Evaluation and validation of chimeric mouse model for testing anti- viral hepatitis compounds t.j. gao, d.l.j. tyrrell and n. w. kneteman kmt hepatech inc, edmonton, alberta, canada

Evaluation and Validation of the KMT Mouse Model
for Testing Anti-Viral Hepatitis Therapies
N. Kneteman*#, T.J. Gao#, B. His*, D. Schiller*, D. Douglas*, B. Addison** and D.L.J. Tyrrell**#
Departments of Surgery*, Medical Microbiology and Immunology** , University of Alberta and KMT Hepatech Inc.#, Edmonton, Alberta, T6G 2E1, Canada
INTRODUCTION
Study 2: Effect of protease inhibitor BILN 2061 on HCV
Lack of a robust small animal model of hepatitic C virus (HCV) infection has
Study Design
Serum HCV titer change in KMT m ice after BILN2061
impeded the development of effective antiviral therapies and understanding of
Dose: 10 mg/kg per dose
treatment for 4 days
HCV biology. By transplanting normal human hepatocytes into the liver of
Administration: Twice a day, oral gavage
Courses: 4 or 7 days treatment
immunodeficient mice (SCID/Beige) carrying the urokinase type plasminogen
Animals in the study:
activator transgene linked to an albumin promoter (Alb-uPA), we generated mice
4 days treatment: BILN2061: 3, Control: 0 (Fig1)
with chimeric human livers (1, 2, 3). Mice engrafted successfully with
7 days treatment: BILN2061: 6, Controls: INF: 5
and vehicle: 4 (Fig2)
transplanted human hepatocytes screened with ELISA for human alpha-1
antitrypsin (hAAT) are capable of supporting long-term stable infection with
hAAT for monitoring human hepatocyte grafts
Roche Amplicor for HCV RNA quantitative and
qualitative testing
We evaluated therapeutic agents against HCV and HBV infection that have
been clinically validated. These included interferon alpha 2B(4) and a protease
HCV titer change in BILN2061 treated patients, 200 m g b.i.d. by
inhibitor previously reported to decrease HCV titers effectively (BILN2061) (5). In
oral adm inistration
Anim al ID
addition, we studied the impact of Lamivudine on hepatitis B virus (HBV)
infection in this model (6,7).
, l
L
/m
s 5.0 0
Serum HCV titer change in KMT mice after BILN2061 treatment
EXPERIMENTAL STUDIES- MODEL VALIDATION
for 1 w eek
Since this mouse model was published in Nature Medicine in August 2001, we
have validated this system in the context of development of antivirals and HCV
therapeutic strategies. The following studies have been carried out with this
B aseline
animal model system:
Time (hr)
• Interferon alpha 2B treatment of HCV infection- 4 independent experiments
Fig 3. HCV-infected patients were treated with BILN 2061
(Study 1)
200 mg twice daily for 2 days in a proof-of–concept study.
HCV viral load was reduced by 2-3 logs detected by Cobas
• Assessment of anti-HCV activity of a serine protease inhibitor (BILN 2061)
Amplicor HCV Monitor Version 2.0 (adapted from Lamarre
reported to result in decreased HCV viral titres in clinical trials (Study 2)
D., et al. An NS3 protease inhibitor with antiviral effects in
humans infected with hepatitis C Virus. Nature: 426:186 –
BI2061(n=6)
INF(n=5)
Vehicle(n=4)
• Verification of anti-HBV effect of the nucleoside analogue lamivudine on
189, 2003)
established HBV infection in chimeric mice (Study 3)
• Evaluation of gene therapy approach against HCV infection using modified
• HCV viral RNA decreased mean 2.0 log after 4 day- treatment course with BILN 2061
BID (oral presentation O-61, abstract N0. 281, 10th HCV meeting, Kyoto)
• HCV viral RNA decreased 1.2 log(6.7±0.24 to 5.5±0.34) in BILN 2061 and 0.6 log(6.2 ± 0.41 to 5.6 ±0.34) in INF, but increased 0.18 log
(6.8±0.32 to 7.0±0.33) in vehicle group after 7 day-treatment course
• A significant difference in the net decrease of HCV viral RNA exists between BILN 2061 and Vehicle (p<0.0001), INF and vehicle (p< 0.005)
and BILN 2061 and INF (p<0.03), respectively (ANOVA and LSD test)
• Results parallel reported impact of BILN 2061 in HCV-infected patients (Fig 3)
Study 1: Interferon alpha 2B treatment of HCV
Study 3: Anti-HBV effect of Lamivudine in chimeric mice
Serum HCV RNA titer changes during INF treatm ent, INF vs
Study Design
Vehicle control (day 0 to day 14)
Study Design
Dose: 1350 IU/gram body weight/day per animal
HBV DNA titer change during treatment ofKMT mice with
Administration: Once a day, i.m. injection
Dose: 20 mg and 50 mg/kg per animal
Lamivudine for 4 weeks and observation for 2 weeks
Courses: 2 and 4 weeks, one week follow-
Administration: Twice daily, oral gavage
up post-treatment
Courses: 4 weeks treatment and 1 week follow- up
post-treatment
Animals in the study:
Animals in the study:
2 wk treatment: INF: 18, Control: 16
50 mg group: 6
4 wk treatment: INF: 4, Control: 4
20 mg group: 7
l RNA I 4.00
Vehicle (saline) control: 3
hAAT for monitoring human hepatocyte grafts
Assays: hAAT for monitoring human grafts and
Roche Amplicor for HCV RNA titration
Light Cycler PCR for HBV DNA quantitation
IN F t r eat ment
Baseline
INF (n=18)
• HBV viral load reduced consistently in lamivudine
Veh (n=16)
* p<0.01, ** p<0.001
groups during treatment.
