University of Virginia Licensing & Ventures Group BIO 2016 Pain Program Overview University of Virginia Licensing & Ventures Group BIO 2016

Neuropathic Pain Nerve injury or inflammation can produce neuropathic pain Approximately 4 million people in US had neuropathic pain in 2006 (1) Associated with neuronal central sensitization (2) Role of microglial cell priming suggested to play role in pain intensity and duration (3) Activated glial cells release (4): pro-inflammatory cytokines, IL-1β, IL-6, TNFα Chemokines Prostaglandins, ROS and NO Decreasing spinal pro-inflammatory cytokines or increasing anti-inflammatory cytokines reduces allodynia induced by neuropathy (4-6) 1. Taylor 2006, 2. Hanisch 2007, 3. Hains 2010, 4. Watkins 2007, 5. DeLeo 2001, 6. Milligan 2006

Adenosine Actions in Neuropathic Pain Adenosine can reduce pain in some human pain conditions Adenosine binds to four receptor subtypes, A1, A2A, A2B and A3 A1 receptors are expressed in neurones (1) A1 agonists reduced pain in a number of pain models A2A agonists have potent anti-inflammatory effects via suppression of pro-inflammatory cytokines and increased anti-inflammatory cytokines A2A receptor is expressed in neutrophils, macrophages, and T cells but also in basal or activated glial cells from primates and rodents (2-4) A2A agonists reduce pain in preclinical animal models Last 2 bullets is rationale for why A2A agonists 1. Hasko 2007, 2. Boison 2010, 3. Gyoneva 2009, 4. van der Putten 2009

Glial Cells as Target for A2A Agonists A2A receptor expression is increased in glial cells

Chronic Constriction (CCI) Model of Neuropathic Pain . Fukuoka and Noguchi, 2002 . Fukuoka and Noguchi, 2002 BL 1 2 4 8 9 12 Timecourse (wks) sham CCI 50% Withdrawal Threshold (g) 0.3 0.6 1.0 1.8 3.2 5.6 10.0 BL 1 2 4 8 9 12 Timecourse (wks) sham CCI 50% Withdrawal Threshold (g) 0.3 0.6 1.0 1.8 3.2 5.6 10.0 Therapeutic benefit CCI model of neuropathic pain, Filament (plastic) applied, gram threshhold for removal Four ligatures loosely tied around sciatic nerve, induces neuropathic pain Measurement is force in (g) applied to hind paw with a filament before the paw is withdrawn. Filament test is used in podiatrist office to test for diabetic nephropathy in patients for example

ATL313 Reduces pain for 4-5 weeks After Single Injection (1) - no tolerance Repeated injection, no tolerance In week 6 , pain response is going back up, probably due to the turnover in macrophages 1. Loram et al 2009

ATL313 Reduces Pain for Duration of Study - multiple administration maintains anti-allodynia effect Multiple administrations, pain tolerance is almost back to sham animals

Co-administered A2A Antagonist Blocks Effect of ATL313 Antagonist Does Not Reverse Anti-Allodynic action Looking at MOA Left side co administration with antagonist (ZM241385) Right side, 313 administered, time delay, then either ZM2 or vehicle administered Shows that once the switch is flipped you cant block the effect b/c its now a secondary message from or within the glial cells Note these animals still have a normal pain response to heat

IL-10 Neutralization Reverses effects of ATL313 - ATL313 appears to upregulate IL-10 Continue to look at MOA, 313 upregulates IL10 IL10 Ab neutralizes 313 effects

Intrathecal ATL313 in Pain Management Neuropathic pain is associated with glial cell activation A2A receptor expression is increased in glial cells A2A activation reduced pro-inflammatory cytokines and increases anti-inflammatory cytokines ATL313 decreases TNF α and increases IL-10 in CSF cells from CCI rat model and changes expression of TNF α and IL-10 in microglial cells in culture Intrathecal IL-10 reverses neuropathic pain (1) ATL313 reduces pain in three rodent models of neuropathic pain One injection reduces pain for 4 to 5 weeks Injections every 4 weeks maintains efficacy Increased IL-10 production appears to help mediate the ATL313 response 1. Soderquist 2010

Safety Pharmacology and Toxicology Studies Previously licensed to Adenosine Therapeutics, LLC Partnered with Santen in 2010 for glaucoma Full package completed for compound to enter P2a clinical trials in glaucoma Did not lower intraocular pressure Returned to Adenosine Therapeutics, LLC in 2013 Subsequently returned to UVa LVG

Conclusions Strong rationale for ATL313 treatment of neuropathic pain Effect appears to be mediated by increased IL-10 production Toxicology plan can be based on prior studies in rats and dogs with intrathecal adenosine Non-intrathecal toxicology and PK data available Safety pharmacology, hERG, systemic toxicology, PK Human experience with intrathecal adenosine suggests: No major safety issues Reduction in pain can occur if appropriate patient population chosen Strong IP Portfolio Composition of matter patents issued in US, Europe, and Asia (Expires 2028)

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Intrathecal A2A Agonist Effects on Pain ATL313 is a potent and selective A2A agonist Chronic constriction injury [CCI] model Four ligatures loosely tied around left sciatic nerve of rats Neuropathic pain induced ATL313 (1 μl) was injected intrathecally 10-14 days post-CCI A1 A2A (high affinity) (low affinity) A2B A3 Human 26.3 0.3 11.2 3561 122.3 Rat 13.1 *0.7 25.6 3970 65.8 *estimated based on human A2A high to low affinity Ki ratio

Clinical Development of ATL313 - based on discussion with five KOLs Glial cell activation important in chronic neuropathic pains Proof of Concept Study Enroll patients with steady state pain (VAS = 6-10) Unsuccessful pain alleviation with other pain medications, especially narcotics ATL313 + opioid versus opioid alone Endpoints: self administration of opioids (not FDA approved) and pain scores