Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: van der Ree MH, de Vree JM, Stelma F, et al. Safety, tolerability, and antiviral effect of RG-101 in patients with chronic hepatitis C: a phase 1B, double-blind, randomised controlled trial. Lancet 2017; published online Jan 10. http://dx.doi. org/10.1016/S0140-6736(16)31715-9.
Supplementary Material
Supplementary Tables ............................................................................................................................................. 2 Supplementary Figures............................................................................................................................................ 3 Inclusion and exclusion criteria study ................................................................................................................... 10 Listing of laboratory tests...................................................................................................................................... 12
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Supplementary Tables Supplementary Table 1 – Pharmacokinetics RG-101 measured in plasma and urine 2 mg/kg RG-101
4 mg/kg RG-101
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tmax (h)*
4.0, (2.0 – 4.0), (2.0 - 16.0)
4.0, (2.0 – 4.0), (0.50 - 10.0)
Cmax (µg/mL)
0.73 [57.9%], (0.25 - 2.02)
3.03 [85.5%], (0.88 - 16.3)
6.5 [28.4%], (3.5 - 11.7)
28.5 [35.0%], (14.8 - 43.6)
17.0 [214.9%], (6.6 – 76.1)**
7.2 [64.2%], (4.4 – 18.8)***
N= Plasma
AUC0-t (µg∙h/mL) t½ (h) Urine fe0-24 (%)****
0
(0 – 0) (0 - 0)
0
(0 – 0), (0 - 2.07)
For Cmax, AUC0-t and t½, the geometric mean (geometric coefficient of variation [CV%]) and (minimummaximum) are presented. * For tmax and fe0-24, the median (IQR) and (minimum-maximum) is presented. **n=3; estimated t½ values at the higher end of the reported range were not precisely estimated and thus should be interpreted with caution ***n=6 ****: %fe0–24: percentage of the dose excreted in urine (as parent drug or major metabolite) during the first 24 h after receiving the treatment dose
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Supplementary Figures Supplementary Figure 1 (1A) In each dose group (2 or 4 mg/kg), patients were randomised to receive either RG-101 or placebo (ratio 7:1). (1B) Patients received a single subcutaneous injection of RG-101 or placebo at day 1 (=baseline). All patient cohorts were followed 8 weeks after randomisation. Patients with no virological rebound at week 8 were included in the extended follow-up study in which patients were followed up to 76 weeks after dosing.
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Supplementary Figure 2 Change in laboratory values in patients dosed with 2 and 4 mg/kg RG-101 and placebo. (2A): ALT level, (2B) AST level, (2C) alkaline phosphatase level, (2D): total bilirubin level.
2A
2B
2C
2D
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Supplementary Figure 3 Alkaline phosphatase levels of individual patients dosed with 2 mg/kg RG-101 (3A) and 4 mg/kg RG-101 (3B) throughout the entire study period.
3A.
3B.
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Supplementary Figure 4 (4A) Change in total cholesterol level in patients dosed with RG-101 and placebo. (4B) No significant correlation between change in total cholesterol level and change in HCV RNA level in patients dosed with RG-101. 4A.
4B.
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Supplementary Figure 5 Change in HCV RNA levels within the first 7 days in patients dosed with 2 mg/kg RG-101 and placebo (5A) and 4 mg/kg RG-101 and placebo (5B). 5A.
5B.
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Supplementary Figure 6 HCV RNA levels of patients with matched sample (at baseline and viral rebound) available for sequence analysis. In patients with HCV RNA levels below the detection limit for sequence analysis (20,000 IU/mL) at time of viral rebound (indicated by a solid square), an additional follow-up sample was used for sequence analysis. (6A) HCV RNA levels of individual patients with viral rebound and HCV genotype 1. Identification of C3U RAV in miR-122 binding sites (open dots with X), C3U RAV in combination with C27U/C RAV (open squares with X), or wild-type sequences (solid dots) are indicated. (6B) HCV RNA levels of individual patients with viral rebound and HCV genotype 3 and 4. Identification of C2G and C3U RAVs in miR-122 binding sites (open dots with X) or wild-type sequences (solid dots) are indicated. 6A
6B.
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Supplementary Figure 7 Sequence analysis of the 5’UTR (nucleotides 1 to 270) were performed by 5’RACE from 14 patients who experienced a virological rebound (VR) and whom matched samples were available for analysis. Sequence data of nucleotide 1-50 are shown for each patient (VR 1-14) aligned to the HCV genotype specific consensus sequence (GT 1, GT 3 and GT 4). miR-122 binding regions are indicated by the boxes (S1: seed site 1, S2: seed site 2). 5’UTR binding site mutations are shown in red, and viral polymorphisms are shown in orange. Y stands for mixed population of C and U.
