# Presentation: 179431

## NMI-1182, a  Novel, Cyclooxygenase- Inhibiting Nitric Oxide Donor (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.
- 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.

## 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

## Structures

_***NMI-1182***_

_***HCT 3012***_

- Naproxen

- linker

*** Esterase sensitive hydrolysis**

**** Reductase sensitive bioactivation/biotransformation**

*****

******

******

*****

******

***Comparison of a dinitrate and a mononitrate***

_***Structures***_

## Human Whole Blood COX-1 and COX-2 Inhibition Assay: NMI-1182 and HCT 3012

***NMI-1182 equivalent to HCT 3012 for COX-1 and COX-2 Inhibition***

**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 B****2**** by EIA.**

## cGMP Synthesis by NMI-1182, HCT 3012, ISMN and GTN in LLC-PK1 Cells

**LLC-PK1 cells (8 x 10****5****/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****** ***

## Rate of cGMP Synthesis by NMI-1182, HCT 3012, ISMN and GTN in LLC-PK1 Cells

**LLC-PK1 cells (8 x 10****5****/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***

## Effect of CINODS on Isolated Rat Aorta

***NMI-1182 produces relaxation at ************M concentrations (NMI-1182>HCT 3012)***

**Rat aortic ring with endothelium isolated and mounted in tissue bath ******** test compound.**
**Contraction induced by phenylephrine (1 ********M).**
** ****Results expressed relative to  maximal relaxation by 10 ********M S-nitrosoglutathione.**

## 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.

## Effect of Orally Administered CINODS on 

_***Carrageenan-Induced Paw Edema in the Rat***_

***Dosed at Naproxen Na equivalent in PEG 400 (n=7-9/group)***

***All tested compounds exhibit anti-inflammatory activity***

**Male SD rats (~250 g) treated with p.o. test compound or vehicle 1 h prior to carrageenan** **injection (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.**

## Effect of Orally Administered CINODS on Carrageenan-Induced Inflammation in Rat Air Pouch Model

***All three compounds inhibit prostaglandin production.***
***Only NMI-1182 and naproxen inhibit leukocyte infiltration.***

**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, PGE****2****, etc.).**

## Gastric Tolerance of Orally Administered

_***CINODS in the Rat***_

***Dosed at Naproxen Na equivalent in PEG 400 (n=10-12/group)***
***Plasma Naproxen at 40 mg/kg were 52 and  66 ************g/ml for NMI-1182 and HCT 3012, respectively.***

***NMI-1182 shows GI tolerance comparable to HCT 3012***

**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.**

## Effect of Orally Administered Compounds on 

_***Blood Pressure in Conscious Wistar Rats***_

***There are no apparent compound related  BP effects in normal rats.***

**Mean ± SEM**

## Effect of Orally Administered Compounds on Blood Pressure in Conscious Hypertensive SHR Stroke Prone Rats

*                                                         **	**	**	**	*
***** ††† ‡‡‡ p < 0.001**                  ‡‡‡ **Significantly Different from HCT 3012**

_***Effect of Orally Administered Compounds on Blood Pressure in Conscious Hypertensive SHR Stroke Prone Rats***_

**NMI-1182 ******** MABP vs. HCT 3012, vehicle and Naproxen**

**Dose 30 mg/kg naproxen Na eq.**

## Effect of Orally Administered Compounds on Flow Rate in Conscious Hypertensive SHR Stroke Prone Rats

**NMI-1182 ~ Naproxen ****** **FR vs. HCT 3012**

**       ****Mean ± SEM***      ** ***  **Significantly Different from Vehicle**   ††† **Significantly Different from Naproxen**** ****Na**
***       *******  *****p < 0.01*** **	**	**	**	*
***** ††† ‡‡‡ p < 0.001**                  ‡‡‡ **Significantly Different from HCT 3012**

## Effect of Orally Administered Compounds on Peripheral Vascular Resistance (MBP/FR) in Conscious Hypertensive SHR Stroke Prone Rats

**NMI-1182, Naproxen and Vehicle ******** PVR vs. HCT 3012, **

** ****Mean ± SEM***      *
**‡‡‡ ****p < 0.001**                      ‡‡‡ **Significantly Different from HCT 3012**

## 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

## 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***

## HCT 3012-Induced GI Mucosal Naproxen Formation in vitro

_***HCT 3012-Induced GI Mucosal Naproxen Formation in vitro***_

- Important interspecies differences in Naproxen release in rat and human stomach mucosa.

- Rat is not necessarily a predictive species!!

## 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***

_***Metabolism of HCT 3012 and NMI-1182 in Human Stomach S9 Fractions: Naproxen Release***_

## 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***

## 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***

_***Metabolism of HCT 3012 and NMI-1182 in Human Liver S9 Fractions: NOx Release***_

## 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***

## 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 NO****x**** 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.**

## Summary:  Biological Properties of NMI-1182 and HCT 3012

_***Summary:  Biological Properties of ***__***NMI-1182 and HCT 3012***_

## Schematic of NMI-1182 and HCT 3012

## 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

## 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 **