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;371:2200-6. DOI: 10.1056/NEJMoa1400284
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 304801 treatment on (A) apob-48 levels and (B) relationship between total plasma TG and apob-48 levels.. 4 Figure S2. Effect of ISIS 304801 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 304801. 6 Table S1. Patient Characteristics. 7 Table S2. Lipid and Lipoprotein Profiles.... 8 Table S3. Clinical Laboratory Tests by Patient at Each Scheduled Visit... 10 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.... 13 References..... 14 1 P a g e
Additional Details on Methods ISIS 304801 (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 304801 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 < 1.006 g/ml) TGs, total cholesterol, HDL-C, non-hdl-c (calculated), LDL-C (isolated by ultracentrifugation), chylomicron/vldl- C (d < 1.006 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
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 155-255 ng LPL/mL or 25-50% of normal. The 95% confidence interval for LPL activity in healthy subjects is 104-336 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 38-200 nmol FFA/min/mL of post-heparin plasma. Hepatic lipase activity for males and preadolescent females is 102-236 nmol FFA/min/mL. The control range of HL activity for adult females is 44-167 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 304801 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
Figure S1. Effect of ISIS 304801 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
Figure S2. Effect of ISIS 304801 treatment on total free fatty acid levels. Solid blue triangles indicate dosing days. 5 P a g e
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 304801. 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
Table S1. Patient Characteristics Characteristic Patient 1 Patient 2* Patient 3* Gender Male Female Female Age, yrs 45 67 28 BMI, kg/m 2 23.7 29.0 23.4 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
Table S2. Lipid and Lipoprotein Profiles Parameter (mg/dl) Patient Primary No. Baseline * Endpoint Change from Baseline % Change from Baseline APOC3 Triglyceride 1 18.9 5.5-13.4-71 2 35.1 3.4-31.7-90 3 19.8 3.5-16.3-83 1 1406 616.5-789.5-56 2 2083 287.5-1795.5-86 3 2043 734.5-1308.5-64 Chylomicron-TG 2 1641 151.5-1489.8-91 1 1054 447.0-607.4-58 3 1511 622.4-888.7-59 VLDL-TG 2 262.3 55.4-206.9-79 1 108.1 60.2-47.8-44 Total-C Chylo/VLDL-C LDL-C HDL-C Non-HDL-C ApoB-48 3 125.4 27.5-97.9-78 1 230 138.5-91.5-40 2 335 105.0-230.0-69 3 258 128.0-130.0-50 1 197 97.0-100.0-51 2 314 54.0-260.0-83 3 231 75.5-155.5-67 1 17 17.5 0.5 3 2 13 30.0 17.0 131 3 13 35.5 22.5 173 1 16 24.0 8.0 50 2 8 21.0 13.0 163 3 14 17.0 3.0 21 1 214 114.5-99.5-46 2 327 84.0-243.0-74 3 244 111.0-133.0-55 1 0.98 0.83-0.15-15 2 1.54 0.28-1.27-82 3 0.72 0.53-0.20-27 8 P a g e
Parameter (mg/dl) Patient Primary No. Baseline * Endpoint Change from Baseline % Change from Baseline Chylo/VLDL APOC3 1 12.2 4.5-7.8-63 2 32.6 2.5-30.1-92 3 16.8 2.3-14.5-86 ApoE 2 11.2 4.6-6.6-59 1 9.2 4.7-4.5-49 3 6.2 4.3-1.9-30 Chylo-ApoE 2 5.5 1.8-3.7-68 1 5.1 2.4-2.7-53 3 2.7 2.1-0.6-22 * 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 0.0259; to convert triglyceride (TG) values to mmol/l multiply by 0.0113; to convert apolipoprotein values (APOC3, ApoB-48, Chylo/VLDL-APOC3, ApoE and Chylo-ApoE) to g/l multiply by 0.01. 9 P a g e
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 20 25 15 15 16 50 36 32 14 AST 25 28 22 19 20 34 31 29 19 Total Bilirubin 0.46 0.57 0.46 0.54 0.59 0.57 0.41 0.40 0.46 Alkaline Phosphatase 66 81 69 75 73 85 70 74 68 1 BUN 15 16 13 15 16 20 17 17 16 Creatinine 0.74 0.80 0.88 0.96 0.82 0.83 0.92 0.94 0.92 Creatine Kinase 35 43 49 40 42 33 38 20 40 Glucose 95 116 100 104 102 99 94 86 90 Uric Acid 6.8 8.5 8.5 8.1 7.4 7.2 8.6 7.4 7.6 ALT 14 14 14 15 14 19 20 16 20 AST 30 23 22 21 18 24 22 19 24 Total Bilirubin 0.43 0.37 0.25 0.31 0.38 0.38 0.32 0.35 0.45 Alkaline Phosphatase 90 84 85 87 83 92 87 103 100 2 BUN 29 39 30 29 35 40 34 44 32 Creatinine 0.84 1.10 1.03 1.01 0.95 1.10 1.20 1.16 1.11 Creatine Kinase 108 126 105 69 55 103 50 55 66 Glucose 109 106 96 106 101 109 96 94 97 Uric Acid 7.6 8.5 8.1 9.2 7.8 8.9 9.7 9.9 8.4 10 P a g e
Patient Test SCR QUAL Day 8 Day 15 Day 29 Day 57 Day 92 Day 127 Day 176 ALT 14 19 24 31 19 19 56 16 20 AST 30 26 31 34 24 24 40 20 21 Total Bilirubin 0.51 0.70 0.46 0.61 0.58 0.53 0.33 0.53 0.46 Alkaline Phosphatase 42 43 48 49 46 47 50 39 41 3 BUN 14 12 16 12 15 15 16 14 15 Creatinine 0.75 0.64 0.81 0.74 0.73 0.84 0.79 0.81 0.81 Creatine Kinase 75 57 52 65 40 33 29 38 50 Glucose 85 87 79 80 81 82 87 82 86 Uric Acid 6.0 5.5 6.0 5.8 5.3 5.8 6.1 5.4 5.6 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, 0.10 1.10 mg/dl; Alkaline Phosphatase, 37 116 U/L. Blood urea nitrogen (BUN) and creatinine values are in mg/dl. Normal ranges: BUN, 5 22 mg/dl; Creatinine, 0.50 1.40 mg/dl. Creatine kinase values are in U/L, glucose and uric acid are in mg/dl. Normal ranges: Creatine Kinase, 25 210 U/L; Glucose, 60 115 mg/dl; Uric Acid, males 4.0 8.5 mg/dl and females 3.0 7.0 mg/dl. 11 P a g e
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
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 1 0.807 1.000 0.948 0.558 patient 2 0.942 0.998 0.973 0.791 patient 3 0.918 0.998 0.970 0.677 p-value APOC3 Chylo-TG non-hdl-c ApoB-48 patient 1 0.0047 <0.0001 <0.0001 0.0596 patient 2 <0.0001 <0.0001 <0.0001 0.0022 patient 3 <0.0001 <0.0001 <0.0001 0.0221 13 P a g e
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