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The cyclophilin inhibitor Debio 025 combined with PEG IFN2a significantly reduces viral load in treatment-naive hepatitis C patients: efficacy & safety
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Hepatology early view April 2009
Robert Flisiak 1, Saya V. Feinman 2, Maciej Jablkowski 3, Andrzej Horban 4, Wieslaw Kryczka 5, Magorzata Pawlowska 6, Jenny E. Heathcote 7, Giuseppe Mazzella 8, Carmen Vandelli 9, Valerie Nicolas-Metral 10, Pierre Grosgurin 10, Jorge S. Liz 10, Pietro Scalfaro 10, Herve Porchet 10, Raf Crabbe 10 *
1Department of Infectious Diseases and Hepatology, Medical University of Bialystok, Bialystok, Poland
2Liver Study Unit, Mt. Sinai Hospital, Toronto, Canada
3Clinic for Infectious Diseases, Medical University, Lodz, Poland
4Hospital of Infectious Diseases, Warsaw, Poland
5Department of Infectious Diseases, Regional Hospital, Kielce, Poland
6Department of Infectious Diseases and Hepatology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland
7Toronto Western Hospital, Toronto, Canada
8Department of Internal Medicine and Gastroenterology, University of Bologna, Bologna, Italy
9Dipartimento di Medicine e Specialita mediche, University of Modena and Reggio Emilia, Modena, Italy
10Debiopharm S. A., Lausanne, Switzerland
email: Raf Crabbe (rcrabbe@debiopharm.com)
*Correspondence to Raf Crabbe, Debiopharm S. A., Forum apres-demain, Ch.. Messidor 5-7, CP 5911, 1002 Lausanne, Switzerland
"the current study seems to confirm previous results indicating that hyperbilirubinemia is an isolated problem of transporter inhibition by high doses of Debio 025 in susceptible patients, and that it is not related to a direct hepatotoxicity.[5] However, because the experience with Debio 025 is still limited, liver function will be carefully followed in future studies to exclude occurrence of rare and more severe forms of drug-induced cholestasis or liver injury."...."The 200- and 600-mg dose were very well tolerated, whereas the 1,000 mg dose revealed isolated and reversible hyperbilirubinemia linked to inhibition of biliary canalicular transporters."
Abstract
The anti-hepatitis C virus (HCV) effect and safety of three different oral doses of the cyclophilin inhibitor Debio 025 in combination with pegylated interferon-a2a (PEG IFN-a2a) were investigated in a multicenter, randomized, double-blind, placebo-controlled escalating dose-ranging phase II study in treatment-naive patients with chronic hepatitis C. Doses of 200, 600, and 1,000 mg/day Debio 025 in combination with PEG IFN-a2a 180 ug/week for 4 weeks were compared with monotherapy with either 1,000 mg/day Debio 025 or 180 ug/week PEG IFN-a2a. In patients with genotypes 1 and 4, the 600- and 1,000-mg combination treatments induced a continuous decay in viral load that reached -4.61 ± 1.88 and -4.75 ± 2.19 log10 IU/mL at week 4, respectively. In patients with genotypes 2 and 3, HCV RNA levels at week 4 were reduced by -5.91 ± 1.11 and -5.89 ± 0.43 log10 IU/mL, respectively, with the same treatment regimens. Adverse events were comparable between treatment groups apart from a higher incidence of neutropenia associated with PEG IFN-2a and an increased incidence of isolated hyperbilirubinemia at the highest dose of Debio 025 (1,000 mg/day).
Conclusion: These results confirm that Debio 025 has a potent activity and an additive effect on HCV RNA reduction in genotype 1 and 4 patients at 600 and 1,000 mg/day when combined with PEG IFN-2a.
Hepatitis C virus (HCV) infection has emerged as a major cause of liver disease worldwide.[1] This potentially progressive disorder is associated with an increasing incidence of cirrhosis and end stage liver disease and is frequently complicated by hepatocellular carcinoma and the subsequent need for orthotopic liver transplantation.[2] Estimates indicate that approximately 130 million people are infected with HCV.[1]
The current standard of care for HCV infection is a combination of injectable pegylated interferon-a (PEG IFN-a) and oral ribavirin administered for 24 to 48 weeks. Treatment duration and ribavirin dose are determined by HCV genotype. The response rate varies according to genotype, virus titer, and patient characteristics. Sustained viral response in genotype 2 and 3 patients after combined PEG IFN-a and ribavirin treatment reaches 80% to 90% but is only 40% to 50% in genotype 1 patients. Few data are available on genotype 4 patients, but it is accepted that their response is intermediate between patients with genotype 1 and genotypes 2 and 3.[2] Clearly, the growing number of individuals diagnosed with chronic HCV infection, including numerous nonresponders and relapsers, calls for new developments in this area.
