Viral Kinetic Parameters in HCV Therapy

3. HCV Therapy Current Standard of Care Pegylated interferon alfa + ribavirin Length of treatment SVR 50-70% overall Worse in GT 1,4 = 40% Intolerance and AEs

4. HCV Research Timeline I think it is important to note the timeline of discovery in HCV as it is quite different that that we know of HIV, primarily due to limitations in cell culture. HCV was first identified in 1989, 6 years after the discovery of HIV. However it was not possible to culture the virus efficiently in vitro, impeding elucidation of the viral life cycle and the development of specifically target antivirals. 4 years after the discovery of HIV the first ART drug AZT was FDA approved. It has been 20 years since the discovery of HCV and yet a directly acting antiviral is still not FDA approved. It was with the discovery of the replicon system in 1999 that for the first time HCV RNA replication could be reproduced in vitro in Huh-7 human hepatocellular carcinoma cell lines. Replicons for GT 1a, 1b and 2a have been constructed. The second great discovery came 2003 when an HCV GT 2a clone isolated from a Japanese patient with a rare case of fulminant HCV designated JFH-1 was found to replicate in Huh-7 and other cell lines without the need for adaptive mutations as prior replicon clones. And for the first time in 2005 JFH-1 genomes transfected into Huh-7 cells produced infectious virus allowing in vitro study of the entire HCV life cycle. There remains a lack of small animal models for infection, which remains a limitation. I think it is important to note the timeline of discovery in HCV as it is quite different that that we know of HIV, primarily due to limitations in cell culture. HCV was first identified in 1989, 6 years after the discovery of HIV. However it was not possible to culture the virus efficiently in vitro, impeding elucidation of the viral life cycle and the development of specifically target antivirals. 4 years after the discovery of HIV the first ART drug AZT was FDA approved. It has been 20 years since the discovery of HCV and yet a directly acting antiviral is still not FDA approved. It was with the discovery of the replicon system in 1999 that for the first time HCV RNA replication could be reproduced in vitro in Huh-7 human hepatocellular carcinoma cell lines. Replicons for GT 1a, 1b and 2a have been constructed. The second great discovery came 2003 when an HCV GT 2a clone isolated from a Japanese patient with a rare case of fulminant HCV designated JFH-1 was found to replicate in Huh-7 and other cell lines without the need for adaptive mutations as prior replicon clones. And for the first time in 2005 JFH-1 genomes transfected into Huh-7 cells produced infectious virus allowing in vitro study of the entire HCV life cycle. There remains a lack of small animal models for infection, which remains a limitation.

5. HCV Genome: Polyprotein HCV contains a positive sense RNA genome of approximately 9600 bases, codes for a single polyprotein precursor of about 3000 amino acides that is co- and posttranslationally cleaved into structural and nonstructural proteins. The structural proteins processed by the endoplasmic reticulum include the core protein (which forms the viral nucleocapsid), the envelope proteins (E1 and E2, which form the viral envelope). The p7 protein is thought to have an important role in viral particle maturation and release. NS3-4A is a serine protease and associated co-factor which is involved in postranslational processing, and small peptides derived from the N-terminal cleavage products of its substrate have been shown to competitively inhibit the protease and thus has emerged as a primary target for directed antivirals known as protease inhibitors. Furthermore, this protease has been shown in vitro to intervene on the intracellular Type I IFN pathway, exerting a suppressive effect on the innate immunologic response to HCV infection by way of the TLR pathway and the RIG-I pathways. NS5B is a RNA dependent RNA polymerase and is another primary target for antiviral therapeutic development. NS5B inhibitors can be classified into active site and allosteric inhibitors. Nucleoside inhbitors (NIs) which resemble HIV nucleosides, act as alternative substrates for the polymerase inhibitor by inserting themselves into the growing RNA chain and binding the NS5B polymerase at the active catalytic site. Nonnucleoside inhibitors (NNIs) are structurally diverse and inhibit the initiation or elongation step of viral replication by binding the polymerase at one of several allosteric binding sites, leading to conformational change. HIV: aspartic protease, HCV contains a positive sense RNA genome of approximately 9600 bases, codes for a single polyprotein precursor of about 3000 amino acides that is co- and posttranslationally cleaved into structural and nonstructural proteins. The structural proteins processed by the endoplasmic reticulum include the core protein (which forms the viral nucleocapsid), the envelope proteins (E1 and E2, which form the viral envelope). The p7 protein is thought to have an important role in viral particle maturation and release. NS3-4A is a serine protease and associated co-factor which is involved in postranslational processing, and small peptides derived from the N-terminal cleavage products of its substrate have been shown to competitively inhibit the protease and thus has emerged as a primary target for directed antivirals known as protease inhibitors. Furthermore, this protease has been shown in vitro to intervene on the intracellular Type I IFN pathway, exerting a suppressive effect on the innate immunologic response to HCV infection by way of the TLR pathway and the RIG-I pathways. NS5B is a RNA dependent RNA polymerase and is another primary target for antiviral therapeutic development. NS5B inhibitors can be classified into active site and allosteric inhibitors. Nucleoside inhbitors (NIs) which resemble HIV nucleosides, act as alternative substrates for the polymerase inhibitor by inserting themselves into the growing RNA chain and binding the NS5B polymerase at the active catalytic site. Nonnucleoside inhibitors (NNIs) are structurally diverse and inhibit the initiation or elongation step of viral replication by binding the polymerase at one of several allosteric binding sites, leading to conformational change. HIV: aspartic protease,

