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The Chimpanzee Model Of HCV: Antiviral Therapy
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Antiviral Therapies in Chimpanzees• PEG‐IFN + Ribavirin
• STAT‐C with DAA–Protease Inhibitors–Nucleoside analogues –Polymerase Inhibitors–NS5A Inhibitors
• Immunomodulators‐ TLR7, TLR9, McAb to cytokines and cytokine receptors
• Antisense‐ siRNA targeting viral RNA
• Therapeutic vaccines
• Essential host targets‐Cyclophilin inhibitors‐miR122 antisense – LNA‐Entry Inhibitors – 4 receptors
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Phase I, PK in uninfected animals• Dosing, acceptability• Serum to liver ratio
Phase II, Efficacy in HCV chronic animals, n=2‐4• Kinetics of decline of vRNA• Evolution of resistance mutations• Persistence of resistance mutations
Dosing limitations Oral: if accepted by chimps indefinite periods, 3‐6 months.
Tang, Kool‐Aid, Applesauce (micro‐encapsulated)Gavage, IV, SQ , IM all require sedation
IACUC normally limits study to 5‐7 doses with follow‐up bleeds progressively spaced out
Chimps can be trained for oral pills and with some effort for SQand IM injection
Design of Antiviral Studies in Chimpanzees
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
STAT-C Therapy in Chimps
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Chen, C.-M. et al. 2007. Antimicrob. Agents Chemother. 51(12):4290-4296
Abbott A‐837093 NNI (benzothiadiazine) HCV G1a and 1b infected chimpanzees
BID, 14 days
• Very rapid decline, G1b decrease 2.5 logs in 2 days• Rapid emergence of resistance in 1b• Day 5, G1b 13% WT; G1a, 74% WT
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Pilot-Matias et al, International Symposium on HCV, Nice France 2009. Abbott
-5.0-4.5-4.0-3.5-3.0-2.5-2.0-1.5-1.0-0.50.0
0 2 4 6 8 10 12 14 16 18 20Time (Days)
Vira
l Loa
d C
hang
e Fr
om B
asel
ine
(Log
10IU
/mL)
Last dose
All clones wt
Dosing period (12.8 mg/kg) BID by oral gavage
46%1%1%5%6%
11%10%20%
R155KA156TA156VD168AD168ED168GD168Vwt
Efficacy of Protease Inhibitor EA‐058 in a Genotype 1a‐Infected Chimpanzee
• >4‐log10 reduction in viral load after 2 days of treatment• Only day 4 contained virus with known resistance mutations; • Short‐term treatment (2 days) did not lead to persistence of resistance mutations
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Carroll, S. S. et al. 2009. Antimicrob. Agents Chemother. 53(3):926-934
Viral loads in HCV-infected chimps dosed at 1 mg/kg MK-0608 orally once daily for 37 days
282 mutation observed after dosing for single day in X6
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
HCV Chronic Infection• 10 HCV chronics; viremia vary by 1000‐fold: 104 to 107 ge/ml
• Expression of 971 genes altered at 99% confidence
• ISG and cytokines also elevated during acute infection
• Elevated hepatic IFN response persists for decades with little pathology
Infected Uninfected
Incr
ease
dD
ecre
ased
Bigger et al. J Virol 78: 13779-92,2004.Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Hepatic ISG Levels Independent of vRNA Levels
Gene Fold Change Avg fold change vs all 6 baselines
ISG27 50 54 59 56 80 76 71 66 82 70
IP-10 CXCL10 18 12 13 8 14 17 2 13 31 46ITAC CXCL11 11 6 11 6 4 23 4 24 19 51MXA 15 13 9 8 13 12 6 11 11 18OAS2 13 13 13 8 11 11 5 8 11 14
Animals selected to have 1000 fold variation in viremiaVariation in liver ISG response not proportional to viremia; maxed-out
Bigger et al. J Virol 78: 13779-92,2004.Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
• Previous studies with Ad gene therapy for IFN failed
• How many HCV‐induced genes are ISGs
• Total genome response to IFN in vivo
• Single dose induction in 3 uninfected chimpanzees
human and chimpanzee IFN tested
5x106 IU IFN SQ Liver and PBMC 0, 4, 8, 24 hrs
Chimpanzee Response to IFNαCHIMPANZEE RESPONSE IFN
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Hepatic ISG Response Rapidly Down-Regulated
0 4 8 24
ISG 15
25
50
100Fo
ld C
hang
e
0 4 8 24
hr post IFN
Ch IFN
Hu peg-IFN
25
50
100
Fold C
hang
e
>500pg/ml IFN
Rapid Down-Regulation of ISG Response in Presence of High IFN Levels
Contrasts to lack of down-regulation during acute and chronic infection.