Debio 025 is a selective cyclophilin inhibitor with well-established in vitro and in vivo anti-HCV properties.[3][4] A recent proof of concept study provided the first evidence that oral administration of Debio 025 monotherapy at a dose of 1,200 mg twice daily for 15 days has a significant anti-HCV effect (mean maximum drop, -3.6 log10) in human immunodeficiency virus/HCV-coinfected patients. Debio 025 was active against the three HCV genotypes represented in the study (1, 3, and 4), and signs of upcoming resistance such as a rebound of HCV viral load in the second phase of viral decay were not observed.[5] This important anti-HCV response, together with preclinical data showing that Debio 025 has an additive anti-HCV effect when combined with IFN-a,[3][4] motivated the study reported here. Debio 025 exerts an inhibiting effect on cyclophilins, which play an important role in HCV replication. It has not yet been fully elucidated which cyclophilins are involved, but cyclophilins A and B have both been proposed as potentially relevant.[6] Given that Debio 025 and PEG IFN-a have different mechanisms of action with different targets within the HCV viral replication cycle,[7][8] we anticipated that HCV-monoinfected patients given lower doses of Debio 025 combined with PEG IFN- would show a stronger viral response than with either of the two treatments alone.
We designed this multicenter, randomized, double-blind, placebo-controlled escalating dose-ranging phase II study to investigate the antiviral potential of Debio 025 combined with PEG IFN-2a in the treatment of naive patients with chronic hepatitis C. The Debio 025 starting dose of 200 mg/day was derived from previous phase I studies showing an excellent tolerance and safety profile at doses between 50 and 1,200 mg/day. Because Debio 025 is mainly metabolized through cytochrome P450 enzymes 3A, which are not affected by IFN-, a pharmacokinetic interaction with PEG IFN-2a was deemed unlikely. In addition, because of the drug's long terminal half-life (about 100-180 hours for the last part of its multiexponential elimination phase) and the importance of rapidly reaching pharmacologically active drug levels, a loading dose regimen was selected for the first week of treatment to reduce time to steady state.
Results
Patient Enrollment.
The study population comprised 90 patients enrolled in 10 centers in Italy, Poland, and Canada between September 2006 and August 2007. A total of 52 men and 38 women were included and treated in four sequential cohorts as shown in Fig. 1. A total of 86 patients completed the study; four patients discontinued because of a TEAE. Patient disposition is presented in Fig. 2. Ninety-six percent of patients were Caucasian, with an average age of 35 years. Sixty patients were included in the potential slow responders class (genotypes 1 and 4) and 30 patients in the potential fast responders class (genotypes 2 and 3). Genotypes 5 and 6 were not represented in this study. Patient baseline characteristics are shown in Table 1.
Response to PEG IFN-a2a/Debio 025 Therapy.
All 90 patients were included in the intention to treat analysis. The most important mean response was observed in the treatment groups combining PEG IFN-a2a and the two highest Debio 025 doses: 600 mg (treatment group 600 Combo) and 1,000 mg (treatment group 1,000 Combo). In both groups, log10 HCV RNA mean (±SD) levels fell rapidly by day 8 from 6.32 and 6.12 log10 IU/mL to 2.89 and 2.06 log10 IU/mL (difference -3.43 ± 1.19 and -4.05 ± 1.28 log10 IU/mL), respectively. HCV RNA levels continued to decline, and the mean value after 4 weeks of treatment dropped below the limit of quantification (difference -5.07 ± 1.73 and -5.09 ± 1.91 log10 IU/mL). The mean reduction of HCV RNA levels after 4 weeks in treatment groups 200 Combo (PEG IFN-a2a and 200 mg Debio 025), Peg Mono (PEG IFN-a2a and Debio 025 placebo), and 1,000 Mono (1,000 mg Debio 025 monotherapy) were -3.30 ± 2.18, -3.56 ± 2.37, and -2.87 ± 2.28 log10 IU/mL, respectively.
