2DG: A Novel Antiglycolytic Anticonvulsant with Disease-Modifying Effects

1. 2DG – A Familiar Antiglycolytic Glucose-analog With Novel Anticonvulsant Properties • Thomas Sutula, MD, PhD • Detling Professor and Chair • Department of Neurology • University of Wisconsin • Chief Scientific Officer • NeuroGenomeX, Inc. • Madison, WI

2. 2DG: a glucose analog differing from glucose only by removal of Oxygen at the 2-position

3. 2DG: a glucose analog differing from glucose only by removal of Oxygen at the 2-position glycolytic inhibitor 2DG transiently inhibits glycolysis by blocking the isomerization step from glucose-6P to fructose-6P

4. 2DG: a glucose analog differing from glucose only by removal of Oxygen at the 2-position focal brain delivery during seizures (18F-2DG PET scan Israel & Fishman 1999) focal brain delivery by stimulation (3H-2DG autoradiogram Sutula et al.) glycolytic inhibitor Activity-dependent uptake loads 2DG into areas of neural circuitry with increased metabolic demands 2DG transiently inhibits glycolysis by blocking the isomerization step from glucose-6P to fructose-6P

5. Completed Preclinical efficacy studies In vivo studies in acute and chronic models of epilepsy • Acute anticonvulsant action • protection against seizures evoked acutely by 6Hz stimulation • (ED50 = 79.5 mg/kg) • protection against audiogenic seizures in Fring’s mice • (ED50 = 206 mg/kg) • 2-fold slowing of latency to status epilepticus onset by pilocarpine • Chronic antiepileptic action • 2-fold slowing of kindled seizure induction and progression from different brain sites (37.5 mg/kg) • Effective against seizure progression when administered as long as 10 min AFTER a seizure!

6. Completed Preclinical efficacy studies In vivo studies in acute and chronic models of epilepsy In vitro studies in hippocampal slices • Acute anticonvulsant action • protection against seizures evoked acutely by 6Hz stimulation • (ED50 = 79.5 mg/kg) • protection against audiogenic seizures in Fring’s mice • (ED50 = 206 mg/kg) • 2-fold slowing of latency to status epilepticus onset by kainic acid • Chronic antiepileptic action • 2-fold slowing of kindled seizure induction and progression from different brain sites (37.5 mg/kg) • Effective against seizure progression when administered as long as 10 min AFTER a seizure! • 2DG reduces epileptic discharges • evoked by : • 7.5 mM K+ (ictal and interictal) • bicuculline (GABAa antagonist) • 4AP (K+ channel antagonist) • DHPG (metabotropic glutamate agonist) Implies that actions of 2DG at the cellular level are potentially “broad-spectrum” against different mechanisms of network synchronization

7. 2DG slows progression of kindled seizures by 2-fold also at 37.5 mg/kg 2DG has “disease-modifying” effects against progression of seizures and long-term consequences of poorly controlled epilepsy

8. “Disease- modifying” actions of 2DG against progressive adverse effects of repeated seizures NUMBER OF SEIZURES SPONTANEOUS SEIZURES 1 5 30 90-100 + + + REDUCED INHIBITION HIPPOCAMPAL SCLEROSIS MEMORY LOSS APOPTOSIS NEUROGENESIS SPROUTING + 2DG NUMBER OF SEIZURES SPONTANEOUS SEIZURES 1 5 30 90-100 + + + REDUCED INHIBITION HIPPOCAMPAL SCLEROSIS MEMORY LOSS APOPTOSIS NEUROGENESIS SPROUTING

9. “disease-modifying” antiepileptic actions with 2DG administration as long as 10 minute AFTER seizures

10. “disease-modifying” antiepileptic actions with 2DG administration as long as 10 minute AFTER seizures implications for novel applications including status epilepticus, seizure clusters, Lennox-Gastaut syndrome

11. Chronic “disease-modifying” antiepileptic effects of 2DG are associated with alterations of seizure-induced gene expression by novel mechanisms of metabolic transcriptional regulation required for kindling progression