6. DAA In Development Most targets in development are shown here, although this changes frequently, more than 50 trials are currently underway in CHC for new drug therapies. In black are new interferon based therapies, viramidine is a RBV like compound, many protease and polymerase inhibitors, a number of immunomodulators, and other including viral entry and maturation. Most targets in development are shown here, although this changes frequently, more than 50 trials are currently underway in CHC for new drug therapies. In black are new interferon based therapies, viramidine is a RBV like compound, many protease and polymerase inhibitors, a number of immunomodulators, and other including viral entry and maturation.

7. PROVE1: Protease Inhibitor TelaprevirN=260, GT1 Treatment Naive PROVE1 was a phase II RCT conducted in the US that examined the efficacy of telaprevir plus pegIFNalpha and RBV in 260 tx naïve patients with CHC GT1, noncirrhotics. Patients were randomized to 1 of 4 arms; IFN dosing was 180 mcg/week, RBV weight based at 1000-1200 mg per day and TVR 750mg PO TID. PROVE1 was a phase II RCT conducted in the US that examined the efficacy of telaprevir plus pegIFNalpha and RBV in 260 tx naïve patients with CHC GT1, noncirrhotics. Patients were randomized to 1 of 4 arms; IFN dosing was 180 mcg/week, RBV weight based at 1000-1200 mg per day and TVR 750mg PO TID.

8. This figure shows the mean log10 HCV RNA decline of all 4 treatment arms in the first 24 weeks of therapy. It is clear the more rapid viral decline noted in all TVR arms as compared to the control arm. This figure shows the mean log10 HCV RNA decline of all 4 treatment arms in the first 24 weeks of therapy. It is clear the more rapid viral decline noted in all TVR arms as compared to the control arm.

9. PROVE2: Protease Inhibitor TelaprevirN=323, GT 1 Treatment Naive Dosing same as PROVE-1 however there are two important parts with regards to the design of PROVE2: (1) there is a no RBV arm and (2) there is no 48 week TPV arm. Dosing same as PROVE-1 however there are two important parts with regards to the design of PROVE2: (1) there is a no RBV arm and (2) there is no 48 week TPV arm.

10. SPRINT-1: Protease Inhibitor BoceprevirN=595, Treatment Naïve GT 1 The SPRINT-1 study is a phase 2b multicenter study conducted in GT1 tx naïve patients, one novel element of the study design was to assess the impact of a lead-in phase in which pegiFN and RBV were initiated for the first 4 weeks prior to the initiation of the PI, with the hypothesis that this would benefit from first phase kinetic declines and thus decrease risk of resistance selection. Boceprevir dosing was TID, pegIFN was given as 180mcg/week and the RBV dosing is noted here, there was a low dose RBV arm. AS you can see there were 6 different arms in this study: The SPRINT-1 study is a phase 2b multicenter study conducted in GT1 tx naïve patients, one novel element of the study design was to assess the impact of a lead-in phase in which pegiFN and RBV were initiated for the first 4 weeks prior to the initiation of the PI, with the hypothesis that this would benefit from first phase kinetic declines and thus decrease risk of resistance selection. Boceprevir dosing was TID, pegIFN was given as 180mcg/week and the RBV dosing is noted here, there was a low dose RBV arm. AS you can see there were 6 different arms in this study:

11. This is the genomic overview of the region where the most significant signal was noted for association with the outcome of interest: response to HCV treatment. On the Y axis are the Pvalues of all genotyped (550000+) SNPs in the region and the structures of the surrounded genes. The SNPs that show genome wide significant association with SVR are here in red, and these all localize to the IL28 region on Chromosone 19. And the SNP that had the strongest association with a Pvalue of 1.37X10-28 (note that 10-9 if the threshold for significance in GWAS studies) is SNP rs12979860. This polymorphism denoted here with red arrow resides 3 kilobases upstream of the IL28B gene, which encodes IFN-lambda-3. This is the genomic overview of the region where the most significant signal was noted for association with the outcome of interest: response to HCV treatment. On the Y axis are the Pvalues of all genotyped (550000+) SNPs in the region and the structures of the surrounded genes. The SNPs that show genome wide significant association with SVR are here in red, and these all localize to the IL28 region on Chromosone 19. And the SNP that had the strongest association with a Pvalue of 1.37X10-28 (note that 10-9 if the threshold for significance in GWAS studies) is SNP rs12979860. This polymorphism denoted here with red arrow resides 3 kilobases upstream of the IL28B gene, which encodes IFN-lambda-3.

