Liprotamase , a Non Porcine Enzyme Therapy, Restores Growth, and Normalizes Lipid Absorption in Pigs With Exocrine Panc

1. Liprotamase, a Non Porcine Enzyme Therapy, Restores Growth, and Normalizes Lipid Absorption in Pigs With Exocrine Pancreatic Insufficiency (EPI) Grujic D,1Piedra J,2,4Szymanczyk S,2,4Szwiec K,2Bala T,2Uschakova G,2,3Osdachenko I,2,3 Kovalenko T,2 Pierzynowski S2,3,4 1Alnara Pharmaceuticals, Cambridge, MA, USA; 2SGPlus, Malmo, Sweden; 3Dept Biology, Lund, Sweden; 4Dept Medical Biology, IMW, Lublin, Poland Figure 4. NEFA Concentration in Blood Taken Before and After the Meal on the Last Day of Control and LipRDT Weeks (n=5-6) RESULTS • 1 week of treatment with liprotamase RDT resulted in significant growth and phenotypic changes shown in Fig. 6 and Fig. 7. INTRODUCTION Adaptation7 days • Fat digestion, expressed as %CFA for 72h collections is shown in Fig.1. Treatment CFA was 84% and 79% with HRDT and LRDT (ext. LRDT) respectively, vs. 93% in healthy pigs. Figure 6. Mean Body Mass Increase in EPI Pigs after 1 Week of Treatment (*p<0.05) • Liprotamase is a novel biotechnology-derived, non-porcine enzyme replacement therapy that contains three (3) highly purified and stable enzymes: crystalline cross-linked recombinant lipase (32,500 USP units/capsule), crystalline protease (25,000 USP units/capsule), and amorphous amylase (3,750 USP units/capsule). The particular enzymes selected for inclusion in liprotamase were based on their stability in the pH of the gastrointestinal tract without enteric coating and their broad substrate specificity without the need for co-factors.ref1,2 Because of its purity and stability, recently we were able to develop a novel liquid formulation, rapid disintegrating tablets (LipRDT), meant for treatment of the pediatric population and others who have difficulty swallowing capsules. • Liprotamase RDT was tested in a pig surgical model of exocrine pancreatic insufficiency (EPI).3,4 Total EPI in pigs dramatically decreases the levels of digestive enzymes and bicarbonate in the small intestine, causing bile salt precipitation and inhibition of micelle formation resulting in poor lipid absorption, steatorrhea and arrested growth, similar to symptoms seen in patients with pancreatic insufficiency (PI). Figure 1. Mean CFA in EPI Pigs During Control, LipRDT Weeks, Wash Out, and Comparison with Healthy Pigs (n=7-10, *p<0.05) Figure 7. Sick EPI Pig vs. “Happy“ Healthy and Alert Liprotamase RDT Pig • As demonstrated, postprandial changes in LI, NEFA and TG very much resemble lipid absorption profiles of healthy pigs, suggesting effective digestion and absorption in the proximal part of the small intestine. OBJECTIVE • To assess the efficacy of a new, RDT formulation as a PERT, we conducted a cross over design study in young EPI pigs. The effectiveness for fat digestion was demonstrated by change in coefficient of fat absorption (%CFA). As a novel way of efficacy expression, we monitored fat and protein absorption, expressed as postprandial changes in lipaemic index (LI), non-esterified fatty acid (NEFA), triglyceride (TG) and somatic growth. Figure 5. Ceco-illeal Loop Showing “Natural” Lipid Absorption in LipRDT-treated Pig • Based on comparison analysis with healthy pigs, liprotamase treatment resulted in effective digestion of fat as shown by normalization of postprandial lipid profiles demonstrated by LI (Fig. 2), TG (Fig. 3) and NEFA (Fig. 4). Figure 2. Lipaemic Index From Blood Taken Before and After the Meal on the Last Day of Control and LipRDT Weeks (n=5-6 pigs) METHODS • Efficacy of two daily doses of liprotamase, high (HRDT: 290,000 U Lipase, 24,000 U Amylase, 194,400 U Protease) and low (LRDT: 146,000 U Lipase, 12,000 U Amylase, 102,400 U Protease) were tested in eight (8) EPI pigs that were fed a high fat diet (HFD, ~40g fat/meal). Prior to dosing, tablets were dissolved into a small amount of water, (4 tablets/meal) and offered in the middle of the morning and afternoon meals. Study included 1 control and 2 treatment periods, each one week. During the fifth week all pigs were placed on LRDT. CONCLUSIONS • This in vivo activity study of a novel formulation of liprotamase in a EPI pig model that mimics PI disease in human subjects resulted in: • Normalization of fat absorption based on postprandial LI, and blood concentrations of NEFA and TG with both tested doses • 2. Reversal of growth retardation from control week, with a mean body mass increase of 6% (0.9kg/week) and 8%(1kg/week) with HRDT and LRDT respectively • 3. Significantly improved fat digestion to: • HRDT: 84% CFA; from basal of 24% to treatment of 84%, (Δ60%) • LRDT: 79% CFA; from basal of 24% to treatment of 79%, (Δ55%) • 4. Evidence that postprandial LI, TG and NEFA, are viable endpoints that better reflect nutritional status and health, and should be explored in human trials instead of %CFA which has limitations in the clinical setting • 5. Successful in vivo activity of a novel RDT formulation of liprotamase designed for the treatment of EPI in infants, children, patients with a G-tube, and others who have difficulty swallowing capsules Study Design: • Comparative analyses between healthy pigs (of the same age and breed) fed high fat feed vs. EPI liprotamase RDT treated pigs (Table 1). Table 1. EPI Pigs Treated With LipRDT vs. Healthy Pigs Figure 3. TG Concentration in Blood Taken Before and After the Meal on the Last Day of Control and LipRDT Weeks (n=5-6 pigs) • For the fecal balance study, stool was collected on days 5, 6, and 7 of the respective control and treatment weeks from EPI and healthy pigs and the %CFA was calculated using a standard gravimetric method.5 • Fat absorption was assessed by postprandial changes in LI, TG and NEFA in blood samples collected before the morning meal and then at 30 min and 1h intervals on the last day of the control and treatment weeks in both EPI and healthy piglets. • Changes in growth (body mass increase kg/week) and overall health were recorded after seven (7) days of treatment. References: • Borowitz D, et al. Study of a novel pancreatic enzyme replacement therapy in pancreatic insufficient subjects with cystic fibrosis. J Pediatr. 2006; 49:658-662. • Borowitz D, et al. An international open-label study of the long-term safety of liprotamase for treatment of pancreatic insufficiency in cystic fibrosis, Poster at NACF meeting 2009. • Donaldson, J et al. The effectiveness of enzymatic replacement therapy measured by turbidimetry and the lipaemic index in exocrine pancreatic insufficient young growing pigs, fed a high –fat diet. (2009) Advances in Medical Science, 54 (1),p 7-13. • Rengman S. et al. An elemental diet fed, enteral or parental, does not support growth in young pigs with exocrine pancreatic insufficiency. (2009), Clinical Nutrition; 1-6. • van de Kamer, J. Total Fatty Acids in Stool, Standard Methods of Clinical Chemistry, vol. 2, (1958). The American College of Gastroenterology (ACG) Annual Scientific Meeting and Postgraduate Course; San Antonio, TX, USA; October 15–20, 2010 Sponsored by Alnara Pharmaceutical Inc., a wholly-owned subsidiary of Eli Lilly and Company