12. TOXICITY PROFILE OF 2DG Previous 2DG experience • Used since 1979 in humans as PET imager (18F-2DG) • >20 investigator-initiated clinical trials in man (~700 normal controls and patients) with no systematic side effects • FDA Approved IND tox packages from U.Iowa and Kettering available to NGX as “right of reference” letters • 2004 completed Phase I clinical trial for adjuvant cancer chemotherapy at doses up to 200 mg/kg was without adverse toxicity (Threshold Pharma) • Hundreds of published animal studies

13. TOXICITY PROFILE OF 2DG Previous 2DG experience Tox Observations • No overt systemic toxicity in rats treated for 6 months at 500 mg/kg/day • No effect on spatial memory in rats after 2 weeks at 1 gm/kg/day • No effect on open field activity at minimal effective dose of 37.5 mg/kg • Used since 1979 in humans as PET imager (18F-2DG) • >20 investigator-initiated clinical trials in man (~700 normal controls and patients) with no systematic side effects • FDA Approved IND tox packages from U.Iowa and Kettering available to NGX as “right of reference” letters • 2004 completed Phase I clinical trial for adjuvant cancer chemotherapy at doses up to 200 mg/kg was without adverse toxicity (Threshold Pharma) • Hundreds of published animal studies

14. TOXICITY PROFILE OF 2DG Previous 2DG experience Tox Observations • No overt systemic toxicity in rats treated for 6 months at 500 mg/kg/day • No effect on spatial memory in rats after 2 weeks at 1 gm/kg/day • No effect on open field activity at minimal effective dose of 37.5 mg/kg • Used since 1979 in humans as PET imager (18F-2DG) • >20 investigator-initiated clinical trials in man (~700 normal controls and patients) with no systematic side effects • FDA Approved IND tox packages from U.Iowa and Kettering available to NGX as “right of reference” letters • 2004 completed Phase I clinical trial for adjuvant cancer chemotherapy at doses up to 200 mg/kg was without adverse toxicity (Threshold Pharma) • Hundreds of published animal studies Based on this favorable prior history, we are hopeful for: FDA approval for an abbreviated pre-IND toxicology package FDA approval for combined Phase I/II studies in epilepsy patients

15. Possible clinical trials/applications for 2DG Relevant 2DG Properties Clinical Trial Options • Efficacy against focal seizures and secondary generalized seizures in preclinical models • Relatively short t1/2 of ~ 40 minutes • Rapid absorption by both oral and parenteral routes • Enhanced activity-dependent focal loading in epileptogenic brain regions maximized around the time of seizure Photosensitivity trial Conventional six month double blind add-on cross-over trial in refractory patients with partial complex and secondary generalized seizures Double blind trial in adult patients with Lennox-Gastaut syndrome (potential orphan drug indication) Administration at onset of seizures in patients experiencing seizure clusters Double blind add on trial in refractory status eplilepticus Implanted device - 2DG combination trials (stimulation-loading of 2DG into epileptogenic circuitry)

16. INTELLECTUAL PROPERTY • Licensed from Wisconsin Alumni Research Foundation (WARF) to NeuroGenomeX • license agreement* includes all WARF “therapeutic use” patents of 2DG 1) “Metabolic-Based Methods for Modulating Gene Expression”P05137US(Priority date- 2/14/2005) claims: USE of 2DG for prevention of cancer metastasis and treatment of other systemic conditions status: patent issued 2) “Compounds and Methods for Treating Seizure and Paroxysmal Disorders” P04134US(Priority date- 6/17/2004) claims: USE of 2DG for treatment of seizures and neuropathic pain status: pending Freedom-to-operate: favorable opinion provided in 2005 3) “Methods and Compounds for Treating Seizure Disorders” P05095US(Priority date - 3/25/2005) claims: Another metabolic-based method for therapeutic development status: awaiting office action * License includes all US and foreign related patent applications

17. 2DG in the drug development pipeline: approaching IND and Phase I/II 2DG

18. 2DG in the drug development pipeline: approaching IND and Phase I/II • novel acute and chronic anticonvulsant mechanisms based on metabolic regulation and long-term alterations • in seizure-related gene expression • broad spectrum of action at the cellular level • against mechanisms of network synchronization • distinctive spectrum of activity against • preclinical screening models • disease-modifying actions against progressive effects of seizures • activity-dependent delivery to regions of epileptic activity • potentially novel methods of delivery: • post-seizure, with device therapies • favorable preclinical toxicity and human use toxicity profile