Statistical analysis using the mixed effect model revealed a significant viral load reduction over time (P < 0.001) and significant differences in viral load reduction between treatment groups (P < 0.001) and HCV genotype classes (P < 0.001). Maximal viral load reduction was also significantly different between treatment groups (P = 0.005) and between HCV genotype classes (P < 0.001). Among pairwise treatment group comparisons in maximal viral load reduction, the 1,000 Mono and 1,000 Combo treatments were significantly different (P < 0.008) using the Holm-Bonferroni adjustment for multiple comparisons. This indicates that the combination 1,000 mg Debio 025 with PEG IFN-a2a is significantly more effective in reducing HCV RNA than 1,000 mg Debio 025 in monotherapy.
In the subgroup of genotype 1 and 4 patients, response profiles in treatment groups 600 Combo and 1,000 Combo showed a rapid reduction in HCV RNA levels in the first week with values of -3.01 ± 1.05 and -3.84 ± 1.47 log10 IU/mL, respectively (Table 2; Fig. 3). Afterward, there was a continuous steady decline of viral load and HCV RNA reductions that reached -4.61 ± 1.88 log10 IU/mL and -4.75 ± 2.19 log10 IU/mL at week 4 in the 600 Combo and 1,000 Combo groups, respectively. These viral load reductions were larger than those achieved at the same time point in treatment groups 200 Combo (-2.65 ±1.65), Peg Mono (-2.49 ± 1.95), and 1,000 Mono (-2.2 ± 2.4 log10 IU/mL) (Table 2; Fig. 3). The maximum log10 reduction in HCV RNA copies was significantly larger in treatment groups 600 Combo and 1,000 Combo versus Peg Mono and in treatment group 1,000 Combo versus 1,000 Mono (Holm-Bonferroni-adjusted P < 0.02). In both monotherapy groups (Peg Mono and 1,000 Mono), undetectable viral load at study end (< 15 IU/mL) was observed in three out of 12 patients. In treatment groups 200 Combo, 600 Combo, and 1,000 Combo, the proportion of patients with undetectable viral load at study end reached 2, 4, and 8 out of 12, respectively.
Viral load reduction in genotype 2 and 3 patients was more pronounced than in genotype 1 and 4 patients. Peg Mono and 1,000 Mono treatments induced HCV RNA reductions of -5.69 ± 1.58 and -4.22 ± 1.33 log10 IU/mL at week 4, respectively; at day 8, levels had dropped by -2.67 ± 1.16 and -3..39 ± 0.77 log10 IU/mL respectively, and at day 22 the large majority of patients had undetectable HCV RNA. In each group, viral load was undetectable in four out of six patients at treatment end (Table 2; Fig. 3). In combination groups 200 Combo, 600 Combo, and 1,000 Combo, HCV RNA reductions at week 4 reached -4.59 ± 2.68, -5.91 ± 1.11 and -5.89 ± 0.43 log10 IU/mL, respectively (Table 2; Fig. 3). The number of patients with undetectable viral load in these groups at treatment end was 3, 5, and 5 out of 6, respectively. The number of genotype 2 and 4 patients in the 1,000 Mono group was very small: two and one, respectively. However, these three patients decreased their HCV RNA by more than 2 log10 IU/mL after the 4-week treatment.
Safety.
