INTRODUCTION

1. A New Formulation of Liprotamase Restores Growth, and Normalizes Lipid Absorption in Pigs With Exocrine Pancreatic Insufficiency (EPI) Grujic, Danica1, Piedra JV2,4, Szymanczyk, S2,4, Szwiec, K2, Bala, T2, Uschakova G2,3, Osdachenko I2,3, Kovalenko T2,and Pierzynowski SG2,3,4 1Alnara Pharmaceuticals Inc, Cambridge, MA, USA, 2SGPlus, Malmo, Sweden, 3 Dept Biology, Lund, Sweden , 4Dept Medical Biology, IMW, Lublin, Poland INTRODUCTION RESULTS Protein digestion expressed as %CNA for 72h collections during control, treatment, and washout periods are shown in Fig 2. Treatment CNA was 60% (Δ26%) and 58% (Δ24%) with HRDT and LRDT respectively, vs.85% in healthy pigs. In the published work with Creon in EPI pigs with daily dose of 900,000 U protease, treatment CNA was 75% with ΔCNA of 16%.3 Standard therapy for EPI in humans, including patients with CF, is a porcine-derived pancreatic enzyme replacement therapy (PERT). However, nutrient absorption remains low, pill burden is high and potential for viral contamination exists. Thus, we posited a new approach for treatment of EPI in children and adults with a microbial product, liprotamase, which contains 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.1,2 Recently we developed a new easy to swallow formulation, a rapid disintegrating tablet (LipRDT). This formulation was tested in young pigs with EPI. The pancreatic duct ligation causes a total lack of pancreatic enzymes and diminished levels of bicarbonate that result in reduced pH of the duodenum, poor nutrient digestion and absorption, steatorrhea and arrested growth, similar to symptoms seen in patients with exocrine pancreatic insufficiency caused by cystic fibrosis.3,4 Fat digestion expressed as %CFA and %CNA for 72h collections during control, treatment, and washout periods are shown in Fig 1. and Fig 2. Treatment CFA was 84% (Δ60%) and 79% (Δ55%) with HRDT and LRDT vs. 93% in healthy pigs. In the published work with Creon in EPI pigs, with daily dose of 240,000 U lipase, treatment CFA was 80%, with ΔCFA of 37%.3 CONCLUSIONS • This efficacy study of a novel formulation of liprotamase in EPI pigs resulted in: • Significantly improved fat digestion to: • HRDT: 84% CFA; from basal of 24% with Δ60% • LRDT: 79% CFA, from basal of 24% with Δ55% • Significantly improved protein digestion to: • HRDT: 60% CNA; from basal of 34% with Δ26% • LRDT: 58% CNA, from basal of 34% with Δ24% • Normalization of fat absorption based on postprandial LI, NEFA and TG with both tested doses, despite the fact that CFA and CNA were not normalized • Significantly improved skeletal muscle performance to levels observed in healthy pigs • Reversal of growth retardation with both tested doses after only one week of treatment, with a mean body mass increase of 6% and 8% • The novel formulation of liprotamase, tablets formulated to be used as a liquid, is an effective alternative to traditional PERT, especially for treatment of EPI in infants, children and patients with G-tubes. Our study demonstrates that although LipRDT treatment didn’t completely restore %CFA and %CNA to values seen in healthy pigs, it normalized postprandial fat absorption, improved skeletal muscle performance, and restored somatic growth - all important nutritional measures. This suggests limitations of the conventional CFA/CNA measures that only present digestion of nutrients, but do not reflect where digestion and absorption occurs that is critical for proper utilization of nutrients. Thus, postprandial LI, TG and NEFA, as a measure of efficacy, are viable endpoints that should be explored in human trials. Figure 1. Mean CFA in EPI Pigs in Comparison with Healthy Pigs (n=7-10, *p<0.05) Figure 2. Mean CNA in EPI Pigs in Comparison with Healthy Pigs (n=7-10, *p<0.05) OBJECTIVE Liprotamase treatment resulted in effective digestion