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Inclusion and exclusion criteria study Main study Inclusion Criteria: 1. Males, or post-menopausal or hysterectomized females; females must be either at least 1 year postmenopausal (amenorrhea duration of at least 12 months and FSH >40 U/L at screening) or hysterectomized; all females must have a negative pregnancy test at screening and each admission 2. Age : 18 - 65 years, inclusive 3. BMI : 18.0 – 38.0 kg/m2 [BMI (kg/m2) = Body weight (kg) ÷ Height2 (m2)] 4. Clinical and laboratory findings consistent with a clinical diagnosis of Chronic Hepatitis C (CHC), including: a) Previous documentation of positive HCV serology (HCV antibody or HCV RNA) at least 24 weeks prior to enrollment, OR b) Positive HCV serology (HCV antibody or HCV RNA) with a prior remote risk factor (more than 24 weeks prior to Screening) for the acquisition of hepatitis C 5. Serum HCV RNA ≥ 375,000 copies/mL or ≥ 75,000 IU/mL at screening 6. Subjects infected with HCV genotype 1, 3 or 4. 7. Treatment-naïve to or relapsed after IFN-alpha based therapies; Relapse is defined as the recurrence of HCV RNA following the termination of a full course of treatment and after having achieved an undetectable HCV RNA during treatment 8. Screening hematology, clinical chemistries, coagulation and urinalysis are not clinically significant and the following criteria are met: a) Platelets >100x109/L b) Total white blood cells >3.0x109/L and absolute neutrophil count >1.5x109/L c) Hemoglobin >6.8 mmol/L for females and >7.4 mmol/L for males d) Total and direct bilirubin within normal limits (except for clearly documented Gilbert’s Syndrome) e) ALAT < 5x ULN f) Serum creatinine within normal limits and estimated creatinine clearance rate as calculated by the CockcroftGault formula1 > 60 mL/min 9. Male subjects must agree to use birth control (condoms) from the time of dosing until 90 days after the followup visit; male patients who are surgically sterile need not employ a method of contraception 10. Ability and willingness to abstain from methylxanthine-containing beverages or food (coffee, tea, cola, chocolate, "powerdrinks") and grapefruit (juice) from 48 hours prior to entry in the clinical research center for an in-house stay until discharge after the in-house stay 11. Ability and willingness to abstain from alcohol from 48 hours prior to entry in the clinical research center for an in-house stay until discharge after the in-house stay, and to limit the alcohol intake to a maximum of 5 units per week during the whole study period 12. Negative results on the following screening laboratory tests: urine drug screen (except for cannabinoids and methadone), HBsAg, and HIV antibody 13. Willingness to sign the written ICF Exclusion Criteria: 1. Other known cause of liver disease except for CHC 2. History or symptoms of decompensated liver disease: Child-Pugh Class B or C, including ascites, hepatic encephalopathy, esophageal variceal bleeding, fibrosis or other signs of hepatic insufficiency or portal hypertension 3. History of hepatocellular carcinoma on imaging studies or serum alpha-fetoprotein (AFP) >50 ng/mL at screening 4. Concurrent clinically significant medical diagnosis (other than hepatitis C-related conditions) that would potentially interfere with the subjects study compliance or confound study results 5. Treatment with ribavirin within 21 days prior to entry; treatment with ribavirin while on study is prohibited 6. Concurrent social conditions (e.g., drugs, alcohol) which would potentially interfere with the subject’s study compliance 7. Mental handicap or history of or current significant psychiatric disease (which might impair ability to provide informed consent) 8. Clinically significant illness within 30 days preceding entry into the study 9. Have used an investigational drug or has participated in an investigational study with a licensed drug within 30 days or 5 half-lives, whichever is longer, prior to study drug administration 10. History of relevant drug and/or food allergies
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11. Donation of more than 500 mL of blood within 60 days prior to drug administration. Donation of more than 1.5 liters of blood (for men) / more than 1.0 liters of blood (for women) in the 10 months preceding the start of this study (this is administration of study drug).
Extended Follow-up Study Inclusion Criteria: 1. Decrease from baseline (Day 1 pre-dose) in HCV viral load of at least 2 logs at any time during the main study (up to Day 57) Exclusion Criteria: 1. Confirmed virological breakthrough, defined as an increase in viral load of >1 log from nadir, at Day 57 of the main study or at any visit during the extended follow-up period. Virological breakthrough has to be confirmed in a re-test within 14 days (+/- 5 days) after the visit.
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Listing of laboratory tests Complement
Clinical chemistry
Hematology
Coagulation Urinalysis Serology Drug screen Pregnancy test Additional assessments at screening Liver enzymes Renal safety Lipid profile
Factors C5a, C3a, Bb creatinine, urea, total bilirubin (conjugated and non-conjugated bilirubin), gamma glutamyl transferase (GGT), ASAT, ALAT, alkaline phosphatase (ALP), amylase, creatine phosphokinase (CPK), lactate dehydrogenase (LDH), sodium, potassium, chloride, calcium, inorganic phosphate, total protein, albumin, globulin, albumin/globulin (A/G) ratio, C-reactive protein (CRP), glucose Hemoglobin (Hb), red blood cell count (RBC), hematocrit (Hct), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), reticulocytes, white blood cell count (WBC), differential white blood cell count(neutrophils, lymphocytes, monocytes, eosinophils, basophils, platelets) Prothrombin time (PT), fibrinogen, coagulation factor V, aPTT. pH, specific gravity, protein, glucose, ketones, urobilinogen, bilirubin, β-2-microglobulin, microscopy (in case of abnormal findings only) HBsAg, anti-HCV and anti-HIV Opiates, methadone (not for HCV patients), cocaine, amphetamines (including ecstasy), cannabinoids (not for HCV patients), barbiturates, benzodiazepines, tricyclic antidepressants and alcohol Beta-human chorionic gonadotropin (serum) HCV genotype, IL28B genotype, alpha-fetoprotein Total bilirubin (conjugated and non-conjugated bilirubin), GGT, ASAT, ALAT, and ALP Urine: quantitative protein, creatinine, protein to creatinine ratio, creatinine clearance, beta-2-microglobulin and N-acetyl-beta-Dglucosaminidase (NAG) Total cholesterol, HDL-C, LDL-C, VLDL-C, triglycerides, apoA1, apoB, and apoB/apoA1ratio
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