Lanford et al, Hepatology 43:961-972, 2006.Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
HCV Chronic Chimps Null IFNα Response
Time After First Dose of IFN
-4 wk
-2 wk
4 hr8 hr24 hr
1 wk
2 wk
4 wk
6 wk
8 wk
9 wk
10 wk
11 wk
12 wk
13 wk
14 wk
18 wk
GE/
ml
10 3
10 4
10 5
10 6
10 7
10 8
10 9
Genotype 1b
Genotype 3a Genotype 1a
Peg IFN
Lanford et al, Hepatology 46:999-1008, 2007.
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Uninfected
0 4 8 24
Fold
Inc
reas
e
0
200
400
600
800
Liver
Hours Post-IFN0 4 8 24
Fold
Inc
reas
e
0
100
200
300
400
500
HCV Chronic
0 4 8 240
20406080
100120
Hours Post-IFN0 4 8 24
020406080
100120
Fold Change IP-10 Transcription
Lanford et al, Hepatology 46:999-1008, 2007.
PBMC
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Uninfected
0 4 8 24
Fold
Inc
reas
e
0
200
400
600
800
Liver
Hours Post-IFN0 4 8 24
Fold
Inc
reas
e
0
100
200
300
400
500
HCV Chronic
0 4 8 240
20406080
100120
Hours Post-IFN0 4 8 24
020406080
100120
Fold Change IP-10 Transcription
Lanford et al, Hepatology 46:999-1008, 2007.
PBMC
FC vs uninfected
FC vs HCV baseline
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Conclusions from IFN Studies• ISG response highly elevated in chronic chimpanzee liver
• May be maximally induced
• Exogenous IFN does not further induce liver ISG response
• Chimps maybe be representative of human null responders
• Humans: two phenotypes high or low ISG prior to therapylow ISG correlates with protective IL28B alleles
• Potential of antivirals to convert null response phenotypereduced vRNA reduce ISGs = responder phenotype?
• Only 35% of chimps progress to chronic infection, protective allele• All chimps examined have high ISG phenotype and lack response to IFN,
non‐protective allele• Chimps are not polymorphic at same sites in IL28B as humans
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
miR122 liver specific miRNA
70% of all miRNA in liver - 50,000 copies per cell
miR122 regulates cholesterol and fatty acid synthesis
Jopling et al demonstrated HCV requires miR122Two miR122 binding sites in 5’ NCR,
Santaris developed LNA technologymiR122 knockdown in vivo in AGMreduced TSC ~40%
Potential for HCV therapy
Targeting Host Factors for Antiviral Therapy
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
• SPC3649: 15-mer high-affinity LNA-antimiR122
•(8 LNAs, Tm=80 oC, PS backbone)
UGUUUGUGGUAACAGUGUGAGGU 5’ miR122CcAttGTcAcaCtCC 3’ SPC3649
CGACACUCCACCAUGAAUCACUCCC HCVGUGAGGU miR‐122GUGAGGU5’
CGACACUCCACCAUGAAUCACUCCC HCV
SPC3649 treatment
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Safety–Efficacy Trial in HCV Infected Chimpanzees
4 HCV genotype 1 infected chimpanzees: 2 high dose - 5 mg/kg2 low dose - 1 mg/kg
4 weeks pre-study12 weeks IV dosing in saline14 weeks follow up
Proof of Concept Study
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
• 2.6-log reduction in HCV RNA• No viral rebound during dosing• Maximum HCV suppression extended 11 weeks after dosing• miR122-LNA duplex still present in liver at 14 weeks after dosing• Total serum cholesterol reduced by 45%.
SPC3649-Prolonged Antiviral Activity
Lanford et al, Science 327:198-201, 2010.
HCV VIRAL RNA
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
-4 -2 0 2 4 6 8 10 12 14 17 21 25
week
GE/
ml
placebo dosing
-100
-80
-60
-40
-20
-10 1 2 3 4 5 6 7 8 910111213141517192123
Chn
age
in T
SC
Total Serum Cholesterol
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
No Selection of Adaptive Changesin miR-122 Seed Matches
• Clonal and Deep sequencing of high dose animals• No increase in nt changes in 5’ NCR during dosing
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Normalization of Hepatic ISG Expression
• Affymetrix microarray data – Supervised analysis of ISGs• ISGs decline during treatment or normalize• HCV infected chimpanzees are Null responders to IFN • Conversion of Null Phenotype to Responder Phenotype
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
CONCLUSIONS
•SPC3649 provides long term sequestration of miR122.
•No rebound of virus and no selection of adaptive variants.
•Very long half life in liver
•SPC3649 provides a high barrier to resistance.
•TSC was reduced by up to 44% in chimpanzees and 30% in humans.
•The ISG response was normalized suggesting the opportunity to more effectively treat IFN null responders.
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA
Collaborators
Southwest FoundationDeborah ChavezBernadette GuerraHelen LeeLena NotvallYunmi ChungSNPRCKathleen Brasky DVM
Stan LemonChris WalkerDave Thomas
Santaris PharmaLisa Hildebrandt-EriksenAndreas PetriSakari KauppinenHenrik Oerum
Presented at the 5th International Workshop on Hepatitis C, Resistance and New Compounds24-25 June 2010, Boston USA