133 Sebelipase Alfa Improves Atherogenic Biomarkers in Adults and Children With Lysosomal Acid Lipase Deficiency Don P Wilson,1 Sachin Marulkar,2 Radhika Tripuraneni,2 and Barbara K Burton3 1Cook Children’s Medical Center, Fort Worth, TX; 2Alexion Pharmaceuticals, Inc, New Haven, CT; 3Northwestern University Feinberg School of Medicine, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL

•• Management with lipid-lowering medications10: INTRODUCTION RESULTS Table 4. Effect of Sebelipase Alfa on Atherogenic Biomarkers in LAL-D Subjects –– HMG CoA reductase inhibitors disrupt synthesis of FC, proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors Stratified by Use of Lipid-Lowering (LLM) at 20 Weeks of Treatment •• Lysosomal acid lipase deficiency (LAL-D) is a rare, autosomal recessive lysosomal storage disorder (Online Mendelian prevent degradation of LDL receptors, and both types of lipid-lowering can markedly increase LDL uptake and increase lysosomal accumulation of LDL (Figure 2)2,10 Subject Characteristics Subjects Using LLM Subjects Not Using LLM Inheritance in Man [OMIM] #278000), historically called Wolman disease in infants and cholesteryl ester storage disease (n=24) (n=37) in older children and adults, that is associated with accumulation of cholesteryl esters and triglycerides in the and –– Although therapy may improve lipid parameters, clinical reports have noted progression of liver disease in •• Complete data on atherogenic biomarkers were available from 61 of 66 subjects (N=33 for sebelipase alfa and N=28 for 1 other organs1-3 patients with LAL-D despite long-term treatment with HMG-CoA reductase inhibitors placebo) Mean (SD) Percentage Mean (SD) Percentage –– Hydroxymethylglutaryl-coenzyme A (HMG CoA) reductase inhibitor (statin) therapy may improve lipid parameters but Change From Baseline Change From Baseline 2 •• Subjects had marked dyslipidemia at baseline (Table 2), and 39% (24/61) were using lipid-lowering medication –– LAL maintains cholesterol homeostasis in hepatocytes (Figure 1A) does not improve liver disease associated with LAL-D, and, in some patients, it may be associated with progression of Sebelipase Percentage Sebelipase Percentage ŸŸ LAL catalyzes hydrolysis of low-density lipoprotein cholesterol (LDL-C)–derived cholesteryl esters to free liver disease over time1,4 –– Mean LDL-P, LDL-C, and apoB levels were elevated compared with normal ranges; mean apoA1 level was at the low Atherogenic Alfa Placebo Point Alfa Placebo Point cholesterol (FC) and triglycerides to free fatty acids (FFA) end of the normal range Biomarker (n=14) (n=10) Difference P Value (n=19) (n=18) Difference P Value ŸŸ Increased levels of cytosolic FC and FFA downregulate sterol regulatory element–binding protein (SREBP) Figure 2. Lipid-Lowering Medications May Exacerbate the Underlying Mechanism of –– Overall, the mean total LDL-P concentration at baseline was 2151 nmol/L (reference value: very high, >2000 nmol/L) LDL-P number, −34.2 (13.6) −1.6 (11.3)a 32.6 0.0002 −21.7 (20.6) 1.9 (20.7) 23.6 0.0017 transcription factors to decrease LDL uptake and production of FC and fatty acids Disease in LAL-Da nmol/L –– LAL-D is characterized by lysosomal accumulation of lipids enhanced by increased LDL uptake and upregulation of LDL-C, mg/dL −36.1 (16.4) −10.3 (15.9) 25.8 0.0034 −27.4 (18.8) −4.6 (11.8) 22.8 0.0003 LDL LDL Table 2. Atherogenic Biomarkers and Lipid-Lowering