Antisense Mediated Lowering of Plasma Apolipoprotein C-III by Volanesorsen Improves Dyslipidemia and Insulin Sensitivity in Type 2 Diabetes

Transcription

1 Antisense Mediated Lowering of Plasma Apolipoprotein C-III by Volanesorsen Improves Dyslipidemia and Insulin Sensitivity in Type 2 Diabetes Digenio A, et al. Table of Contents Detailed Methods for Clinical HE Clamp Procedure Supplementary Figure S1. Study Design Treatment and Procedure Schema...3 Supplementary Figure S2. Flow of Study Participants...4 Supplementary Table S1. Hyperinsulinemic Euglycemic Clamp Results...5 Supplementary Table S2. Significant Correlation between Change in ApoC-III and Triglycerides and Change in (A) Whole-Body Insulin Sensitivity, SI clamp and (B) HbA1c Supplementary Table S3. Effect of Volanesorsen 300 mg on Body Weight. 7 Supplementary Table S4. Summary of Pharmacokinetic Parameters for Volanesorsen 300 mg..8

2 Detailed Methods for Two-step HE Clamp Procedure The two-step hyperinsulinemic-euglycemic clamp procedure was performed at Baseline (Day 1) and End-of-Treatment (Day 92). Glucose was stabilized at 110 mg/dl for at least 2 hours prior to stating the clamp procedure. At the same time the subject was connected to the Biostator (MTB Medizintechnik, Amstetten, Germany), which automatically calculates the appropriate glucose infusion rate (GIR) to keep blood glucose at the target level once the clamp procedure is initiated. Patients fasted except for sips of water and remained supine or semi supine throughout the clamp. To measure glucose, we inserted an 18-gauge PTFE double lumen catheter retrograde in a vein into the hand or forearm, which connected to the glucose sensor of the Biostator. The catheter was maintained with a low dose heparin solution (10,000 Units heparin/100 ml saline) to prevent clotting. The hand was warmed with a heating pad throughout the clamp to arterialize venous blood. A second 18-gauge catheter in the antecubital vein of the same arm provide blood samples for serum insulin analyses, kept patent with 0.15 mm saline. Another vein in the contralateral forearm was cannulated with an 18-gauge PTFE catheter to infuse insulin by infusion pumps. Two serial insulin infusion rates comprised each insulin infusion phase of 180 minutes each: Step 1 at a low rate and Step 2 at a high rate. The last 30 minutes of each phase was taken as steady-state. A low insulin dose was used in Step 1 to raise plasma insulin without maximally suppressing HGP, infusing insulin (Humulin R U100, Eli Lilly& Co., Indianapolis, IN, USA) at only 30 mu/m 2 /min. The high dose of Step 2 is more suited to stimulate glucose disposal. Thus, after Step 1 (180 minutes) the insulin infusion accelerated to 150 mu/m 2 /min for a further 180 minutes. This dose provides supra-physiological insulin concentrations (approximately 400 /ml) to near-maximally stimulate glucose disposal, primarily into skeletal muscle. The hepatic glucose production (HGP) from Step 2 was expected to be near maximally suppressed. The glucose infusion rate (GIR; 20% v/v) required to maintain arterialized venous blood glucose at the target level of 110 mg/dl was recorded throughout the clamp. The steady-state period for insulin sensitivity measurements was defined as the time from minutes following the initiation of the continuous insulin infusion during each step, and the GIR recorded during the last 30 minutes of each insulin infusion step to determine insulin sensitivity. Blood samples to determine plasma glucose were drawn before and during the clamp. The Biostator was be recalibrated at regular intervals by the reference method (YSI 2300 Stat) approximately every 30 minutes and approximately every 10 minutes during steady state, using simultaneous or nearsimultaneous blood draws ( 5 min). The clamp procedure ended six hours after the initiation of Step 1 by stopping the insulin infusion after the last blood sample, then patients ate a standardized lunch and blood glucose was monitored as needed. 1 - Steele R. Influences of glucose loading and of injected insulin on hepatic glucose output. Ann NY Acad Sci. 1959;82:

