PM-015 Frédérique Menzaghi, Robert H

DEVELOPMENT OF THE NOVEL, PERIPHERALLY-SELECTIVE KAPPA OPIOID AGONIST CR665 FOR THE TREATMENT OF ACUTE PAIN PM-015 Frédérique Menzaghi, Robert H. Spencer, Michael E. Lewis and Derek T. Chalmers Cara Therapeutics Inc., One Parrott Drive, Shelton CT 06484, USA

Introduction Peripheral Selectivity of CR665 CR665 Suppresses TNFα Release in Safety and Tolerability of CR665 in The aversive cognitive effects caused by activation of kappa opioid Enadoline CR665 Mouse Model of Sepsis Human receptors in the CNS (dysphoria, hallucinations) have hindered the 100 5000 3000 100 development of compounds acting by this mechanism. However, Tolerability 80 activation of kappa receptors located outside of the CNS (peripheral 80 4000 9 CR665 was well tolerated in both male and female healthy 60 2000 nerve endings and immune cells including T-cells, macrophage, mast Writhing 60 Writhing 3000 * volunteers at dose levels through 0.42 mg/kg (1-h) and through pg/mL Rotarod pg/mL

Rotarod α cells) appears sufficient to modulate the transmission of pain and 40 40 α 0.09 mg/kg (5-min) 2000 % Activity

% Activity 1000 * inflammatory signals. Activation of kappa opioid receptors with kappa- 20 20 9 No severe or serious adverse effects (AEs) observed

↓ 41% TNF- selective agonists is also known to produce pharmacological effects TNF- 1000 9 No report of hallucination, dysphoria or emesis that differ from activation of mu opioid receptors (e.g., no inhibition of 0 0 ↓ 74% 0 0 9 Most frequently reported AEs were mild, transient nervous Vehicle CR665 Vehicle Prednisolone GI transit, water retention, itch or induction of respiratory depression). 0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 10 (Saline, S.C.) (8.5 mpk) (CES, I.P.) (3 m pk) system symptoms (paresthesia, dizziness, somnolence) Therefore, peripherally-restricted kappa agonists may represent a (mg/kg, i.v.) (mg/kg, i.v.) 9 LPS-induced cytokine release in female Balb/C mice novel, better tolerated, class of anti-inflammatory and analgesic drugs 9 No treatment-related trends or significant clinical findings in for the treatment of acute and chronic pain. Writhing Rotarod 9 Cytokine levels in serum measured by ELISA, 2 hr post-LPS serum biochemistry, haematology or urinalysis data (A50, mg/kg) (A50, mg/kg) Peripheral Selectivity α Evidence from knockout mice suggest that kappa receptors play an 9 Significant reduction in circulating TNF levels following 9 No changes observed in serum electrolytes Enadoline 0.002 0.004 2 pre-treatment with CR665 Safety important role in the modulation of visceral pain. Here we are CR665 0.007 3.8 543 9 Suggests direct action of KOR on inflammatory cells 9 CR665 was determined to be safe at all doses and infusion presenting CR665, the clinical lead of our first generation of 9 0.6% acetic acid induced writhing in male CD-1 mice peripherally-acting kappa agonists as a novel approach for the rates tested (1 hr or 5-min infusion) 9 CR665 IV has significantly larger therapeutic index treatment of acute visceral pain without the complications and side- Pharmacokinetics of CR665 in Human compared to small molecule kappa agonists like Enadoline effects of traditional opioid therapies. CR665 Attenuates Moderate Visceral 1200 1 Hr Intravenous Infusion Low Brain Penetration of CR665 Pain in Human Subjects Development of Peripherally-Restricted 1000 0.015 mg/kg Low CNS Penetration of CR665 0.03 mg/kg Plasma and Brain Exposure for CR665 800 Kappa Agonists (10 mg/kg, subcutaneous bolus in mice) 0.06 mg/kg (10 mg/kg, subcutaneous in mice) 9 CR665 was evaluated in a model of visceral pain in human 0.12 mg/kg Peripheral Analgesia 4000 600 subjects using esophageal distension (see PM-178) 3500 0.24 mg/kg Peripheral Nerve 400 0.36 mg/kg 9 Randomized, double-blind, double-dummy, active and