Medication Use at Baseline in lipid production in response to decreased cytosolic FC (Figure 1B)2 ARISE Trial Subjects apoB, mg/dL −29.1 (12.3) −6.5 (10.4) 22.6 0.0005 −26.2 (14.3) −3.2 (7.3) 23.0 <0.0001 a Figure 1. Lipid Metabolism in Normal and LAL-D Hepatocytes Lysosome Sebelipase Alfa Placebo apoA1, mg/dL 10.2 (14.3) −2.5 (10.7) 12.7 0.0224 8.9 (9.2) −2.1 (13.2) 11.0 0.0041 Parameter N=33 N=28 Reference Value LDL-P size, nm −1.0 (2.2)b 0.4 (2.8)a 1.4 0.1927 −0.8 (3.9) 0.1 (2.3) 0.9 0.3302 LDLR A oral epatocyte B AD epatocyte LAL a b Mean LDL-P number, nmol/L 1942 2414 Very high, >2000 nmol/L apo, apolipoprotein; LAL-D, lysosomal acid lipase deficiency; LDL-C, low-density lipoprotein cholesterol; LDL-P, low-density lipoprotein particle. an=9; bn=13. LDL LDL LDL LDL Mean LDL-C, mg/dL 189.1 233.0 Normal, <130 mg/dL Increased LDLR uptake Mean apoB, mg/dL 148.0a 168.9 ULN, 120 mg/dL Lysosome FA synthesis pathway Lysosome SREBP CONCLUSIONS a LDLR Mean apoA1, mg/dL 102.5 102.1 LLN, 100 mg/dL LAL Nucleus LDLR LAL ACAT TG •• Dyslipidemia in LAL-D resembles heterozygous familial hypercholesterolemia (HeFH) and familial combined HMG - Mean LDL-P size, nm 21.6a 21.7b — FC & FFA CoA FC CE hyperlipidemia (FCH) Feedback inhibition FA synthesis pathway Increased LDLR uptake and cellular production LLM use, n (%) 14 (42) 10 (36) –– Given their similar lipid profiles, LAL-D should be considered in children and adults thought to have HeFH or FCH SREBP FA synthesis pathway SREBP VLDL Nucleus ACAT FC FC TG Nucleus ABCA1 Statin 14 (42) 8 (29) •• Baseline atherogenic markers were elevated in LAL-D patients, often beginning in childhood. This life-long exposure HMG - ACAT TG FC CoA CE HMG - — represents significant atherosclerotic risk in patients with this disease FC CoA CE LCAT Other LLM 5 (15)c 3 (11)d VLDL apoA1 HDL FC FC •• Sebelipase alfa treatment resulted in statistically significant decreases in mean LDL-P number, LDL-C level, and apoB ABCA1 FC FC VLDL ABCA1 ABCA1, adenosine triphosphate–binding cassette transporter A1; ACAT, acyl-coenzyme A:cholesterol acyltransferase; apoA1, apolipoprotein A1; CE, cholesteryl esters; Combination therapy 5 (15) 1 (4) level, and a significant increase in mean apoA1 level, representing an improved cardiovascular risk profile for these LCAT apoA1 HDL LCAT FA, fatty acid; FC, free cholesterol; HDL, high-density lipoprotein; HMG-CoA, hydroxymethylglutaryl-coenzyme A; LAL, lysosomal acid lipase; LAL-D, lysosomal acid lipase apo, apolipoprotein; ARISE, Acid Lipase Replacement Investigating Safety and Efficacy; LDL-C, low-density lipoprotein cholesterol; LDL-P, low-density lipoprotein particle; patients compared with placebo recipients apoA1 HDL deficiency; LCAT, lecithin-cholesterol acyltransferase; LDL, low-density lipoprotein; LDLR, LDL receptors; SREBP, sterol regulatory element–binding proteins; TG, triglycerides; LLM, lipid-lowering medication; LLN, lower limit of normal; ULN, upper limit of normal. –– These improvements were observed even in LAL-D patients who were receiving concomitant lipid-lowering VLDL very-low-density lipoprotein. aN=34; bN=27; cOther LLM category included (n=3), cholestyramine (n=1), colestilan (n=1), (n=1), and fish oil (n=1); dOther LLM category included ABCA1, adenosine triphosphate–binding cassette transporter A1; ACAT, acyl-coenzyme A:cholesterol acyltransferase; CE, cholesteryl esters; FA, fatty acid; FC, free aAdapted