3 Supplementary Figure S1 Study Design Treatment and Procedure Schema SCREEN 28 to 4 days before Dosing Day 1 BASELINE: IN-HOUSE PERIOD 1 Days-2 to Day 1 Day -2: Standard Meals, Lipid and Diabetes Panel Day -1: MMTT Day 1: Two-step HE Clamp and Glucose Turnover, First Dosing OUTPATIENT DOSE TREATMENT PERIOD Days 1 to Weeks Treatment, PK Blood Draws EARLY TREATMENT TERMINATION IN-HOUSE PERIOD 2 Days 90 to Day 92 Equal to Baseline In-House Period POST-TREATMENT EVALUATIONS Days 86 to176 END-OF-STUDY PROCEDURES Early Termination or Day 176 ( ± 5 days)

4 Supplementary Figure S2 Flow of Study Participants Abbreviations: PD, pharmacodynamics; PK, pharmacokinetics Analysis of PD outcomes was performed on the per-protocol population as defined in the methods section *Administrative reasons

5 Supplementary Table S1 Hyperinsulinemic Euglycemic Clamp Results, Mean (SD) Placebo (n=5) Volanesorsen (n=8) Group Comparison SI Clamp : Insulin Sensitivity Index Ratio (Primary Measure of Whole-Body Insulin Sensitivity) Day (0.0074) (0.0043) Day (0.0063) (0.0046) %Change -7.0% (25.1%) +50.3% (48.6%) +57.3% (41.6%) P-Value * M : Glucose Disposal at High Insulin Rate (mg/kg/min) Day (4.10) 8.14 (2.43) Day (3.91) 9.41 (1.87) %Change +0.2% (18.8%) +24.4% (42.1%) +24.2% (35.4%) P-Value MCR : Metabolic Clearance at High Insulin Rate (mg/kg/min per mg/dl) Day (3.73) 7.45 (2.19) Day (3.60) 8.61 (1.62) %Change -0.7% (19.4%) +24.5% (43.9%) +25.2% (36.9%) P-Value M/I : Glucose Metabolism:Insulin Ratio at Insulin at High Insulin Rate (mg/kg/min per uiu/ml) Day (0.0258) (0.0048) Day (0.0185) (0.0057) %Change +1.5% (22.9%) +31.5% (33.5%) +30.0% (30.1%) P-Value Results shown are the unadjusted values for patients who had valid clamp data (N=13). P-values were derived from the mixed effect regression analysis.; Results shown for the treatment group, placebo and volanesorsen, are the within group comparison of Day 1 and Day 92. * p 0.05 for % change group comparison by Wilcoxon Rank Sum test

6 Supplementary Table S2 Significant Correlation between Change in ApoC-III and Triglycerides and (A) Change in Insulin Sensitivity and (B) HbA1c demonstrated by the Pearson Correlation test. A) SI Clamp, Day 92 N r p-value Apolipoprotein CIII Triglyceride VLDL-ApoCIII VLDL-C B) HbA1c, Day 176 N r p-value Apolipoprotein CIII Triglyceride VLDL-ApoCIII VLDL-C

7 Supplementary Table S3 Effect of Volanesorsen 300 mg on Body Weight Weight Placebo Volanesorsen P-Value N 5 10 Baseline Mean (SD), kg 89.7 (18.5) 85.6 (16.7) End of Treatment, Day 99 Mean (SD), kg 89.8 (18.2) 83.9 (16.9) Change from Baseline, % 0.23 (1.81) (1.62) Post-treatment F/U, Day 176 Mean (SD), kg 89.7 (17.7) 82.5 (16.3) Change from Baseline, % 0.28 (1.84) (2.52) 0.040

8 Supplementary Table S4 Summary of Pharmacokinetic Parameters for Volanesorsen 300 mg Dose Route C trough, Day 92 (ng/ml) C trough at Steady State (ng/ml) b t ½ (day) N 7 a c 300 mg SC C trough denotes plasma trough concentration; SC, subcutaneous; t ½, apparent elimination half-life Data presented are the mean standard deviation. a Three patients terminated the study early and did not have plasma samples collected on Day 92. b C trough at steady-state was calculated as the mean values of C trough between Days 29 and 92, where available. c For the three patients who terminated treatment early, t was determined following their last administered dose on Day 29 (n=1) and Day 64 (n=2).