- Top of page
- Competing Interests
Chronic hepatitis C (HCV) infection is the leading cause of liver disease, affecting over 180 million people worldwide [1, 2]. The prevalence of HCV infection in the United States between the years 1999 and 2002 was 1.6%, equating to about 4.1 million people . Calculations indicate that mortality related to HCV infection will continue to increase over the next two decades .
The current standard of care (SoC) for HCV treatment is a combination of pegylated interferon-alpha (Peg IFN-α) and ribavirin (RBV). Complete removal of the virus occurs in more than 75% of individuals with genotype 2 and genotype 3 infections but less than 50% of individuals with genotype 1 infections . Treatment is often poorly tolerated with many adverse effects being reported, e.g. flu-like symptoms, neuropsychiatric events and neutropenia which are often dose and treatment limiting . To address these issues, a number of oral direct acting antiviral agents are being investigated as combination treatments with Peg IFN-α. The availability of an oral combination regimen could improve convenience of HCV therapy and subsequently compliance, improve response rates and reduce adverse events.
PF-04878691 is a novel, selective and potent agonist of toll-like receptor 7 (TLR7) being developed as an oral therapy for the treatment of HCV infection. TLRs are expressed by immune cells and recognize specific microbial molecular patterns that initiate and direct immune responses . Successful host defence against HCV viral infections relies on the production of specific immunomodulatory cytokines and chemokines including interferons (IFN) . Some of these cytokines, in particular IFN-α, can be activated by TLR7 agonists . Much of the interferon produced comes from plasmacytoid dendritic cells . As well as activating anti-inflammatory cytokines (e.g. IFN), TLR7 agonists also activate pro-inflammatory cytokines (e.g. IL6) which are thought to be responsible for the dose limiting side effects . For any new HCV therapies a wider therapeutic window between the anti-inflammatory (reducing HCV infection) and inflammatory cytokines (side effects) would be required.
Several TLR7 agonists have been evaluated in clinical studies and have been shown to reduce viral load [11–14]. This is believed to be mediated via stimulation of the IFN-α pathway, as measured by IFN-α and markers of IFN induction, e.g. 2′,5′-oligoadenylate synthetase (OAS). Imiquimod, an imidazoquinoline with TLR7 agonist activity, is approved as a topical treatment for anogenital warts caused by human papilloma virus . However, when administered orally little effect was observed in HIV-infected patients . An intravenous isatoribine (TLR7 agonist) dose of 800 mg administered once a day for 1 week and an oral resiquimod dose (TLR7/8 agonist) of 0.02 mg kg−1 administered twice a week for 4 weeks resulted in an increase in IFN-α concentrations and a transient decrease in HCV viral load of 0.76 and 1–3 log10, respectively. However, several patients reported adverse events consistent with elevated cytokine concentrations [13, 14].
PF-04878691 has been studied in preclinical and clinical studies sponsored by 3 M Pharmaceuticals and the Coley Pharmaceutical Group . Several phase 1 and 2 studies were conducted in healthy volunteers and cancer patients. In order to determine its therapeutic index, we conducted in our laboratories in vitro human peripheral blood mononuclear cell stimulation experiments with PF-04878691, evaluating its antiviral efficacy in an HCV replicon assay. From these experiments, a separation of the antiviral response from the induction of pro-inflammatory cytokines was seen, supporting the hypothesis that repeated doses of PF-04878691 would activate the antiviral response in vivo without giving rise to significant side effects. Therefore we performed a 2 week ‘proof of pharmacology’ (POP) study with PF-04878691 in healthy volunteers to determine clinically whether sufficient pharmacology (as measured by OAS) could be achieved in the presence of a suitable side-effect profile. If successful, PF-04878691 would be taken forward to a randomized, double-blind, placebo-controlled ‘proof of concept’ (POC) study in HCV infected patients to investigate its pharmacodynamics (as measured by reduction in HCV RNA from baseline), pharmacokinetics (PK), safety and toleration following 4 weeks of monotherapy.
