Here we report the virologic characterization of variants emerging in the Phase IIa C210 study, the first clinical trial investigating telaprevir in treatment-naïve, chronically-infected HCV G4 patients. Telaprevir combined with Peg-IFN/RBV had greater antiviral activity against HCV G4 than telaprevir monotherapy or Peg-IFN/RBV alone . Telaprevir monotherapy had limited antiviral activity over 15 days; HCV RNA levels decreased only slightly and five of eight patients had vBT (no patients in the TPR group had vBT during this time). The remaining three patients treated with telaprevir monotherapy had detectable HCV RNA at Day 15.
As mentioned above, all vBTs in the investigational, telaprevir treatment phase occurred in the T group. HCV RNA levels only decreased slowly with telaprevir monotherapy compared with combination therapy. These findings are in line with the results of other studies investigating telaprevir monotherapy versus telaprevir plus Peg-IFN/RBV in HCV G1, G2, and G3 patients, and show better results with combination therapy compared to telaprevir monotherapy to control the emergence of resistant variants [10, 12, 17]. In this regard, 4/5 patients with vBT after 2 weeks of telaprevir monotherapy in our study achieved undetectable HCV RNA with subsequent Peg-IFN/RBV treatment and three achieved SVR (one patient had missing follow-up Week 24 data but had undetectable HCV RNA at follow-up week 12).
Analysis in this study focused on detecting previously reported HCV G1 amino acid mutations in the NS3•4A region; including single change V36A/M, T54A, R155I/K/M/T, and A156S which have been associated with lower level in vitro resistance to telaprevir (3- to 25-fold increase in replicon 50% IC50), and single change A156T/V and double change at positions V36M + R155K which have been associated with higher level in vitro telaprevir resistance (>25-fold increase in replicon IC50) [10, 18, 19]. None of the 23 patients with sequences obtained by population sequencing in this study had mutations associated with decreased telaprevir susceptibility at baseline, although a leucine (L) substitution in HCV G4 patients was detected in place of a V present in HCV G1 patients at position 36. The functional ramifications of this substitution are as yet unknown, though the V36L substitution in HCV G1 has been shown to exhibit a 2-fold change in telaprevir susceptibility .
Sequences were available for four of the five patients who had vBT during the investigational phase of the study (baseline to Day 15). In two of these patients, T54A/T emerged, which is a mutation known to moderately (FC ~6) increase telaprevir resistance in patients with G1 HCV . Two other T54A/T mutations were also detected, one in a patient in the T group who did not have vBT but who had detectable levels of HCV RNA at the end of telaprevir treatment, and one in a patient in the TPR group who also had detectable levels of HCV RNA at the end of telaprevir treatment. Overall, paired phenotype data at baseline and time of vBT or end of telaprevir treatment indicated that the emergence of the T54A/T mutation in this study population was associated with a 2- to 4-fold increase in telaprevir FC compared with baseline. Importantly, this mutation was not associated with failure of subsequent Peg-IFN/RBV treatment and all patients with a T54A/T mutation subsequently achieved SVR.
Taken together, these findings suggest that vBT and the emergence of resistant variants following telaprevir monotherapy had little or no impact on the efficacy of subsequent treatment with Peg-IFN/RBV. However, additional data on the clinical relevance of resistance with DAAs are required. In this regard, interim results from EXTEND, a multinational, 3-year follow-up study of patients treated with telaprevir-based regimens in Phase II and Phase III clinical trials, showed that, after ceasing telaprevir treatment, levels of resistant variants can rapidly decline as they are outcompeted by other, potentially more fit wild-type variants . Additional research is needed to ascertain whether these patients can be successfully retreated with the same DAA class. Nevertheless, the presence of some mutations can still be detected years later , and it is therefore important to limit potential for resistant variants . The rationale for combination therapy is due to the non-competing mechanisms of actions of the different agents used, which means that viruses associated with resistance to one class of agent are often susceptible to another class . This is supported by data in this study, as no variants known to be associated with resistance to telaprevir were detected in patients treated with TPR therapy.
Several other mutations were also detected in HCV G4 infected patients in this study. The variant V170A (HCV G4a) was observed in one patient at the same time as vBT in the T arm. This mutation has been previously observed in the HCV G1 population, although not at high frequency. It has also been linked with low-to-moderate levels of resistance to boceprevir [10, 21]. Another patient (HCV G4c) had three mutations detected at the time of vBT (including I170I/M and T54A/T). However, the importance of the combination of these mutations is not well defined and needs to be investigated further. Other mutations identified as emerging from baseline in patients with vBT were: V18I, T63I, I64M, A95T/V, P129A, and L135F. Due to the limited sample size, the lack of consistency of the mutations across patients and the scarcity of HCV G4 comparator sequences, the relevance of these mutations is currently not understood. Consequently, on the basis of this study, new HCV G4-specific mutations could not be identified.
Although few mutations known to be associated with reduced susceptibility to telaprevir in HCV G1 were observed in this study, this may be due to sample size limitations. Other viral mutations that affect DAA activity in HCV G1 may also be relevant for patients with HCV G4, since active site residues in HCV G1 and HCV G4 in NS3 protease domains are similar. Genotypic polymorphisms in the HCV NS3•4A proteases inter- and intra-genotypes are in general located on the protease surface, far from the telaprevir binding site .
Telaprevir FC at baseline ranged from 2.5 to 8.9 using the replicon-based assay and from 1.7 to 11.9 using the biochemical phenotypic assay. A correlation was observed between the telaprevir FC at baseline and change in HCV RNA from baseline to Day 3. However, as mentioned below, this finding needs to be interpreted with caution.
Conclusions inferred from the result of this study are limited due to small sample size and insufficient power to perform appropriate statistical analysis. The small population of this study also means that discrepancies in baseline data (i.e., gender and race) may exert an effect on study results, along with baseline differences in HCV G4 subtype between treatment groups. Unfortunately, sequencing and virologic response data could not be obtained for some patients in this study, further compounding the issue of small sample size. Despite these limitations, it should be noted that the size of this study is not unusual for an exploratory, proof-of-principle study investigating molecular consequences of antiviral activity.
In summary, the results of this study indicates that the in vivo intrinsic activity of telaprevir monotherapy against genotype 4 HCV is modest after 2 weeks of treatment, and suggests synergy between telaprevir and Peg-IFN/RBV against genotype 4 HCV compared with Peg-IFN/RBV alone. Whether the synergy between telaprevir and Peg-IFN/RBV translates in higher SVR rates than Peg-IFN/RBV alone or not, needs to be further investigated. During the telaprevir/placebo treatment phase of this study, all cases of vBT occurred with telaprevir monotherapy, highlighting the importance of combination TPR therapy. Despite this, most patients who experienced vBT with telaprevir monotherapy achieved SVR, indicating that vBT and the emergence of resistant variants do not preclude successful Peg-IFN/RBV treatment. The most common mutation selected in G4 patients during telaprevir therapy was T54A, a mutation previously described for telaprevir in the context of G1 infection.