Overall, 610 TEAEs were reported in 87 (97%) patients. The highest TEAE incidence was found in treatment group Peg Mono (152 TEAEs) and the lowest in treatment group 1,000 Mono (87 TEAEs). A total of 109, 125, and 137 TEAEs were recorded in treatment groups 200 Combo, 600 Combo, and 1,000 Combo, respectively (Table 3). Flu-like symptoms (headache, pyrexia, myalgia, and chills), nausea, and fatigue were the most frequently reported TEAEs (>/=20% of patients). Flu-like symptoms, which were less reported in treatment group 1,000 Mono (no PEG IFN-a2a), were mostly deemed to be PEG IFN-a2a-related. Headache, nausea, fatigue, and hyperbilirubinemia were the most frequent TEAEs (>10% of patients) considered to be related to study medication. Most TEAEs were resolved by the end of the study. No serious TEAEs were reported. Four patients discontinued treatment because of TEAEs. One subject developed a dental abscess after 10 days of 600 Combo, which required treatment with antibiotics; the second subject experienced chills, dysgeusia, tachycardia, pain, nausea, dehydration, wheezing, dyspepsia, and photophobia in the first week of 1,000 Combo. The third subject was diagnosed with hypertension (160/110 mm Hg) at day 21 of treatment (1,000 Combo) that was not present at the start of the study. The fourth subject developed hyperbilirubinemia after 1 week of 1,000 Mono (1,000 mg Debio 025 twice daily). The finding persisted after a dose reduction to 100 mg once daily, and the patient was withdrawn from the study after 15 days. At that time, total bilirubin was 138.6 umol/L (normal range, 3.8-21.9 umol/L). There was no associated increase in aminotransferases, alkaline phosphatases, or Y-glutamyltransferase. A liver biopsy taken 30 days after the last drug administration revealed signs compatible with mild HCV-related inflammation, but no signs of intrahepatic cholestasis. In all four cases, symptoms subsided rapidly after treatment discontinuation, and all patients recovered completely. Hypertension and hyperbilirubinemia were considered as treatment-related. Dose-related increases in conjugated and total bilirubin levels were observed in the Debio 025-treated groups and lead to reports of hyperbilirubinemia in 13 out of 36 patients receiving the 1,000-mg dose (treatment groups 1,000 Combo and 1,000 Mono). Increases were most prominent during the first week when patients received the 1,000-mg loading dose twice daily, and returned to normal levels in most patients. Only four out of 36 (11%) patients maintained bilirubin levels above 34.2 mol/L (range, 41.1-138.6 umol/L). Hyperbilirubinemia was associated with clinical signs (icterus) of respectively mild and moderate severity in two patients. There was no associated increase in aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, or y-glutamyl transferase. Bilirubin returned to baseline values after treatment cessation. No hyperbilirubinemia was reported with treatments 200 Combo and 600 Combo. Neutropenia (lowest value 0.4 X 109/L), a well-known side effect of PEG IFN-a2a, was reported in 16 out of 54 (30%) patients receiving combined PEG IFN-2a and Debio 025 treatment versus 5 out of 18 (28%) in the PEG IFN-2a and none in the Debio 025 monotherapy group. Thrombocytes decreased to a mean value of 149 ± 38 X 109/L after 4 weeks of treatment with Peg Mono and 198 ± 47 X 109/L after 4 weeks of treatment with 1,000 Mono. Values after 4 weeks in treatment groups 200 Combo, 600 Combo, and 1,000 Combo were 165 ± 38 X 109/L, 138 ± 27 X 109/L, and 149 ± 76 X 109/L, respectively, at the same time point. The lowest value recorded was 41 X 109/L, which occurred after 3 weeks of treatment with Peg Mono. In all other cases, thrombocyte levels remained above 50 X 109/L. Mean values of main hematological parameters over time per treatment group are shown in Fig. 4.
Discussion
Debio 025 is a cyclophilin inhibitor with a potent in vitro effect on HCV replication.[4] A recent study in human immunodeficiency virus/HCV-coinfected patients demonstrated that the drug was able to induce a mean reduction in HCV RNA viral load of 3.6 log10 IU/mL after 14 days of monotherapy (1,200 mg twice daily) in this particularly difficult-to-treat population. The individual data from this small proof of concept study also revealed that the drug was active against genotypes 1, 3, and 4, all of which were represented in the study.[5]
With the present study, we intended to confirm previous results in a population of treatment-naive chronic HCV-monoinfected patients. In addition, we wanted to investigate whether Debio 025 combined with a standard dosing regimen of PEG IFN-a2a (180 ug/week) would induce an additive reduction in HCV RNA levels when compared with both treatments given individually. Ribavirin was not added to the dosing scheme because the contribution of each compound to the efficacy and safety profile of the combination treatment would be easier to assess with a two-way rather than a three-way combination. Because clinical experience with Debio 025 is still limited, this approach also appeared safer. PEG IFN was preferred over ribavarin in this short-term study because its contribution to the antiviral effect in the first weeks of treatment is more important than that of ribavirin. Finally, to explore a differential effect on several genotypes, the study population was stratified into two groups, one including genotypes 2 and 3, known to better respond to IFN-based therapy, and one including genotypes 1 and 4. As could be expected from HCV epidemiology, the former group consisted mainly of genotype 3 and the latter of genotype 1 patients, with only a minor contribution of genotypes 2 and 4 (Table 1).