The aim of this work was to predict the likely outcome of PF-04878691 in a POC study in HCV patients in order to support further decision making around this compound. This was achieved by firstly developing a model using available POP clinical data to describe PF-04878691 PK and its relationship to OAS (marker of pharmacology) and lymphocyte levels (marker of safety) following multiple doses in healthy subjects. To date this compound has not been dosed in HCV patients. Therefore in order to predict possible HCV viral RNA profiles in patients, a model was developed to describe the relationship between change from baseline OAS expressed as fold change and HCV viral RNA concentrations using clinical data from HCV patients for a separate compound, CPG-10101 (ACTILON™), a TLR9 agonist . Using these relationships and assuming that the relationship between OAS and HCV viral RNA is the same for TLR7 and TLR9 agonists, PF-04878691 exposure and HCV viral RNA concentrations were simulated in HCV patients receiving twice weekly administration for 4 weeks over a range of doses. The likelihood of a positive POC study (>1 log10 decrease in HCV viral RNA concentrations) was assessed.
- Top of page
- Competing Interests
A multiple dose escalation study where male and female healthy volunteers were administered PF-04878691, a TLR7 agonist, orally twice a week for 2 weeks has been performed. Population PK, OAS and lymphocyte models were developed that adequately described the observed clinical data from this study. In addition a model was developed using the individual level data following administration of a TLR9 agonist, CPG-10101 (ACTILON™) subcutaneously once/twice weekly for 4 weeks to describe the relationship between OAS and HCV viral RNA concentrations. These models were used to predict the likely outcome of PF-04878691 in HCV patients in order to support further decision making around this compound.
Observed PF-04878691 plasma exposure increased over time in a manner that was not consistent with the clearance of the compound. A standard two-compartmental model over-predicted exposure on day 1 (the first dose) and under-predicted exposure on day 11 (the fourth dose). An empirical model was used to describe the time dependent increase in exposure and provided an adequate description of PF-04878691 plasma concentrations. In vitro data suggest that PF-04878691 is partially metabolized by CYP1A2. Drug–drug interactions have been reported between theophylline (CYP1A2 substrate) and IFN, where IFN increases the exposure of theophylline . The production of IFN over time as a result of TLR7 agonism, could result in the inhibition of CYP1A2 and consequently the time dependent increase in exposure of PF-04878691. Another IFN inducer, tilorone hydrochloride, has been shown to reduce drug metabolizing activity in rat liver .
The OAS response observed in the clinical study at doses at and above 6 mg is in a similar range to those observed with other TLR7 agonists and with IFN treatment in the clinic where therapeutic reductions in HCV viral load were obtained [13–15, 23–29]. Data from IFN treatment indicate that a maximum increase in OAS of approximately 8-fold can be expected at efficacious doses [23–29]. Isatorabine showed a 7.6 fold increase in OAS from baseline with moderate changes in antiviral activity (0.75 log10 reduction from baseline) . In our multidose study with PF-04878691, OAS increases of 8-fold or more from baseline were seen in 3/6 individuals at 3 mg and 6/6 at 6 mg and 5/6 at 9 mg (a non-responder was identified in the 9 mg cohort). Two healthy volunteers who received 9 mg of PF-04878691 developed serious adverse events consistent with IFN and cytokine production, following the second and fourth doses of the compound, respectively. A less severe but similar event was observed in one volunteer following the fourth dose at 6 mg. Transient dose and time dependent decreases in lymphocyte counts were observed in the 6 and 9 mg dose groups. The adverse effects were similar to what was seen in a single dose healthy volunteer study with PF-04878691, evaluating oral doses of 2, 10, 15 and 20 mg, where transient, dose-dependent decreases in mean absolute lymphocyte count were observed . The only dose therefore with a safety/tolerability profile similar to SoC (IFN-α/ RBV) was the 3 mg dose. There was significant overlap in exposure between the 3 and 6 mg doses, resulting in an extremely small safety window (<2-fold). Some of the side effects such as flu-like symptoms, are associated with, the current SoC for the treatment of HCV [30, 31]. The degree of lymphopenia observed in the 6 mg and 9 mg cohorts of PF-04878691 was more severe than is normally seen with IFN-α/RBV treatment .
In order to determine whether other dosing regimens could be explored to provide an adequate OAS fold increase while maintaining adequate safety and tolerability, a model was developed to describe the OAS and lymphocyte level data. As a result of the limited number of doses studied with PF-04878691, the typical Emax style indirect response model could not adequately identify the model parameters and a power function was used to describe the relationship between PF-04878691 and OAS as well as total lymphocyte levels. This was deemed appropriate for fitting and further simulations as simulations were to be performed within the dose range already studied.
