Impact of Lipidized Neuropeptide Analogs on Food Intake and Metabolic Changes in Rodents

1. Impact of novel lipidizedanalogs of neuropeptides on food intake decrease and metabolic changes in rodents Lenka Maletínská Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Prague, Czech Republic ENDOCRINOLOGY-2014 Chicago, October 22nd, 2014

2. Food intake regulation by peripheral hormones and central neuropeptides hypothalamus brainstem PrRP CCK PP Amylin anorexigenic = decreasing food intake orexigenic = increasing food intake Schwartz, Nature 404 (2000) Zanella, Arq Bras Endocrin Metab 53 (2009)

3. Neuropeptides as potential anti-obesity agents Central administration Advantages: specific, centrally acting Disadvantages: not able to cross BBB from the periphery Peripheral administration – how to cross blood brain barrier? Lipidization of neuropeptides: possibility of transport through BBB Peripheral administration

4. PrRP – prolactin-releasing peptide • centralanorexigenic neuropeptide (hypothalamus, brainstem) • receptor GPR10 – PrRPdiscovered 1998 (Hinuma et al., Nature 393:272 (1998)) • affinityalso to neuropeptide FF receptor (NPFF2) • effect on prolactinreleasequestioned ↑ PrRP PrRP KO mice are obese PrRP receptor KO mice are obese Takayanagi et al., J Clin Invest 118: 1 (2009) Bjursell et al., BBRC 363: 633 (2007)

5. PrRP – lipidization „RF-amide peptides“ 2 natural forms with equal biological activity: PrRP31, PrRP20 - identical C-terminus important for biological activity Phe

6. PrRP – lipidization „RF-amide peptides“ Lipidization with fatty acid attached by amid bound at N-terminal amino acid of PrRP31 Fatty acid: Octanoyl Decanoyl Dodecanoyl Myristoyl Palmitoyl Stearoyl Fatty acid Phe Solid phaseFmocsynthesis, lipidization on resin

7. PrRP – lipidization „RF-amide peptides“ Lipidization with fatty acid attached by amid bound at N-terminal amino acid of PrRP20 Fatty acid: Octanoyl Decanoyl Dodecanoyl Myristoyl Fatty acid Phe Thr Solid phaseFmocsynthesis, lipidization on resin

8. High affinity binding of lipidized PrRP analogs to human PrRP and NPFF2 receptors

9. Lipidized PrRP31 analogs: Food intake after peripheral administration Food intake in fasted mice (dose 5 mg/kg SC) * Statistika:One-way ANOVA, Dunnett’s post hoc test, vs saline (n = 6-8 )

10. Lipidized PrRP20 analogs: Food intake after peripheral administration Food intake in fasted mice (dose 5 mg/kg SC) Statistika:One-way ANOVA, Dunnett’s post hoc test *** vs saline (n = 6-8 )

11. Lipidized PrRP analogs with anorexigenic activity increase neuronal activity in the brain areas regulating food intake PVN PrRP31 Oct-PrRP31 PVN PVN Paraventricular nucleus 3V saline Myr-PrRP31 Palm-PrRP31 Imunohistochemistry: Fos 90 min after lipo-PrRP injection to fasted mice (s.c. administration 5 mg/kg)

12. Palm-PrRP31 lowers food intake in free fed rats Wistar rats, repeated IP administration – once daily (before lights out) (n=5, 10mg/kg in saline) t-test: *** vs saline

13. Lipidization increases stability of PrRP in rat plasma (Measured by PrRP EIA kit)

14. Long-term effect of lipidized PrRP analogs in mouse DIO (diet-induced obesity) model (repeated SC administration) C57BL mice: 3 months on high-fat diet (60% fat) 14 days s.c. administration 2-times per day: • saline • Myr-PrRP20, 5mg/kg • Palm-PrRP31, 5mg/kg

15. Anorexigenic lipidized PrRP analogs lower body weight of DIO mice DIO mouse model: body weight decrease Food intake, body weight, fat, liver weight Glucose, insulin, leptin in blood plasma Statistics:One-way ANOVA, Dunnett’s post hoc test (n = 9-10 )

16. Lipidized PrRP analogs: Summary • New PrRPanalogslipidizedat N-terminus are stableagonistswithhighaffinity to PrRP and NPFF2 receptors • Palmitoylated and myristoylatedPrRPanalogsafteracuteperipheraladministrationshowed long-lastingdecrease in food intakeandincreasedneuronalactivityin hypothalamus and brainstenoffastedmice • In leanfree fedrats, repeated IP treatmentwith palm-PrRP31 very significantlydecreased food intake • In micewith diet-induced obesity, repeated SC treatmentwith myr-PrRP20 and palm-PrRP31 causedsignificantdecrease in food intake, body weight and improvementofmetabolicparametersconnectedwith obesity • No possiblesideeffectsfound: • no anxiety, sedation, analgesia • no signsof toxicity • no prolactinreleaseafterperipheraladministration

17. Conclusions • Palmitoylated PrRP31 and myristoylated PrRP20 • are: • Stable, selective and long-lastinganorexigenicpeptides • Centrallyactiveafterperipheraladministration • Potentialcandidatesforantiobesitytreatment

18. Acknowledgements IOCB Group of Antiobesity peptides Blanka Železná, Martina Holubová, Andrea Špolcová, Veronika Nagelová Jana Zemenová, Barbora Mikulášková, Zdeno Pirník, Hedvika Vysušilová Peptide synthesis: Mirka Blechová Radioiodination: Tomáš Elbert Stability and pharmacokinetics by LC-MS: David Sýkora University of Chemical Technology, Prague Food intake – rat models: Jaroslav Kuneš Institute of Physiology Insulin resistance, clinical diabetology and obesitology: Martin Haluzík, Zdena Lacinová First Faculty of Medicine, Charles University, Prague Grant Czech Science Foundation P303/12/0576 TACR TE01020028 / Center for Development of Original Drugs and RVO:61388963