from Reiner et al.2 medication, suggesting that use of lipid-lowering medication alone is not sufficient cholesterol; FFA, free fatty acids; HDL, high-density lipoprotein; HMG-CoA, hydroxymethylglutaryl-coenzyme A; LAL, lysosomal acid lipase; LAL-D, LAL deficiency; LCAT, lecithin- cholestyramine (n=2) and fish oil (n=1). cholesterol acyltransferase; LDL, low-density lipoprotein; LDLR, LDL receptors; SREBP, sterol regulatory element–binding proteins; TG, triglycerides; VLDL, very-low-density •• The observed reduction in LDL-C with sebelipase alfa appears to be associated with a reduction in LDL-P number, lipoprotein. •• Sebelipase alfa is a recombinant human LAL that catalyzes the hydrolysis of cholesteryl esters to FC and triglycerides to aAdapted from Reiner et al.2 glycerol and FFA; it is indicated for treatment of patients of all ages with a diagnosis of LAL-D11 Efficacy suggesting that cardiovascular disease risk in LAL-D patients may be reduced with sebelipase alfa treatment •• Clinical manifestations of LAL-D include severe dyslipidemia and hepatic steatosis, fibrosis, and cirrhosis1,4 •• In the Acid Lipase Replacement Investigating Safety and Efficacy (ARISE) study, sebelipase alfa normalized serum •• Treatment with sebelipase alfa significantly improved key atherogenic biomarkers from baseline compared with placebo •• Treatment with sebelipase alfa improves lipid biochemistry and other atherogenic biomarkers in LAL-D by addressing –– The dyslipidemia may cause accelerated atherosclerosis in LAL-D patients, increasing the risk of premature ischemia, aminotransferase levels; improved LDL-C, non-high-density lipoprotein cholesterol (HDL-C), triglyceride, and HDL-C levels; (Table 3) the underlying pathogenetic mechanism of disease and is the recommended treatment for LAL-D patients of all ages 8 myocardial infarction, and stroke1 and decreased hepatic fat content in children and adults with LAL-D –– The atherogenic biomarker profile associated with LAL-D is similar to that observed in heterozygous familial Table 3. Effect of Sebelipase Alfa on Atherogenic Biomarkers in LAL-D Subjects at hypercholesterolemia (HeFH) and familial combined hyperlipidemia (FCH, Table 1) REFERENCES METHODS 20 Weeks of Treatment Table 1. Atherogenic Biomarker Profiles in HeFH, FCH, and LAL-D 1. Bernstein DL, et al. J Hepatol. 2013;58:1230-43. 2. Reiner Z, et al. Atherosclerosis. 2014;235:21-30. 3. Lysosomal acid lipase deficiency (#278000). Mean (SD) Percentage OMIM database. 2015. http://omim.org/entry/278000. Accessed April 27, 2016. 4. Grabowski GA, et al. In: Valle D, et al, eds. Metabolic and Molecular LAL-Da Objective Change From Baseline Bases of Inherited Disease. 8th ed. New York, NY: McGraw-Hill; 2012. 5. Kusters DM, et al. JAMA. 2014;312:1055-7. 6. Wiegman A, et al. JAMA. HeFH FCH SA Placebo •• To evaluate the effects of sebelipase alfa on measures of atherogenic biomarkers in LAL-D 2004;292:331-7. 7. Veerkamp MJ, et al. Circulation. 2004;109:2980-5. 8. Burton BK, et al. N Engl J Med. 2015;373:1010-20. 9. Expert Panel on 5,6 7 8 8 Sebelipase Alfa Placebo Percentage Point Characteristic (Mean) (n=194) (n=121) (n=36) (n=30) Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents. Pediatrics. 2011;128:S213:56. 10. Rader DJ. N Engl J Med. Atherogenic Biomarker N=33 N=28 Difference P Value 2015;373:1071-73. 