All patients treated with mericitabine were characterized by a relatively slow viral decline in the first 4 days of treatment compared to rates previously observed in treatment naïve patients during daily IFN-based therapy (15
) and during therapy with NS3 inhibitors (17
), non-nucleoside NS5B inhibitors (23
) or NS5A inhibitors (24
). However, monotherapy with these agents are limited due to the rapid emergence of viral resistance, which was not observed following 14 days of mericitabine. Whereas patients treated with these other classes of agents typically exhibit a biphasic viral decline (14
), about 40% of the patients in our study presented a novel pattern of viral decline, that we identified as “monophasic”, i.e., characterized by a slow first phase of viral decline that extended throughout the 14 days of treatment. We showed that a model assuming mericitabine’s main mode of action was to reduce the rate of virus production, with an effectiveness that increases over time, could describe the data well. The observation that the pyrimidine nucleotide PSI-7977 induces a more rapid first phase of viral decline (25
) than mericitabine, even though the active species of PSI-7977, a uridine triphosphate, is the same uridine triphosphate produced by mericitabine (26
) suggests that the first phosphorylation may be the step limiting the rapid build up of mericitabine’s antiviral effectiveness (13
The estimated final treatment effectiveness was strongly associated with the drug regimen, and the BID regimens had a final effectiveness in blocking viral production (mean 750-mg and 1500-mg: 98% and 99.8%, respectively, P=0.018), significantly higher than the QD regimens (mean 99% and 90%, P<10−7
). How fast the antiviral effectiveness built up was also drug regimen dependent, and we predicted that 12/16 patients in the BID regimens reached 90% of their final antiviral effectiveness by day 4 (Table S1
In all patients, the second phase slope of viral decline was modest. This was attributed, in our model, to a low intrinsic rate of loss of infected cells, δ, which might be causally related to the fact that these patients had previously failed IFN-based therapy. However, other interpretations are possible. In models that allows target cell levels to vary, there exists a certain patient-specific antiviral effectiveness level that needs to be exceeded or the virus will not be eliminated (27
). In this case, the second phase slope of viral decline will be minimal and will not reflect the loss rate of infected cells (27
). From that perspective, the fact that our study population consisted of patients that had previously failed peg-IFN/RBV suggests they may have had a high critical effectiveness, due for instance to more advanced disease with a higher baseline proportion of infected cells (28
By fitting HCV RNA kinetics after treatment cessation, we could estimate parameters related to the drug pharmacodynamics. We estimated that after drug withdrawal the drug effectiveness, after a delay of 0.37 days, declined with a mean half-life in the QD and BID regimens of 30.2 h and 13.9 h, respectively. Since RG7128 requires intracellular uptake and phosphorylation to two active species, a cytidine triphosphate and a uridine triphosphate, with intracellular half-lives of ~5 h and 38 h, respectively (13
), our estimate tends to support that the uridine triphosphate form contributes to maintaining some antiviral effectiveness for a day or two after treatment cessation.
Several assumptions and limitations of our model need to be addressed. The assumption of a gradual increase in antiviral effectiveness that explains the initially slow decrease in viral load still needs to be validated, even though it is supported by the observation that the active forms of mericitabine in vitro
take ~48 hours to accumulate to steady-state triphosphate levels (13
). It is noteworthy that ribavirin, which needs to be phosphorylated to its mono-, di- and tri-phosphate analogs, when given as monotherapy also induces a monophasic viral decline consistent with the variable effectivenss assumption (29
). Second our model does not distinguish between the cytidine and the uridine triphosphates, which could have slightly different potencies and are expected to accumulate at different rates. Third, it is hard to precisely estimate ε1
and δ as they have overlapping effects on the viral load decline. At least one additional sampling measurement between days 1 and 4 would be necessary to estimate more precisely the initial antiviral effectiveness, ε1
. However, the fact that the CE and the VE models provided very similar estimates of ε and δ (see & ) is an indication that these parameters were precisely estimated, and consequently that infected cell loss/death may be playing a minor role in the overall viral load decline. Lastly, for the sake of parameter identifiability, the target cell level was assumed constant throughout the study period. The kinetics of HCV RNA rebound after the end of treatment may be affected by the increased availability of target cells (30
) and hence our estimates of the rate at which antiviral effectiveness decays after the end of treatment may not be as reliable as we would wish.
Recent developments in viral dynamic modeling have emphasized the interplay between the kinetics of intracellular viral RNA (vRNA) and the extracellular viral kinetics measured by serum levels of HCV RNA (31
). Within the context of such models it has been shown that the initial rate of decline of serum HCV RNA is proportional to the ability of drug to block the late stages of virion production (i.e., assembly/secretion) (32
). If a drug does not block virion assembly/secretion, there may be release of preformed virions during the first phase of viral decline that masks the intrinsic plasma HCV clearance rate (32
). Thus the slow initial viral decline observed with mericitabine may reflect that blocking NS5B has only a minimal effect on blocking virus assembly/secretion into the circulation. However, even if a minimal effect in blocking virus assembly/secretion is taken into account using a model that incorporates intracellular events (32
), a gradual decrease in the virus production rate, as in the VE model, is still required to fit the data (not shown). On the other hand the action of the RNA dependent RNA polymerase is needed for the production of negative-strand RNA intermediates and thus mericitabine may block the formation of replication complexes (RC). Guedj and Neumann (31
) proposed a model that takes into account the kinetics of RC and showed that blocking RC formation leads to the progressive depletion of RC with a rapidity that depends on the level of blockage and the intrinsic turnover rate of RC. However, if only extracellular levels of virus are measured, the contribution of this mechanism to the progressive reduction in virus production can not be distinguished from an increase in antiviral effectiveness.
How the viral kinetics results in the present hard-to treat patients would translate to treatment naïve patients is unknown, as are the effects to be seen when mericitabine is given in combination with peg-IFN/RBV or other direct-acting antivirals. However, interim results from a large cohort of treatment naïve patients receiving mericitabine and peg-IFN/RBV (PROPEL study) showed that >80% of patients had undetectable HCV RNA in all cohorts receiving the 12 week triple regimen (33
). Further, 91% of genotype 1 and 4 patients receiving 1000 mg mericitabine BID and Peg/RBV for 24 weeks had undetectable HCV RNA in the JUMP-C trial (34
Because nucleoside analogues appear to have a high barrier to resistance, they are very attractive as part of IFN-free combination therapy. Indeed, when mericitabine was combined with the HCV protease inhibitor danoprevir for 13 days, a 5-log decrease in HCV RNA was achieved, in the highest dosed treatment arms (35
). During monotherapy with NS3/4 protease inhibitors early treatment-resistance viral strains rapidly emerge that lead to viral rebound (8
). Nonetheless, when danoprevir was used in combination with mericitabine or Peg/RBV the viral kinetics were similar (35
) with no evidence of treatment-emergent resistance. This may indicate that the viral kinetics of protease-inhibitor based combination regimens may be primarily driven by the more potent protease inhibitor, with mericitabine or Peg/RBV acting to prevent the emergence of protease resistance.
To summarize, our viral dynamic analysis predicts that mericitabine administered BID achieves a high (final) antiviral effectiveness of 0.98 or greater. Our prediction of a high antiviral effectiveness together with the lack of clinical resistance to mericitabine (12
) support the idea that mericitabine administed BID offers a valuable candidate for IFN-sparing DAA combination regimens.