The Debio 025 doses selected for this study (200, 600, and 1,000 mg/day) were substantially lower than in our previous study (2,400 mg/day).[5] We expected the combination with PEG IFN to maintain a relevant anti-HCV effect while precluding the dose-limiting adverse events seen with the 2,400-mg dose (hyperbilirubinemia and platelet reduction). During the first week of treatment, a loading regimen was administered that consisted of twice the allocated dose daily; without the loading regimen it would have taken more than 1 week for most patients to reach pharmacokinetic steady state due to the long terminal half-life of the drug; it would also have been substantially more difficult to draw the right dose-response conclusions from efficacy and safety data within the 4-week treatment period. Although the importance of trough plasma levels on the anti-HCV effect and/or the development of viral resistance is unknown for Debio 025, it seemed logical that rapidly building up and maintaining effective drug levels would increase the chances of establishing a dose-effect relationship.
Results show that Debio 025 monotherapy at a dose of 1,000 mg/day (1,000 Mono) has a similar effect on HCV RNA load as PEG IFN-a2a monotherapy (Peg Mono). This was not only observed for the entire study population, but also for the subgroups, of which genotype 3 is more sensitive to Debio 025 than genotype 1. Because only one genotype 4 and two genotype 2 patients were in the 1000 Mono arm, it is impossible to draw conclusions about a differential sensitivity of these strains. However, because these three patients were found to have a more than 2 log10 drop in HCV RNA during treatment, the drug seems to be active against genotype 2. The single genotype 4 patient confirmed activity already detected in the previous study.[5]
Debio 025 at a dose of 600 and 1,000 mg/day (600 Combo and 1,000 Combo) induced a statistically significant additive HCV RNA drop in genotype 1 and 4 patients when compared to individual treatments. The maximum HCV RNA reduction was 4.67 and 5.05 log10 IU/mL, respectively, compared with 2.56 log10 IU/mL with Peg Mono and 2.46 log10 IU/mL with 1,000 Mono. The difference in reduction of viral load between 600 Combo and 1,000 Combo was limited (0.38 log10 IU/mL), but the number of patients who fell below the limit of detection increased from four out of 12 in the former group to eight out of 12 in the latter. As expected, viral load reduction with the individual drugs was so important in genotype 2 and 3 patients that it was not possible to demonstrate a significant difference with combination treatment in this small-sample, short-term study. However, the study succeeded in confirming that both genotype 2 and 3 subtypes are responsive to Debio 025.
Sequencing of viral strains to detect mutations conferring for resistance is still ongoing at the time of submission. This is particularly challenging, because the mechanism of action of the drug is not yet fully understood. The individual viral load data in this study did not show any case of viral breakthrough during treatment, confirming previous in vitro and in vivo data indicating Debio 025 to have a high barrier for the development of resistance.[4][5]
Safety analysis revealed that most of the reported adverse events were typical of IFN-based treatment.[9-11] The addition of 200 and 600 mg/day Debio 025 did not change the safety profile of PEG IFN-a2a monotherapy. Although thrombocyte decreases were observed with Debio 025 monotherapy, the three Debio 025 doses combined with PEG IFN-2a did not induce a more important thrombocyte reduction than both monotherapies after 4 weeks. None of the patients in this trial needed a dose reduction of either PEG IFN-a2a or Debio 025 because of thrombocytopenia. At the dose of 1,000 mg, Debio 025 caused isolated hyperbilirubinemia in individual patients without associated increase of other liver enzymes such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, or Y-glutamyl transferase. Hyperbilirubinemia was most pronounced during the first week (loading dose), normalized or diminished thereafter in all but one patient, and was completely normalized in all subjects 3 weeks after the end of treatment. In one patient, hyperbilirubinemia was pronounced and reached 138.6 umol/L at day 15. Other liver enzymes remained within normal limits and hyperbilirubinemia normalized rapidly after treatment cessation. A liver biopsy performed in this patient 30 days after the last drug administration confirmed lesions compatible with chronic HCV infection (Ishak score 3 for inflammation and 0 for fibrosis), but no signs of cholestasis. Isolated hyperbilirubinemia, mainly consisting of conjugated bilirubin without histological signs of cholestasis, may be explained by inhibition of the biliary canalicular transporter multidrug resistance-associated protein 2. This transporter is responsible for the excretion of conjugated bilirubin and also plays a role in the elimination of some beta-blockers (such as atenolol and carvedilol), antiretrovirals (such as indinavir and saquinavir), and mycophenolic acid[12-15]; multidrug resistance-associated protein 2 was significantly inhibited by Debio 025 in preclinical in vitro studies. The current study seems to confirm previous results indicating that hyperbilirubinemia is an isolated problem of transporter inhibition by high doses of Debio 025 in susceptible patients, and that it is not related to a direct hepatotoxicity.[5] However, because the experience with Debio 025 is still limited, liver function will be carefully followed in future studies to exclude occurrence of rare and more severe forms of drug-induced cholestasis or liver injury.