3000 (ng/ml) CR665 0.42 mg/kg placebo-controlled, cross-over study in 18 subjects 2500 200 D-amino acid 2000 0.48 mg/kg 9 CR665 (0.36 mg/kg,1 hr IV infusion) produced significant Tetrapeptide 1500 0 efficacy within a similar range as oxycodone (15 mg, oral) 1000 Plasma 01234 relative to placebo (double-dummy, IV and oral) 500 CR665(ng/ml) Levels Brain Time post-dose (h) Limited Brain Penetration 0 9 CR665 was safe and well-tolerated with no significant AEs 0 15304560 9 Dose-proportionality increase in systemic exposure to CR665 Time After Injection (mins) with concentrations well above the estimated therapeutic Broad Efficacy with concentrations in both male and female subjects Improved Safety Profile 9 Plasma half-life ranging from 1-2 hours across species Conclusions 9 Plasma half-life of 1-2 hours with rapid elimination 9 Brain-to-Plasma Ratio less than 3% 9 CR665 is potent and efficacious in a variety of rodent 9 70-85% Protein binding across species Duration of action similar Pharmacodynamic Effect of CR665 on models of visceral pain, and inflammation 9 No oral bioavailability to small molecules Release of Prolactin in Male Subjects 9 Phase I study completed in normal healthy volunteers Efficacy of CR665 in Rat Models of In Vitro Profile of CR665 9 CR665 was safe and well-tolerated 60 Prolactin CR665 1200 Visceral Pain (ng/ml) Cmax CR665 9 No evidence of dysphoria or hallucination 50 1000 Pancreatitis Model Jejunal Distension Model 9 No evidence of Mu opioid related side effects Compound hKOR hMOR hDOR Veh (n=9) 40 800 100 (Ki, nM) (Ki, nM) (Ki, nM) CR665 (0.1 mg/kg, ip, n=6) * 9 CR665 has demonstrated efficacy for attenuating 30 600 CR665 (0.3 mg/kg, ip, n=9) 80 moderate visceral pain in humans (see PM-178) Peptide CR665 0.24 4050 20300 CR665 (1 mg/kg, ip, n=8) 20 400 10 * 9 Efficacy in preclinical and human models suggest Enadoline 1.25 272 707 60 10 200 8 CR665 may be effective for the treatment of acute Small Asimadoline 0.17 581 322 40 (ng/ml)Prolactin Cmax 0 0 6 ED50 ≈ 0.1 mg/kg visceral pain molecules * 0.02 0.03 0.06 0.12 0.24 0.36 0.42 0.48 TRK-820 0.36 0.71 50 **** 20 4 9 Longer-acting and orally active kappa agonist CR845 ***ED = 0.47 mg/kg Dose (mg/kg) 50 0 Morphine 14.7 4.4 150 2 0.01 0.1 1 also being developed for treatment of chronic *** Response to Jejunal% Distension

Number of withdrawals CR665 (mg/kg, i.v.) 9 Opioids including kappa agonists are known to induce inflammatory and neuropathic pain (see PW-231) 0 BL 0 30 60 90 release of Prolactin 9 Best-in-class non-narcotic opioids Efficacy determined by cardiovascular Time after compound administration (min) response in anesthetized animals 9 Activation of kappa receptors in anterior pituitary (outside of Acknowledgement 9 Full agonist (EC50 hKOR: 0.03 nM, cAMP assay) blood-brain barrier) likely involved 9 High affinity for both human and rodent KOR (rat Ki: 0.42 nM) 9 CR665 is effective in multiple models of visceral pain including 9 Prolactin release observed at lowest dose tested (0.02 mg/kg) ¾ The Authors thank Drs. Frank Porreca, Todd Vanderah and 9 No off-target activity on >94 receptors/channels/transporters acetic-acid writhing, pancreatitis and jejunal distension reaching a maximal effect at 0.12 mg/kg dose Lionel Bueno for their contributions to these studies.