Patient population
The study was conducted from November 2019 to November 2020 at 35 centers across China. Of the 421 patients screened, a total of 327 patients were enrolled in the study, and 326 patients received at least one dose of alfosbuvir plus daclatasvir. The main reason for screening failure was that patients did not meet inclusion criteria or met exclusion criteria (Figure1).
Overall, there were similar proportions of men and women (52.8% vs 47.2%), with a median age of 50 years (range: 23, 72). The genotype of the patients was as follows: genotype 1 accounted for 51.8% of the patients, genotype 2 accounted for 29.1%, genotype 3 accounted for 6.7%, and genotype 6 accounted for 12.3%. Because of the very low prevalence of genotypes 4 and 5 in China, no genotype 4 or 5 was detected or enrolled in this study. 41 (12.6%) patients had compensated cirrhosis at baseline, and 23 (7.1%) had previously failed treatment with interferon-based therapy. A majority (83.1%) of patients had the IL28B CC allele, and approximately half (55.2%) of patients had a baseline HCV RNA≥2,000,000 IU/mL. 99.4% of patients had pre-existing NS5A and/or NS5B RASs at baseline. The baseline demographics and disease characteristics are shown in the Table 1.
Efficacy
Of the 326 patients who received at least one dose of study drugs, 321 (98.5%) achieved the primary endpoints of SVR12, which was superior to the historical SVR rate of 88% (p<0.0001), meeting the primary efficacy endpoint of the study. All the patients who had a SVR12 rate achieved SVR24, except for one patient who did not return for a visit at post-treatment week 24. Virologic response rates during and after treatment with alfosbuvir plus daclatasvir are summarized in Table 2. The virological breakthrough rate at Weeks 2, 4, 8 and 12 of the treatment periods were 0.6% [95% confidence interval (CI): 0.1, 2.2], 12.9% (95% CI 9.4, 17.0), 15% (95% CI 11.3, 19.4), and 3.7% (95% CI 1.9, 6.3), respectively. The virologic relapse rate at post-treatment weeks 4 and 12 were both 1.5% (95% CI 0.5, 3.5). Relative to post-treatment week 4, no new viral relapse occurred at 12 and 24 weeks after the end of the treatment. Plasma levels of HCV RNA declined rapidly with the treatment. The HCV RNA level (Log10 IU/mL, Mean±SD) declined to 1.9029±0.6732 after 1 week of treatment, and to 1.1461±0.0000 after 12 weeks of treatment. The decrease of HCV RNA relative to baseline at different test time points is shown in Figure 2.
The results of the subgroup analysis of SVR12 rate showed that there were some differences in the SVR12 rate between different HCV genotype subgroups. GT-1a, GT-2a and GT-3b groups showed the best efficacy, with the SVR12 rate of 100.0%. The regimen of alfosbuvir plus daclatasvir seemed to show a similar or more potent effect on patients with HCV genotype 3 than the current standard treatment. Patients with GT-1b and GT-6a also responded well to the treatment, with a SVR12 rate of 98.8% and 97.1%, respectively. The SVR12 rate was also different among subgroups of patients with different cirrhosis status. In patients without cirrhosis, the SVR12 rate of the treatment was 99.3%; Compared with patients without cirrhosis, the efficacy on patients with compensatory cirrhosis was slightly worse, with the SVR12 rate of 92.7%. In addition, the SVR12 rates were similar regardless of most baseline characteristics, such as prior HCV treatment history, IL28B genotype, viral load, and the presence of pre-existing RASs. Subgroup analysis of baseline characteristics and virological response rates at week 12 post-treatment is shown in Table 3.
Multivariate analysis of predictors of virologic response rate (Supplement 3) showed that the independent variables entered into the model in the multivariate analysis of SVR12 rate were cirrhosis status (P=0.0055) and baseline viral load (P=0.0679), the odds ratio OR were 0.0693 (95% CI 0.0105, 0.4557) and 0.1723 (95% CI 0.0261, 1.1380), the results suggest that cirrhosis was a risk factor for treatment failure, and subjects with cirrhosis are 0.0693 times more likely to achieve SVR12 than those without cirrhosis, while the baseline viral load has no statistically significant effect on SVR12 .
Resistance-associated substitutions
A total of 5 subjects did not achieve SVR12 during the study period. The drug resistance mutations before and after treatment in 5 patients who have failed treatment were tested. The results showed that all subjects developed new resistance mutations in NS5A region after treatment compared with baseline, but none of the subjects developed new resistance mutations in NS5B region after treatment (Supplement 4). All amino acid mutations at positions 28, 31, 62 and 93 in NS5A region were common drug resistance mutation sites. Among the subjects enrolled in this trial, all subjects with baseline amino acid mutations at positions 28, 31, 62, and 93 achieved SVR except for the 5 subjects who had failed treatment. We therefore speculated that the treatment failure of the subjects in this study may not be clearly correlated with the NS5A mutations.
Safety
The overall incidence of treatment-emergent AEs (TEAEs) was 82.2% (268/326), comprising 232 patients with grade 1 (71.2%), 106 patients with grade 2 (32.5%), 33 patients with grade 3 (10.1%) and 3 patients with grade 4 (0.9%) (Table 4). Grade 4 AEs were increased creatine phosphokinase (0.6%) and hypertriglyceridemia (0.3%). A total of 124 patients (38.0%) experienced TEAEs related to study drug, and the most often reported (≥1%) were hypercholesterolemia (10.1%), hypertriglyceridemia (5.8%), hyperlipidemia (6.4%), increased blood bilirubin (2.5%), increased amylase (2.1%), increased aspartate aminotransferase (1.5%), increased total bile acid (3.7%), hypothyroidism (1.2%). Only one patient prematurely discontinued the study treatment due to hepatic cancer, and no deaths were reported. 25 (7.7%) patients experienced serious AEs, none of which was judged to be related to alfosbuvir plus daclatasvir treatment. Grade 3 or 4 laboratory abnormalities were observed in 14 (4.3%) patients. All of them were asymptomatic and required no specific medical intervention. No patients had a grade 3 or 4 elevations in alanine aminotransferase, aspartate aminotransferase or bilirubin. In the hepatic function test, aspartate aminotransferase, alanine aminotransferase and gamma-glutamyl transferase decreased gradually after the subjects take the drug (Supplement 5). During the follow-up period after the treatment, it remained in a decreasing state without an upward trend. The result showed a trend of normalization in hepatic function throughout the study period.