ZP7570: A NOVEL GLP-1/GLP-2 DUAL ACTING PEPTIDE PT01.2 WITH POTENTIAL AS THE NEXT GENERATION THERAPY FOR SHORT BOWEL SYNDROME Jolanta Skarbaliene1; Wayne Russell1; Jon Griffin1, Per-Olof Eriksson1 1Zealand Pharma A/S, Smedeland 36, 2600 Glostrup, Denmark

INTRODUCTION CONCLUSION The GLP-2 analog teduglutide is approved for use in adult short bowel syndrome (SBS), where it improves intestinal adaptation by The novel dual acting GLP-1/GLP-2 peptide ZP7570 increasing intestinal trophicity, causing reduction in parenteral possesses specific and potent GLP-1 and GLP-2 receptor support requirements. The GLP-1 analogs exendin-4 and agonist effects in vivo, evidenced as reduced intestinal motility liraglutide have shown positive effects in small clinical studies in and anti-hyperglycemic actions, and intestinotrophic effects in SBS patients, possibly through reducing intestinal motility and / or rodents. improving intestinal absorptive capacity. Our hypothesis is that combining GLP-1 and GLP-2 activity in one molecule will provide The motility and intestinotrophic effects are expected to result additional benefits for patients, such as reducing the rate of in increased absorption of fluid and nutrients in SBS patients. intestinal transit and thus improving nutritional uptake. Furthermore, due to its GLP-1 agonist activity ZP7570 is also expected to protect against hyperglycemia and liver injury, which are both frequently associated with parenteral support. AIM To evaluate the intestinotrophic, gastrointestinal motility and glycemic effects of the GLP-1/GLP-2 dual acting peptide ZP7570 These findings warrant clinical evaluation of ZP7570 as a in rodents. future treatment for SBS.

RESULTS

ZP7570 is a potent GLP-1/GLP-2 dual acting Pharmacokinetics of ZP7570 in rats and mice peptide

Peptide hGLP-1R hGLP-2R ZP7570 Rat (sc) Rat (iv) Mouse (sc) Mouse (iv) EC50 EC50 (cAMP; nM) (cAMP; nM) Variable Mean Mean Mean Mean

ZP7570 0.19±0.029 (n=3) 0.21±0.054 (n=4) Vdss (mL/kg) 60.8 69.3 CL (mL/hr/kg) 5.64 8.76

Liraglutide 0.03±0.001 (n=4) ND* T½ (h) 7.7 8.8 3.6 5.5

Tmax (h) 9.0 4.5 Teduglutide ND* 0.019±0.01 (n=121) F (%) 58 71

Table 1. Effect of reference compounds and ZP7570 on the GLP-1 and GLP-2 receptors Table 2. Pharmacokinetic profiles of ZP7570 in rats and mice after sc and iv administration of measured as cAMP formation in HEK293 cells stably expressing human (h) GLP-1 or GLP- 10 nmol/kg. 2 receptor. Data are mean ± SD. *Not Determined.

Dose-dependent effects of ZP7570 on intestinal growth in rats

A B C 20 2.5 200 * *** *** *** *** *** 15 *** 2.0 150 * 1.5 10 100 %↑ 1.0 vs Vehicle 5 0.5 50 -1% 4% 2% 32% 97% 100%113% -4% -2% 2% 3% 8% 23% 16% -1% 4% -1% 7% 17% 20% 19%

Intestinal length (cm) length Intestinal

Small Intestine weight (g) weight Intestine Small 0 (g) weight intestine Large 0.0 0

Vehicle Vehicle Vehicle

ZP7570 6nmol/kg ZP7570 6nmol/kg ZP7570 6nmol/kg ZP7570 25nmol/kg ZP7570 ZP75700.1nmol/kgZP7570 0.4nmol/kg 1.5nmol/kgZP7570 25nmol/kg ZP7570 25nmol/kg ZP7570ZP7570 0.1nmol/kg ZP75700.4nmol/kg 1.5nmol/kg ZP7570ZP7570 100nmol/kg 225nmol/kg ZP7570ZP7570 100nmol/kg 225nmol/kg ZP7570 ZP75700.1nmol/kgZP7570 0.4nmol/kg 1.5nmol/kg ZP7570ZP7570 100nmol/kg 225nmol/kg

Figure 1. Evaluation of small intestine wet weight (A), large intestine wet weight (B), and total intestinal length (small and large intestine; C) after treatment with vehicle or ZP7570 for 14 days. Data were compared by 1-way ANOVA followed by Dunnett’s multiple comparison test and presented as mean +SEM. *p < 0.05 or ***p < 0.001 vs. Vehicle group, n=6.

