Supplementary Appendix
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1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Gaudet D, Brisson D, Tremblay K, et al. Targeting APOC3 in the familial chylomicronemia syndrome. N Engl J Med 2014;371: DOI: /NEJMoa
2 SUPPLEMENTARY APPENDIX Targeting APOC3 in Familial Chylomicronemia Syndrome Gaudet D, et al. Table of Contents Additional Details on Methods. 2 Figure S1. Effect of ISIS treatment on (A) apob-48 levels and (B) relationship between total plasma TG and apob-48 levels.. 4 Figure S2. Effect of ISIS treatment on total free fatty acid levels. 5 Figure S3. Postprandial lipid levels of (A) total plasma TG, and (B) chylomicron-tg in patient 2 before (Day -1) and after (Day 103) treatment with ISIS Table S1. Patient Characteristics. 7 Table S2. Lipid and Lipoprotein Profiles Table S3. Clinical Laboratory Tests by Patient at Each Scheduled Visit Table S4. Serious Adverse Events.. 12 Table S5. Adverse Events Related or Possibly Related to Study Drug. 12 Table S6. Correlation of total plasma TG levels to APOC3, chylomicron-tg, non-hdl-c, and apob-48 levels by patient References P a g e
3 Additional Details on Methods ISIS (5 -AGCTTCTTGTCCAGCTTTAT-3 ) incorporates several chemical modifications to improve potency, duration of action, and tolerability. All of the internucleotide phosphates are chemically modified with a phosphorothioate substitution, in which one of the nonbridging oxygen atoms is substituted with sulfur. Additionally the drug incorporates five 2 - O-(2-methoxyethyl) (2 -MOE) modified ribonucleosides (underlined) at the 3 and 5 ends while retaining ten 2 -O-deoxyribonucleosides within the central portion of the molecule. ISIS was supplied in 2-mL stoppered glass vials as a 1 ml solution (200 mg/ml) for single use only by Isis Pharmaceuticals, Inc. (Carlsbad, CA). Total plasma APOC3 and chylomicron/vldl APOC3 (d < g/ml) TGs, total cholesterol, HDL-C, non-hdl-c (calculated), LDL-C (isolated by ultracentrifugation), chylomicron/vldl- C (d < g/ml), and apob-48 were determined at MedPace Reference Labs (Cincinnati, OH). Chylomicron-TG and VLDL-TG were isolated by ultracentrifugation and measured at Ecogene 21 (Chicoutimi, QC Canada). Plasma apoe and chylomicron-apoe were also measured at Ecogene 21 (Chicoutimi, QC Canada). Safety evaluations included blood chemistry, hematology, coagulation, and urinalysis. Other assessments included vital signs, ECGs, and physical examination. Standard laboratory tests were performed by MedPace Reference Labs (Cincinnati, OH). Data were analyzed by representatives of the study Sponsor, Isis Pharmaceuticals. SAS v.9.2 or higher software (SAS Institute Inc., Cary NC) was used for analyses. Correlation coefficients (r) were derived from Pearson product-moment correlations. 2 P a g e
4 Lipoprotein Lipase (LPL) and Hepatic Lipase (HL) activity Post-heparin LPL activity and mass have been assessed in more than 20 LPL P207L and G188E homozygotes or compound heterozygotes (HoLPL, null alleles) as well as in tens of heterozygotes (HeLPL). These measurements were performed in two independent laboratories, using established radiochemical methods. 1-4 Combining both published and unpublished data, LPL activity is approximately 50% of normal in HeLPL and below 5% of normal in P207L or G188E HoLPL. 5-8 P207L and G188E HoLPL have a post-heparin LPL mass, which ranges between ng LPL/mL or 25-50% of normal. The 95% confidence interval for LPL activity in healthy subjects is nmol FFA/min/mL and is similar for males and females. 