In conclusion, the results of the present study confirm that Debio 025 has a potent activity against the four most prevalent genotypes of HCV in treatment-naive patients. When combined with a standard dosing regimen of PEG IFN-2a, Debio 025 at a dose of 600 or 1,000 mg/day shows a clear additive reduction of HCV RNA in the subgroup of patients with genotype 1 and 4. In genotype 2 and 3 patients, HCV RNA reduction with Debio 025 and PEG IFN-2a was so important that a majority of patients achieved undetectable plasma levels, making it impossible to demonstrate an additive effect of the combination therapy. The 200- and 600-mg dose were very well tolerated, whereas the 1,000 mg dose revealed isolated and reversible hyperbilirubinemia linked to inhibition of biliary canalicular transporters. Studies of Debio 025 in combination with PEG IFN-2a and ribavirin aiming at achieving sustained viral response are needed to establish the full efficacy and safety profile of this drug as well as its potential position in future anti-HCV treatment combinations.
Patients and Methods
Study Design.
This was a multicenter, randomized, double-blind, placebo-controlled escalating dose-ranging study. Treatments were administered via four sequential cohorts of treatment-naive patients with chronic hepatitis C: three double-blind, placebo-controlled cohorts of 24 patients each (cohorts I, II, and III) and one open noncomparative cohort of 18 patients (cohort IV) (Fig. 1). Within cohorts I to III, patients were randomly assigned to one of the following treatments: cohort I received PEG IFN-2a combined with Debio 025 (2 X 200 mg followed by 1 X 200 mg), defined as 200 Combo, or PEG IFN-a2a combined with Debio 025 placebo, defined as Peg Mono; cohort II received PEG IFN-a2a combined with Debio 025 (2 X 600 mg followed by 1 X 600 mg), defined as 600 Combo, or Peg Mono; cohort III received PEG IFN-a2a combined with Debio 025 (2 X 1,000 mg followed by 1 X 1,000 mg), defined as 1,000 Combo, or Peg Mono. In cohort IV, all patients received Debio 025 monotherapy (2 X 1,000 mg followed by 1 X 1,000 mg), defined as 1,000 Mono. Debio 025 was administered twice daily from day 1 to day 7 and once daily from day 8 to day 29. PEG IFN-a2a was given subcutaneously at 180 ug/week, including on day 29. Within cohorts, patients were stratified by HCV genotype class as potential slow responders (genotypes 1, 4, 5, or 6) and potential fast responders (genotypes 2 or 3) in a 2:1 unbalanced ratio. Within cohorts I to III, patients were randomized to Combo treatment versus Peg Mono in a 3:1 ratio. Debio 025 dose escalation from one cohort to the other was recommended by an independent data-monitoring committee after review of safety and efficacy data. Treatment duration (29 days), follow-up (3 weeks), and assessments were identical in all treatment groups.
A sample size of 18 patients per treatment group was required to detect between the combination Debio 025 and PEG IFN-a2a and PEG IFN-a2a alone a difference of at least 1.0 log10 copies/mL in HCV viral load reduction assuming a common standard deviation of 1.0 log10 IU/mL, with a nominal two-sided a risk of 0.05 and a power of at least 0.80 (beta risk of 0.20). The actual power was 0.83.