11. Kanuma [package insert]. Cheshire, CT: Alexion Pharmaceuticals Inc; December 2015. Age (years) 13 48 17 15 Study Design Total Chol (mg/dL) LDL-P number, nmol/L −27.0 (18.8) 0.8 (18.0)b 27.8 <0.0001 302 267 253 297 •• A prespecified subanalysis of prospectively collected data on atherogenic biomarkers from ARISE (NCT01757184) in (acceptable: <170)9 which patients were randomized to sebelipase alfa 1 mg/kg or placebo LDL-C (mg/dL) LDL-C, mg/dL −31.1 (18.1) −6.6 (13.4) 24.5 <0.0001 SUPPORT 9 239 173 190 230 –– Subjects were randomized to sebelipase alfa 1 mg/kg or matched placebo via intravenous infusion every other week (acceptable: <110) apoB, mg/dL −27.4 (13.3) −4.4 (8.5) 23.1 <0.0001 This study was sponsored by Alexion Pharmaceuticals, Inc. Editorial assistance was provided by Michael Morren, MBA, RPh of Peloton Advantage, LLC and was HDL-C (mg/dL) during a 20-week, double-blind treatment period funded by Alexion Pharmaceuticals, Inc. 47 38 32 33 (acceptable: >45)9 •• Atherogenic biomarkers analyzed included serum low-density lipoprotein particle (LDL-P) number, LDL-C, apolipoprotein apoA1, mg/dL 9.5 (11.5) −2.2 (12.1) 11.7 0.0002 TG (mg/dL) 70 213 153 174 B (apoB), apolipoprotein A1 (apoA1), and LDL-P size (assessed using nuclear magnetic resonance–based lipoprotein (acceptable: <90)9 LDL-P size, nm −0.9 (3.3)a 0.2 (2.4)b 1.1 0.1083 apoB (mg/dL) analysis) DISCLOSURES b c apo, apolipoprotein; LAL-D, lysosomal acid lipase deficiency; LDL-C, low-density lipoprotein cholesterol; LDL-P, low-density lipoprotein particle. 9 141 137 148 169 D Wilson is a speaker for the Osler Institute, Insulet, and Alexion Pharmaceuticals, Inc; has participated in an advisory board for Aegerion Pharmaceuticals and (acceptable: <90) –– Apolipoprotein and lipoprotein particle analyses were conducted by LipoScience, Inc, Raleigh, NC aN=32; bN=27. apoA1 (mg/dL) for Alexion Pharmaceuticals, Inc; has participated in an advisory board for and has been a speaker for Synageva BioPharma Corp; and has received research 126 NA 102b 102c –– LDL-C analyses were conducted by Covance Inc, Princeton, NJ (acceptable: <120)9 funding from Merck Sharp & Dohme and Novo Nordisk. B Burton has received funding for the conduct of clinical trials from Synageva BioPharma Corp, Alexion Pharmaceuticals, Inc, BioMarin Pharmaceutical, Shire, Genzyme, and Ultragenyx Pharmaceutical; funding for independent research and/or education apo, apolipoprotein; Chol, cholesterol; FCH, familial combined hyperlipidemia; HeFH, heterozygous familial hypercholesterolemia; HDL-C, high-density lipoprotein cholesterol; •• Between-group differences from baseline to Week 20 were assessed using a Wilcoxon rank-sum test for the overall •• Improvements in atherogenic biomarkers occurred with sebelipase alfa regardless of concomitant lipid-lowering from BioMarin Pharmaceutical and Shire; and consulting fees and honoraria from BioMarin, Shire, Alexion Pharmaceuticals, Inc, Genzyme, and Regenxbio. LAL-D, liposomal acid lipase deficiency; LDL-C, low-density lipoprotein cholesterol; TG, triglycerides; SA, sebelipase alfa. LAL-D cohort and separately for subjects receiving and not receiving lipid-lowering medications during the study medication usage (Table 4) a39% (26/66) LAL-D subjects were receiving lipid-lowering medication; bn=34; cn=28. S Marulkar and R Tripuraneni are employees of Alexion Pharmaceuticals, Inc.

Presented at the National Lipid Association 2016 Scientific Sessions, May 19–22, 2016, New Orleans, Louisiana.