4 Hepatic Lipase activity in G188E and P207L HoLPL is normal or, most often, lower than normal, ranging between nmol FFA/min/mL of post-heparin plasma. Hepatic lipase activity for males and preadolescent females is nmol FFA/min/mL. The control range of HL activity for adult females is nmol FFA/min/mL. In the current study, we measured both LPL and HL activity at the end of the study in two patients (#2 and #3). Post-treatment LPL activity in patient 2 and 3 was 1.95 and 3.8 nmol FFA/min/mL respectively (<3% of normal), similar to pretreatment values. Thus, administration of ISIS was not associated with any measureable increase in LPL activity. Post-treatment HL activity in patient 2 and 3 was 44.6 and 38.4 nmol FFA/min/mL respectively, lower than normal levels. 3 P a g e
5 Figure S1. Effect of ISIS treatment on (A) apob-48 levels and (B) relationship between total plasma TG and apob-48 levels. Solid blue triangles indicate dosing days. 4 P a g e
6 Figure S2. Effect of ISIS treatment on total free fatty acid levels. Solid blue triangles indicate dosing days. 5 P a g e
7 Figure S3. Postprandial levels of (A) total plasma TG, and (B) chylomicron-tg in patient 2 before (Day -1) and after (Day 103) treatment with ISIS Prior to the postprandial evaluations, the subject consumed standardized pre-cooked meals and abstained from alcohol consumption for 2 days. Lunch and dinner meals were provided, with instructions for breakfast and snacks. The subject remained fasted after consuming dinner on the evening prior to postprandial assessment. The following morning, she consumed a standardized liquid meal over a 30 min period, which was then followed by serial blood sampling. The standard liquid meal (500 ml) contained soybean oil (15 g/l), safflower oil (9 g/l), dried non-fat milk (280 g/l), egg phospholipids (0.18 g/l) and water with the addition of chocolate syrup (190 ml/l) to provide 896 kcal with 13 g of fat (13% of energy) and 141 g of carbohydrates (65% of energy). 9,10 6 P a g e
8 Table S1. Patient Characteristics Characteristic Patient 1 Patient 2* Patient 3* Gender Male Female Female Age, yrs BMI, kg/m LPL Genotype P207L/P207L P207L/G188E P207L/P207L APOE Genotype E3/E3 E3/E4 E3/E3 *participated in LPL activity measurements 7 P a g e
9 Table S2. Lipid and Lipoprotein Profiles Parameter (mg/dl) Patient Primary No. Baseline * Endpoint Change from Baseline % Change from Baseline APOC3 Triglyceride Chylomicron-TG VLDL-TG Total-C Chylo/VLDL-C LDL-C HDL-C Non-HDL-C ApoB P a g e
10 Parameter (mg/dl) Patient Primary No. Baseline * Endpoint Change from Baseline % Change from Baseline Chylo/VLDL APOC ApoE Chylo-ApoE * Baseline is defined as Day -8 for measurements performed by MedPace; and as the average of Day -8 and Day -1 (or pre-dose Day 1) for measurements performed by Ecogene21 (Chylomicron-TG, VLDL-TG, ApoE and Chylo-ApoE). The primary endpoint is defined as the average of Day 85 (pre-dose) and Day 92 results. Ecogene-21 SI units: To convert cholesterol (C) values to mmol/l, multiply by ; to convert triglyceride (TG) values to mmol/l multiply by ; to convert apolipoprotein values (APOC3, ApoB-48, Chylo/VLDL-APOC3, ApoE and Chylo-ApoE) to g/l multiply by P a g e
11 Table S3. Clinical Laboratory Tests by Patient at Each Scheduled Visit Patient Test SCR QUAL Day 8 Day 15 Day 29 Day 57 Day 92 Day 127 Day 176 ALT AST Total Bilirubin Alkaline Phosphatase BUN Creatinine Creatine Kinase Glucose Uric Acid ALT AST Total Bilirubin Alkaline Phosphatase BUN Creatinine Creatine Kinase Glucose Uric Acid P a g e