The placebo formulation was similar to that of Debio 025 except for the active compound. Visual inspection did not allow differentiation between treatments, which were packaged in a blinded fashion prior to study start. A computer-generated randomization was issued prior to study initiation by the study biostatistician. Study treatments were assigned centrally via an Internet-based case report form application. Ethical committees at participating centers approved the study protocol. All patients provided written informed consent. The study was conducted according to the principles set forth in the 2000 Declaration of Helsinki and Good Clinical Practices guidelines.
Participants.
Male and female patients were eligible for inclusion if they were between 18 and 70 years of age; presented with treatment-naive HCV infection and detectable plasma HCV RNA levels >/=1,000 IU/mL; were negative for hepatitis B surface antigen and anti-human immunodeficiency virus antibody; had normal or compensated liver function (supported by albumin >/=35 g/L, total bilirubin /=3.0 X 109/L, neutrophil count >/=1.5 X 109/L, hemoglobin >/=100 g/L, and platelets >/=150 X 109/L. Patients were excluded in cases of previous antiviral treatment; clinically significant coexisting medical condition; documented liver biopsy indicating advanced inflammation, necrosis, or fibrosis (Scheuer grade 4 for necroinflammation and fibrosis, Metavir score of A3F4 or higher); major liver impairment; a-fetoprotein >50 ug/L; arterial hypertension; or significant kidney impairment.
Endpoint Measures.
Antiviral activity was assessed as the maximum log10 reduction of HCV RNA copies from baseline to on- and post-treatment (primary endpoint), the change in log10 HCV RNA copies from baseline to day 29, and the proportion of patients with a 2 log10 reduction in HCV RNA copies and/or undetectable viral load (<15 IU/mL) 4 weeks after treatment. Safety was assessed on changes in vital signs, 12-lead electrocardiograms, standard hematology, chemistry and urine laboratory tests, and reports of treatment-emergent adverse events (TEAEs).
Procedures.
After prestudy evaluation, eligible patients were started on PEG IFN-a2a/Debio 025 treatment as described above. Patients were seen on five occasions during the 29-day treatment period and on one occasion during follow-up. Study medication was administered under visual supervision at the study centers and documented in a patient diary when taken at home. Blood was sampled for plasma HCV levels pre-dose on day 1 and in the morning pre-dose on days 8, 15, 22, and 29 and during follow-up on day 50. Safety assessments were performed at the same time points.
Determination of HCV RNA Levels and HCV Genotyping.
Polymerase chain reaction was used to measure plasma HCV RNA (limit of quantification, 45 IU/mL; limit of detection, 15 IU/mL) (Roche COBAS TaqMan, Roche Laboratories, Basel, Switzerland) and to determine HCV genotyping (Abbott ABI Prism 7000, Abbott Diagnostic Division, Santa Clara, CA). HCV RNA determination and HCV genotyping were performed by MDS Pharma Services (Paris, France and North Brunswick, NJ).
Statistical Analysis.
Distributional assumptions underlying the statistical analyses were assessed for normality by normal probability plots and for homogeneity of variance by plotting student residuals against the predicted values for the model. Log10 reduction in HCV RNA copies (IU/mL) from pretreatment to end of treatment (day 29) was analyzed using the mixed effects model analysis of variance, including the factors treatment group and HCV genotype class (stratification factor), and treatment by genotype interaction. To determine the minimum effective dose, adjustment for multiple comparisons was performed using the closed testing procedure by Holm-Bonferroni when comparing each Debio 025 Combo treatment group with the Peg Mono treatment and 1,000 Combo versus 1,000 Mono. Treatment differences were tested using least-squares means from analysis of variance. HCV viral loads below the limit of quantification were considered as half the limit of quantification, and viral loads below the limit of detection were given the value 1 IU/mL for the purpose of log transformation. The proportion of patients with a viral load reduction by at least 2 log10 after 4 weeks of treatment (day 29) was analyzed using Fisher's exact test. The proportion of patients with an undetectable viral load (below limit of detection) after 4 weeks of treatment (day 29) was analyzed similarly. The treatment by genotype interaction was assessed using the Breslow-Day test. If the genotype class turned out to be statistically significant at the 10% level, a separate Fisher's exact test was performed for each genotype class and genotype. The baseline value was the value taken on day 1 prior to first study drug administration. Statistical analysis was performed with SAS version 8.2. The a-risk P values reported were two-sided, and the statistical significance nominal limit was set at P < 0.05. Safety analysis was conducted in all randomized patients who received any dose of study medication.
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