To date PF-04878691 has not been dosed in HCV patients. Therefore in order to predict possible HCV viral RNA profiles in patients, a model was developed to describe the relationship between change from baseline OAS expressed as fold change and HCV viral RNA concentrations using clinical data available in patients for a separate compound, CPG-10101 (ACTILON™), a TLR9 agonist . The OAS data reported by McHutchison et al.  were measured using an activity assay, whereas OAS data in our study were measured using a gene expression assay . Based on literature evidence, the relationship between OAS expressed as fold change and HCV viral RNA concentrations seems largely independent of the OAS assay type [13–15, 23–29].
Traditionally the correlation between PK (dose), HCV RNA level and OAS or other biomarkers, such as IFN and IP-10, has been determined using the maximum change from baseline approach [13, 16]. This approach only uses the maximum biomarker level and correlates it with the maximum change from baseline HCV RNA concentration. The biggest disadvantage to this is that the variation of biomarker levels with time, as well as within-subject and between-subject variabilities are ignored. In the current analysis, an attempt to model the relationship between OAS and HCV RNA concentration was made.
There are several challenges when attempting to determine the relationship between OAS and HCV RNA concentrations. The first is the precision and sensitivity of the assay used to determine the OAS expression. The second is the heterogeneity of the HCV patient population in terms of disease status and baseline HCV RNA concentrations. The third is the highly variable HCV RNA levels even within an individual. All these could be the key factors for explaining the relatively high IIV and %CV for parameters such as Imax and VO50. In general the VPC plots suggested that the random effects parameters were over predicted for all the models used in this study. However the 50% percentile indicates the models captured the central tendency correctly. Among the 60 HCV patients in study CPG-10101, there were two prior treatment naive patients, 10 patients prior treatment intolerant, nine partial responders (≥1 log10 decrease in viral titre reported on prior treatment, but did not clear virus), 30 treatment relapsers (cleared virus on previous treatment, but relapsed), and nine uncharacterized responders (≥1 log10 decrease in viral titre reported on prior treatment, uncertain if ever cleared virus, but viral positive on screening before study) . Though there is a wide range of baseline HCV RNA concentration across the HCV patients (4.73–7.73 log10 copies ml−1) the model based estimate appears to be relatively precise, 7.26 log10 copies ml−1 with a CV% of 1.38%.
For the current translational modelling approach, across mechanisms (TLR7 and TLR9) and across populations (healthy volunteers and HCV patients), the following key assumptions were necessary. Firstly, the subsequent intracellular signalling pathways are similar after binding of both TLR7 and TLR9 agonists to their receptors; thus the magnitude of OAS and IFN response required for an antiviral effect by each of the pathways would be comparable. As both TLR7 and TLR9 work through the same pathway, this is a reasonable assumption . Secondly, there is no PK or biomarker difference between the healthy volunteer and HCV patient populations. There is some evidence, however, to suggest that biomarker level production could be reduced in HCV patients compared with healthy subjects . The simulations made could therefore represent a ‘best case’ scenario in terms of pharmacological activation.
Using these relationships, PF-04878691 exposure and clinical outcome was simulated in HCV patients receiving twice weekly administration of PF-04878691 using a range of doses for 4 weeks. The likelihood of achieving adequate HCV viral RNA concentration reduction (>1 log10 decrease in HCV viral RNA concentrations) at a dose/dosing regimen that was considered safe was assessed. Results indicate that significant reductions (1–2 log drop) in viral load would be expected only at doses >6 mg. However at these doses, grade 3 lymphopenia would also be predicted (Figure 10). POC criteria were agreed as demonstration of a mean reduction of >1 log10 in HCV RNA following 4 weeks of dosing with a dose that has a safety profile that is no worse than SoC. Population models to describe the PK and PD response of PF-04878691 indicate that this compound is unlikely to achieve POC criteria. The models developed during this exercise support the discontinuation of this compound. This, together with subsequent data indicating that in vitro activity against HCV was only observed at doses where mechanism-related adverse events were seen, raises concerns regarding the therapeutic window and potential utility of this compound class for the treatment of HCV. A robust approach for translation of anti- and pro-inflammatory responses from ex vivo to in vivo could also have aided in this decision process. However the translation of such responses from ex vivo data has not been widely studied.