30
NRC
NRM
Control
25
20
of T Lymphocytes
15
p0.007t

• At baseline, responders, NRM had higher CD56
  CD8/CD56 than RM

10
5
0
pre-treatment
post-treatment
follow-up
Control
T Lymphocyte Subset-CD8CD56
RC
RM
20
NRC
18
NRM
Control
16
14
12
of T Lymphocytes
10
p0.047t
8
6
4
2
0
pre-treatment
post-treatment
follow-up
Control
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T cell proliferation
against cAg

• Responders had higher T cell proliferation than
  non-responders at baseline

plt0.034t
against eAg
plt0.034t
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Cytokine secretion assays

• Responders had dramatically higher TNF-a
  secretion than non-responders
• However all HBV pts had poor IL2 response
  compared to controls

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IFN-? ELISPOT assay for T cell cytotoxicity
ELISPOT -IFN-?
140
120
100
80
60
40
20
pre-treatment
post-treatment
follow-up
Control
ELISPOT assay was similar in RM RC during
therapy but in RC continued to increase after
end of treatment period.
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Summary

• CD4 was higher in RC vs RM
• CD4/CD25high was higher in RM vs RC
• Baseline levels of CD56, CD8/CD56 were higher in
  NRM vs RM
• Baseline T cell proliferation to HBcAg was higher
  in RC vs RM, and in RM vs NRM
• T cell proliferation was higher in RC vs RM at
  baseline and end-of-treatment
• TNF-? showed dramatic but non-significant
  increases during treatment
• There was overall poor IL2 response in all HBV
  patients
• ELISPOT to IFN-? increased during treatment in
  responders that continued in RC but returned to
  baseline in RM

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Conclusions

• It could not be ascertained definitive immune
  discriminators of seroconversion
• However, combination Ta1 IFN increases ELISPOT
  after end of treatment compared to IFN alone
• The increase in CD4/CD25high at end of FU in IFN
  monotherapy pts suggests that T regulatory cells
  may be reduced in thymosin-treated pts,
  permitting cytotoxic T cells to regain function
• Overall IL2 response in HBV was very poor

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Acknowledgements

• Immunovirology Lab Centre for Molecular
  Medicine ASTAR Singapore
• Research Nurses
• Clinical Trial Unit
• Dept of Gastro Hepatology
• National University Hospital
• Singapore

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Questions Answers