of fat shown by normalization of postprandial lipid profile expressed as LI (Fig. 3), TG (Fig. 4) and NEFA (Fig. 5) based on comparison analysis with healthy pigs (n=5-6).Blood samples were taken on the last day of control and treatment weeks. AUCs are shown in Table 1. To assess the efficacy of a new, liquid formulation of liprotamase as a PERT, we conducted a cross over design study in young EPI pigs. As a novel way of efficacy evaluation, that better reflects nutritional status and overall health than conventional CFA and CNA, we monitored postprandial changes in fat and protein absorption expressed as lipaemic index (LI), trigliceride (TG), non-esterified fatty acid (NEFA), skeletal muscle performance and resulting somatic growth. LRDT 7 days n=4 HRDT 7 days n=4 Figure 5. Plasma NEFA Figure 3. Lipaemic Index Figure 4. Plasma TG Wash out 7 days n=8 Adaptation 7 days Control 7 days n=8 End LRDT 7 days n=4 HRDT 7 days n=4 METHODS Two daily doses of liprotamase were tested in eight (8) EPI pigs, 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, 102,000 U Protease) as 4 tablets/meal. All pigs were fed a high fat diet (HFD~40g fat/meal). Prior to dosing, tablets were dissolved in a small amount of water mixed with yogurt and offered in the middle of the morning and afternoon meal. As a comparator, 10 healthy pigs of the same age and breed were maintained on HFD. References: Table1. AUC for LI, TG and NEFA (*p<0.05) • Borowitz D, Goss CH, Limauro S, Konstan MW, Blake K, Casey S, Quittner AL, Murray FT. Study of a novel pancreatic enzyme replacement therapy in pancreatic insufficient subjects with cystic fibrosis. J Pediatr. 2006; 49:658-662 • Borowitz D, Stevens C, Fratazzi C, Cohen D and Campion M. 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 ipaemic 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, enternal 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) • Bradstreet RB. Kjeldahl method for organinc nitrogen. (1954) Anal Chem;26(1):185-6 • Andersen, NK, Ravn LS, Guy JH, Edwards SA & Harrison AP. Postnatal changes in electromyographic signals during piglet growth, and in relation to muscle fiber types. Livestock Science, 115:301-312, 2008. • Brüggemann, DA, Risbo J, Pierzynowski SG & Harrison AP. Muscle contraction and force: the importance of an ancillary network, nutrient supply and waste removal. Int. J. of Molecular Science, 9:1472-1488, 2008 Study Design sEMG is a safe and easy method to measure skeletal muscle performance. Signal amplitude indicates strong muscle contraction and improvement in neuronal control. On 200Hz frequency stimulation, EPI pigs treated with LRDT have similar response as healthy pigs on the high fat diet. Figure 6. Skeletal Muscle Performance on 200Hz Stimulation Corrected for bw (kg), p<0.05* For the fecal balance study, stool was collected on days 5, 6, 7 of the respective control and treatment weeks from all EPI and healthy pigs and %CFA and %CNA were calculated using standard gravimetric and Kjeldahl methods, respectively.5,6 Fat absorption was assessed by postprandial changes in LI,TGand NEFA in blood samples collected before the morning meal and then at 1h intervals on the last day of the control and treatment weeks in both EPI and healthy piglets.3 As a new parameter, that indirectly demonstrates nutritional status of EPI piglets fed liprotamase, skeletal muscle strength (L. dorsi) was measured after 7 days of treatment, using a non-invasive technique, surface Electromyography (sEMG), that records electrical signals in active muscle fibers through the skin surface.7,8 Changes in somatic growth (body mass increase, kg/week) and overall health were recorded at the end of the respective weeks. 24th Annual North American Cystic Fibrosis Conference (NACFC); Baltimore, MD, USA; October 21–23, 2010 Sponsored by Eli Lilly and Company