12 Patient Test SCR QUAL Day 8 Day 15 Day 29 Day 57 Day 92 Day 127 Day 176 ALT AST Total Bilirubin Alkaline Phosphatase BUN Creatinine Creatine Kinase Glucose Uric Acid SCR denotes screen visit; QUAL, qualifying visit. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase values are in U/L; total bilirubin in mg/dl. Normal ranges: ALT, 6 41 U/L; AST, 9 34 U/L; Total Bilirubin, mg/dl; Alkaline Phosphatase, U/L. Blood urea nitrogen (BUN) and creatinine values are in mg/dl. Normal ranges: BUN, 5 22 mg/dl; Creatinine, mg/dl. Creatine kinase values are in U/L, glucose and uric acid are in mg/dl. Normal ranges: Creatine Kinase, U/L; Glucose, mg/dl; Uric Acid, males mg/dl and females mg/dl. 11 P a g e
13 Table S4. Serious Adverse Events Adverse Event Pt # Severity (# events) Pancreatitis 1 Severe (1)* * Not related to study drug Table S5. Adverse Events Related or Possibly Related to Study Drug * Adverse Event Pt # Severity (# events) Abdominal discomfort 3 Mild (1) Diarrhoea 3 Mild (5) Flatulence 3 Mild (1) Frequent bowel movements 3 Mild (1) Headache 3 Moderate (1) Hypoesthesia 3 Mild (2) * Events at the injection site are excluded. 12 P age
14 Table S6. Correlation of total plasma TG levels to apoc-iii, chylomicron-tg, non-hdl-c, and apob-48 levels by patient. r APOC3 Chylo-TG non-hdl-c ApoB-48 patient patient patient p-value APOC3 Chylo-TG non-hdl-c ApoB-48 patient < < patient 2 < < < patient 3 < < < P a g e
15 References 1. Mantha L, Palacios E, Deshaies Y. Modulation of triglyceride metabolism by glucocorticoids in diet-induced obesity. Am J Physiol 1999;277:R Auwerx JH, Babirak SP, Hokanson JE, et al. Coexistence of abnormalities of hepatic lipase and lipoprotein lipase in a large family. Am J Hum Genet 1990;46: Watson TD, Tan CE, McConnell M, Clegg SK, Squires LF, Packard CJ. Measurement and physiological significance of lipoprotein and hepatic lipase activities in preheparin plasma. Clin Chem 1995;41: Babirak SP, Iverius PH, Fujimoto WY, Brunzell JD. Detection and characterization of the heterozygote state for lipoprotein lipase deficiency. Arteriosclerosis 1989;9: Gaudet D, Methot J, Dery S, et al. Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial. Gene Ther 2013;20: Julien P, Vohl MC, Gaudet D, et al. Hyperinsulinemia and abdominal obesity affect the expression of hypertriglyceridemia in heterozygous familial lipoprotein lipase deficiency. Diabetes 1997;46: Murthy V, Julien P, Gagne C. Molecular pathobiology of the human lipoprotein lipase gene. Pharmacol Ther 1996;70: Carpentier AC, Frisch F, Labbe SM, et al. Effect of alipogene tiparvovec (AAV1- LPL(S447X)) on postprandial chylomicron metabolism in lipoprotein lipase-deficient patients. J Clin Endocrinol Metab 2012;97: Normand-Lauziere F, Frisch F, Labbe SM, et al. Increased postprandial nonesterified fatty acid appearance and oxidation in type 2 diabetes is not fully established in offspring of diabetic subjects. PLoS One 2010;5:e Gaudet D, Methot J, Gagné C, al. e. Modifications in triglyceride-rich lipoprotein metabolism induced by alipogene tiparvovec (AAV1-LPLS447X Gene Therapy) correlate with clinical benefit in patients with lipoprotein lipase deficiency (LPLD). Circulation 2010;122:A P a g e
Supplementary Appendix
Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Gaudet D, Alexander VJ, Baker BF, et al. Antisense inhibition
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