• HBV DNA was reduced 2.1 logs(6.9±0.31 to
Lamivudine Treatment
Serum HCV RNA titer change during INF treatm ent for 4 w eeks
4.8±0.51) in Lamivudine 20 and 50 mg groups and
w ith follow -up observation for 1 w eek
0.6 log (7.6±0.38 to 7.0±0.38) down in saline control
group at 4 weeks
Baseline
• Serum HCV viral RNA decreased by mean 0.7 log
• Significant differences of HBV viral DNA decreases
* P<0.02, ** P<0.05
(5.2±0.53 to 4.5 ±0.54) after 1 week , and 1.0 log (5.2
exist between treatment and control group
±0.53 to 4.1±0.57) after 2 week therapy in INF group
• HCV viral RNA decreased 2.0 log (6.2±0.41 to 4.2
±0.56) in INF group after 4 weeks treatment
• HCV viral RNA level remained stable in control
• HCV viral RNA rebounds after stopping INF
administration (partial)
We evaluated and validated our SCID/bg/uPA chimeric mouse model (KMT mouse)
• Differences in viral titer between treatment and
INF Treatment
for testing anti-HCV and anti-HBV agents (INF alpha 2B, protease inhibitor BILN 2061,
control groups were statistically significant at all time
Baseline
points during treatment (weeks 1, 2, 3 and 4; ANOVA
Lamivudine) and therapeutic strategies (gene therapy for HCV and passive
and LSD analysis)
immunotherapy for HBV). HCV viral load was reduced 0.7, 1.0 and 2.0 log (means) after
* P<0.01, ** P<0.05
1, 2 and 4 week treatment with INF alpha 2B (1350 IU/gm/d), respectively. A small
molecule protease inhibitor (BILN2061, 10mg/kg/bid) resulted in 2.0 and 1.4 log
REFERENCES
reduction of HCV viral load after 4 and 7 day treatment courses. Differences of
Mercer D, et al. Hepatitis C virus replication in mice with chimeric human livers. Nature Medicine 7: 727 -
reduction of HVC viral load between INF and BILN2061 treatment groups and
933, 2001.
corresponding controls were statistically significant (at level of p<0.05). Chimeric mice
Brass V, et al. Of mice and Men: a small animal model of hepatitis C virus replication. Hepatology 35: 722
-723, 2002.
infected with HBV also demonstrated a positive response to lamivudine treatment
Crabb C. Hard-won advances spark excitement about hepatitis C. Science, 294: 506-507, 2001.
(nucleoside analogue, 20 mg/kg/d). HBV viral load decreased steadily and resulted in
Schiller D, et al. Effect of interferon alpha 2B on HCV- infected SCID/uPA mice. 8th International
Symposium on Hepatitis C virus & related viruses. P-142, Paris, 2001.
2.1 log decrease after 4 weeks treatment. In our repeated tests with INF alpha 2B and
Lamarre D, et al. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C
BILN2061, outcomes of HCV viral load were consistent and reproducible. This data
virus. Nature 426:186-189, 2003.
Morrey J, et al. Utilization of transgenic mice replicating high levels of hepatitis B virus for antiviral
provides validation of the KMT mouse model for testing anti-viral therapies for HCV
evaluation of lamivudine. Antiviral Res 42: 97-108, 1999.
Leung N. Clinical experiences with lamivudine. Seminar Liver Diseases 22 suppl: 15 – 21, 2002.
Kneteman N. Prevention of a primary hepatitis B infection with Hepatitis B immunoglobulin in a mouse
model. 9th International Meeting of HCV and Related Viruses, UCSD, La Jolla, CA, 2002
CONCLUSIONS
• This small animal model has proven predictive of clinical treatment impact for antiviral
ACKNOWLEDGEMENTS
compounds for HCV as demonstrated by positive outcomes in the mouse model with
Interferon alpha 2B and a protease inhibitor (BILN 2061), the only two therapies to date
Aukerman L, Hashash A, Kovelesky R, MacKichan M, Ni ZJ, Plattner J, Shoemaker K, and Weiner A. from Chiron Corporation
are acknowledged for their contributions to the study, and Lewis J, Boudrea J and Yakemchuk A for technical support in these
demonstrated to be effective in clinical application
studies.
• Results with lamivudine therapy for HBV infection in the model also mirror clinical
outcomes. The mice can be infected with both HCV and HBV
• Other therapeutic strategies can also be evaluated with this system, including passive
immunotherapy and gene therapy. We have previously reported the ability of Hepatitis B
immune globulin to prevent infection with HBV in this model( 8)
www.kmthepatech.com
P-187, 10th HCV Meeting, Dec. 2 -6, 2003, Kyoto, Japan

Source: http://www.kmthepatech.com/en/KMTMouseModel/Validation/~/media/kmthepatech/Documents/kmthepatech_validation.pdf