Effects of ZP7570 on intestinal transit and gastric Effects of ZP7570 on glucose tolerance and intestinal emptying in rats growth in mice A 100 A B Vehicle 16 80 * Liraglutide, 50 nmol/kg, bid *** Teduglutide, 250 nmol/kg, bid 14 ZP7570, 6 nmol/kg, qd 60 ZP7570, 250 nmol/kg, qd 1.6 *** 12 *** *** 40 *** 1.4 Figure 2. Evaluation of 10 intestinal transit (A) ** ** Intestinal transit Intestinal 20

(% barium transit) (% barium and gastric emptying 1.2 8 (B) after treatment with 0 vehicle or ZP7570 for 6 1.0 B 14 days. Rats were 4 fasted for 16 hrs and 0.8

compounds (mM) glucose Blood 4 administered prior to *** all treatments (except Teduglutide)

Intestine weight (g) weight Intestine 3 barium sulfate gavage. 2 0.6 Data was compared by 1-way 0 0.4 2 ANOVA followed by 15 30 120 Dunnett’s multiple Time (min) comparison test and Vehicle 1 presented as mean

Gastric emptying Gastric +SEM. *p < 0.05 or (Content (g)/B.W.)(Content ***p < 0.001, vs. Vehicle group, n=6. ZP7570 (6 nmol/kg) 0 ZP7570 (250 nmol/kg) Liraglutide (50 nmol/kg) Teduglutide (250 nmol/kg) Figure 3. Effects of vehicle, GLP-2 receptor agonist (Teduglutide), GLP-1 receptor agonist Vehicle (Liraglutide), or ZP7570 on blood glucose levels during an OGTT (after a single s.c. administration; A), and on total intestine (small and large) wet weight (after 3-days treatment; B) in C57BL/6J mice. Data were compared by 2-way ANOVA followed by Bonferroni posttests (A) or by 1-way ANOVA ZP7570 6nmol/kg followed by Dunnett’s multiple comparison test (B) and presented as mean +SEM. *p < 0.05 or ***p ZP7570 25nmol/kg ZP7570ZP7570 0.1nmol/kg ZP75700.4nmol/kg 1.5nmol/kg ZP7570 ZP7570100nmol/kg 225nmol/kg < 0.001, vs. Vehicle group , n=6.

METHODS Study in rats Wistar rats (n=6/group) were dosed SC (QD) with vehicle or ZP7570 (0.1-225 nmol/kg) for 14 days. Gastric emptying and intestinal transit were determined after an overnight fast using a barium sulfate assay (oral gavage of 1 g/mL barium sulfate in salt-free water). At time 60 minutes post-gavage (PG) the stomach was excised, weighed, and then emptied and re-weighed. Also at 60 minutes post-PG the intestinal tract (pylorus to rectum) was removed, and the length of the intestine and the length travelled by barium sulfate recorded. For trophic effects, small and large intestine were carefully cleaned and weighed. Study in mice C57BL76 mice (n=6) were dosed subcutaneously (SC) once daily (QD) with vehicle or ZP7570 (6 and 250 nmol/kg) for 3 days, and compared to reference compounds liraglutide (50 nmol/kg, bid) and teduglutide (250 nmol/kg, bid). After a 6h fasting period, animals were dosed a single s.c. administration (day 1) and an oral glucose tolerance test was performed (0-120 minutes). At sacrifice (after days 3 treatment) the intestinal tract was carefully removed, cleaned and weighed.

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