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NMI-1182 , a Novel, C yclooxygenase- I nhibiting N itric O xide D onor (CINOD)

NMI-1182 , a Novel, C yclooxygenase- I nhibiting N itric O xide D onor (CINOD). David S. Garvey, Ph.D. NitroMed Inc., Lexington, MA 02421, USA. Introduction. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammation and provide pain relief.

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NMI-1182 , a Novel, C yclooxygenase- I nhibiting N itric O xide D onor (CINOD)

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  1. NMI-1182, a Novel, Cyclooxygenase- Inhibiting Nitric Oxide Donor (CINOD) David S. Garvey, Ph.D. NitroMed Inc., Lexington, MA 02421, USA

  2. Introduction • Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammation and provide pain relief. • Recently described Cyclooxygenase Inhibitory Nitric Oxide Donors (CINODs) have comparable anti-inflammatory activity and less gastrointestinal toxicity than standard NSAIDs. • NO provides multi-organ sparing effects • Gastrointestinal irritation • Renal function • Cardiovascular system: hypertension • A novel CINOD, NMI-1182 has been synthesized and its biological activity compared with HCT 3012 (Phase III trials) and naproxen.

  3. Comparison of a NMI-1182, HCT 3012, and Naproxen • In vitro pharmacology • COX-1/COX-2 enzyme inhibition in human whole blood • cGMP Formation in LLC-PK1 cells • Tissue bath smooth muscle relaxation • In vivo pharmacology in the rat • Paw edema • Air pouch • Gastric lesion • Acute blood pressure • Peripheral Vascular Resistance • BioMetabolism • Stomach • Whole Blood • Liver

  4. Structures NMI-1182 ** ** * • Comparison of a dinitrate and a mononitrate Naproxen linker HCT 3012 * ** * Esterase sensitive hydrolysis ** Reductase sensitive bioactivation/biotransformation

  5. Human Whole Blood COX-1 and COX-2 Inhibition Assay: NMI-1182 and HCT 3012 • Heparinized human blood • COX-2LPS (10 µg/ml) for 5 h. • COX-1calcium ionophore A23187 (25 µM) for 30 min. • Assayed for thromboxane B2 by EIA. NMI-1182 equivalent to HCT 3012 for COX-1 and COX-2 Inhibition

  6. cGMP Synthesis by NMI-1182, HCT 3012, ISMN and GTN in LLC-PK1 Cells • LLC-PK1 cells (8 x 105/well) incubated with IBMX (1 mM; to inhibit cGMP degradation) • Test compounds added for 60 min. • Sampled for subsequent determination of cGMP by EIA. NMI-1182 is significantly more potent than HCT 3012 in synthesizing cGMP

  7. Rate of cGMP Synthesis by NMI-1182, HCT 3012, ISMN and GTN in LLC-PK1 Cells • LLC-PK1 cells (8 x 105/well) incubated with IBMX (1 mM; to inhibit cGMP degradation) • Test compounds added for 60 min. • Sampled for subsequent determination of cGMP by EIA. NMI-1182 has a significantly faster rate of cGMP synthesis than HCT 3012

  8. Effect of CINODS on Isolated Rat Aorta • Rat aortic ring with endothelium isolated and mounted in tissue bath  test compound. • Contraction induced by phenylephrine (1 mM). • Results expressed relative to maximal relaxation by 10 mM S-nitrosoglutathione. NMI-1182 produces relaxation at mM concentrations (NMI-1182>HCT 3012)

  9. Summary: In Vitro Pharmacology • NMI-1182 and HCT 3012 equivalent in human whole blood COX-1 and COX-2 inhibition assay. • NMI-1182 is a more potent and faster synthesizer of cGMP in LLC-PK1 cells compared to HCT 3012. • NMI-1182 elicits rat aortic vasorelaxation at lower concentrations than HCT 3012.

  10. Effect of Orally Administered CINODS on Carrageenan-Induced Paw Edema in the Rat • Male SD rats (~250 g) treated with p.o. test compound or vehicle 1 h prior to carrageenaninjection (50 l 1%) into the subplantar region of the right hind-paw pad. • Paw volume determined at time 0, 1 ,3 and 6 h post-carrageenan. Dosed at Naproxen Na equivalent in PEG 400 (n=7-9/group) All tested compounds exhibit anti-inflammatory activity

  11. Effect of Orally Administered CINODS on Carrageenan-Induced Inflammation in Rat Air Pouch Model • Air pouch formed in male SD rats (~200 g)by subcutaneous injection of sterile air into the intrascapular back area (20 ml on day - 6; 10 ml on day - 3) • On day 0, test compound (or vehicle) dosed p.o. 1 h prior to intrapouch carrageenan (1 ml of 1%) injection. • 4 h post-carrageenan, inflammatory exudate recovered from pouch and analyzed (WBC contents, PGE2, etc.). All three compounds inhibit prostaglandin production. Only NMI-1182 and naproxen inhibit leukocyte infiltration.