12. When applied to the study population, the IL28B SNP is strongly associated with SVR is all patient groups. This graph shows SVR% on Y axis and the X axis has 4 groups, the SNP is a recessive allele thus is denoted CC for homozygous, C/T for heterozygous, and TT for lack of polymorphism. As you can see due mostly to power from the numbers of each ethnic group, the SNP shows overwhelming GW significance in the white american population, the CC genotype is associated with a two fold increase in SVR (CI 1.8-2.3) Thus as you can see the CC genotype significnatly improves the chance of achieving SVR even in those patients of AA ancestry. It is also noteable that AA with CC genotype had significantly better SVR (53%) than white americans with TT genotype (33%). When applied to the study population, the IL28B SNP is strongly associated with SVR is all patient groups. This graph shows SVR% on Y axis and the X axis has 4 groups, the SNP is a recessive allele thus is denoted CC for homozygous, C/T for heterozygous, and TT for lack of polymorphism. As you can see due mostly to power from the numbers of each ethnic group, the SNP shows overwhelming GW significance in the white american population, the CC genotype is associated with a two fold increase in SVR (CI 1.8-2.3) Thus as you can see the CC genotype significnatly improves the chance of achieving SVR even in those patients of AA ancestry. It is also noteable that AA with CC genotype had significantly better SVR (53%) than white americans with TT genotype (33%).

13. By looking at a multi-ethnic population sample with unknown HCV status, the group was able to show that a substantially higher frequency of the C allele in East Asians and a lower frequency in African-Americans. As you can see there is striking concordance between C-allele frequency and SVR rates across different populations. Those with lower frequency achieve lower SVR and it is well established that East Asians have higher SVR than Caucasians. This is estimated to account for approximately 50% of the difference in SVR between the AA and white populations that has been reported in the literature. By looking at a multi-ethnic population sample with unknown HCV status, the group was able to show that a substantially higher frequency of the C allele in East Asians and a lower frequency in African-Americans. As you can see there is striking concordance between C-allele frequency and SVR rates across different populations. Those with lower frequency achieve lower SVR and it is well established that East Asians have higher SVR than Caucasians. This is estimated to account for approximately 50% of the difference in SVR between the AA and white populations that has been reported in the literature.

14. Comparison between IL28B SNP and conventional clinical predictors of SVR

15. As mentioned in the background, approximately 30% of persons exposed to HCV will spontaneously clear the virus with innate immunity. Host genetic variation has always been assumed to explain the heterogeneity in HCV clearance because studies with same HCV innocululm show differences and there are clear differences across ethnic groups. Furthermore, variation in genes involved in immune responses have already been linked to outcome in acute HCV infection. This study sought to determine the role of IL28B polymorphism in spontaneous clearance of HCV. Over 1000 patients were genotyped from 6 independent HCV cohorts, 388 with spont clearance and 620 with persistent infection. As you can see by this chart which shows viral clearance on the Y axis and the genotype frequency on the x axis, patients with CC genotype were three times more likely to clear HCV relative to those with TT and CT genotype. Because some of these patients had HBV or HIV co-infection the influence of these were assessed in a mulitvariable regression analyisis, but did not appear to confound the affect of this polymorphism on natural clearance. It should also be noted that the effect of the IL28 polymorphism on viral load was investigated in this and the original GWAS study and for a dichotomized viral load of <600,000 vs >600,000 there was no relationship.As mentioned in the background, approximately 30% of persons exposed to HCV will spontaneously clear the virus with innate immunity. Host genetic variation has always been assumed to explain the heterogeneity in HCV clearance because studies with same HCV innocululm show differences and there are clear differences across ethnic groups. Furthermore, variation in genes involved in immune responses have already been linked to outcome in acute HCV infection. This study sought to determine the role of IL28B polymorphism in spontaneous clearance of HCV. Over 1000 patients were genotyped from 6 independent HCV cohorts, 388 with spont clearance and 620 with persistent infection. As you can see by this chart which shows viral clearance on the Y axis and the genotype frequency on the x axis, patients with CC genotype were three times more likely to clear HCV relative to those with TT and CT genotype. Because some of these patients had HBV or HIV co-infection the influence of these were assessed in a mulitvariable regression analyisis, but did not appear to confound the affect of this polymorphism on natural clearance. It should also be noted that the effect of the IL28 polymorphism on viral load was investigated in this and the original GWAS study and for a dichotomized viral load of <600,000 vs >600,000 there was no relationship.