  12. 70 Naproxen Na 60 NMI-1182 HCT 3012 50 N =10-12/group 40 LESSION SCORE (mm) 30 20 10 0 10 20 40 5 DOSE (mg/kg) Gastric Tolerance of Orally Administered CINODS in the Rat • Sprague-Dawley rats (220 g) treated with p.o. test compound or vehicle (PEG 400) • Animals sacrificed at 3 h post dosing. • Stomachs removed and digitally photographed. Blood collected for plasma naproxen determination. • Gastric lesions scored by an unbiased observer using image analysis software. Dosed at Naproxen Na equivalent in PEG 400 (n=10-12/group) Plasma Naproxen at 40 mg/kg were 52 and 66 mg/ml for NMI-1182 and HCT 3012, respectively. NMI-1182 shows GI tolerance comparable to HCT 3012

  13. Effect of Orally Administered Compounds on Blood Pressure in Conscious Wistar Rats Mean ± SEM There are no apparent compound related BP effects in normal rats.

  14. Effect of Orally Administered Compounds on Blood Pressure in Conscious Hypertensive SHR Stroke Prone Rats Dose 30 mg/kg naproxen Na eq. Mean ± SEM***Significantly Different from Vehicle ††† Significantly Different from NaproxenNa *** ††† ‡‡‡ p < 0.001‡‡‡ Significantly Different from HCT 3012 NMI-1182  MABP vs. HCT 3012, vehicle and Naproxen

  15. Effect of Orally Administered Compounds on Flow Rate in Conscious Hypertensive SHR Stroke Prone Rats Mean ± SEM* ***Significantly Different from Vehicle ††† Significantly Different from NaproxenNa * p < 0.01 *** ††† ‡‡‡ p < 0.001‡‡‡ Significantly Different from HCT 3012 NMI-1182 ~ Naproxen FR vs. HCT 3012

  16. Effect of Orally Administered Compounds on Peripheral Vascular Resistance (MBP/FR) in Conscious Hypertensive SHR Stroke Prone Rats Mean ± SEM ‡‡‡ p < 0.001‡‡‡ Significantly Different from HCT 3012 NMI-1182, Naproxen and Vehicle  PVR vs. HCT 3012,

  17. Summary: In Vivo Pharmacology • NMI-1182 acts as an anti-inflammatory agent in the rat carrageenan paw edema (Naproxen>HCT 3012>NMI-1182) and air pouch model (Naproxen=NMI-1182>HCT 3012). • NMI-1182 exhibits GI tolerance comparable to HCT 3012 (NMI-1182=HCT 3012>>Naproxen). • NMI-1182, HCT 3012 or Naproxen produced no apparent effects on blood pressure in normal rats. • NMI-1182 lowers mean arterial blood pressure but maintains higher flow rates in SHR SP rats compared to HCT 3012. • Peripheral vascular resistance for NMI-1182 is reduced compared to HCT 3012

  18. CINOD BioMetabolism Studies • In Vitro Stomach (Various species) • In Vitro Whole Blood (Human) • In Vitro Liver (Human) • Examined Naproxen release and NOx (nitrite and nitrate) Formation

  19. HCT 3012-Induced GI Mucosal Naproxen Formation in vitro Rat is not necessarily a predictive species!! • Important interspecies differences in Naproxen release in rat and human stomach mucosa.

  20. Metabolism of HCT 3012 and NMI-1182 in Human Stomach S9 Fractions: Naproxen Release NMI-1182 produces more naproxen than HCT 3012 in human stomach

  21. NOx Release by HCT 3012 and NMI-1182 in Human Stomach S9 Fractions and Human Whole Blood NMI-1182 produces more NOx than HCT 3012 in human stomach and whole blood

  22. Metabolism of HCT 3012 and NMI-1182 in Human Liver S9 Fractions: NOx Release NMI-1182 produces much more nitrite than HCT 3012 in human liver

  23. Effect of NMI-1182 and HCT 3012 on cGMP Synthesis in Human Hepatocytes NMI-1182 produces significantly more cGMP than HCT 3012 in human hepatocytes

  24. Summary: BioMetabolism Studies: • Significant differences in metabolism of HCT 3012 between species. • NMI-1182 is more metabolically active than HCT 3012 in human stomach mucosa and blood. • NMI-1182 is more readily hydrolyzed to release naproxen and reduced to liberate NOx in human stomach mucosal S9 and blood. • In human liver S9 both CINODs are quickly hydrolyzed to release naproxen, but only NMI-1182 forms nitrite and stimulates cGMP synthesis in hepatocytes.

  25. Summary: Biological Properties of NMI-1182 and HCT 3012

  26. HCT 3012: Oily mononitrate NMI-1182: Solid Dinitrate Stomach NMI-1182 “Prodrug” Naproxen Naproxen NO NO HCT 3012 NO2- (bioactive NO) X X NO3- (inactive NO) NMI-1182 HCT 3012 Liver • Similar pharmacology in rodent species HCT 3012: Decreased bioactive NO Decreased clinical efficacy? Schematic of NMI-1182 and HCT 3012

  27. Summary • In vitro and in vivo data differentiates NMI-1182 from HCT 3012 • The reported gastroprotection of HCT 3012 may be largely due to action as a prodrug and not as a NO donor. • NMI-1182 may represent the ideal profile for a CINOD, releasing naproxen in the circulation and generating bioavailable NO. • May be a logical successor to withdrawn Coxibs • GI & CV safe anti-inflammatory analgesic agents

  28. Acknowledgements Biology Michael Augustyniak Greg Dube James Ellis Laura Gordon David Janero Terry Melim Madhavi Murty David Schwalb William Selig Matthew Shumway Mark Trocha Delano Young Rosanne Wexler Irina Zemtseva Brian Zifcak Chemistry Richard Earl Subash Khanapure R&D Gordon Letts

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