NMI-1182, a Novel, Cyclooxygenase- Inhibiting Nitric Oxide Donor (CINOD)

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.

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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

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Structures
NMI-1182

• Comparison of a dinitrate and a mononitrate

Naproxen
linker
HCT 3012


Esterase sensitive hydrolysis
Reductase sensitive bioactivation/biotransforma
tion
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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
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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
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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
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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-1182gtHCT 3012)
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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.

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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 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.

Dosed at Naproxen Na equivalent in PEG 400
(n7-9/group)
All tested compounds exhibit anti-inflammatory
activity
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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.
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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
(n10-12/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
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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.
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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 Naproxen Na

p lt 0.001 Significantly
Different from HCT 3012
NMI-1182 ? MABP vs. HCT 3012, vehicle and Naproxen
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Effect of Orally Administered Compounds on Flow
Rate in Conscious Hypertensive SHR Stroke Prone
Rats
Mean SEM Significantly
Different from Vehicle Significantly
Different from Naproxen Na p lt 0.01
p lt 0.001
Significantly Different from HCT 3012
NMI-1182 Naproxen ? FR vs. HCT 3012
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Effect of Orally Administered Compounds on
Peripheral Vascular Resistance (MBP/FR) in
Conscious Hypertensive SHR Stroke Prone Rats
Mean SEM p lt 0.001
Significantly Different from HCT 3012
NMI-1182, Naproxen and Vehicle ? PVR vs. HCT
3012,
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Summary In Vivo Pharmacology

• NMI-1182 acts as an anti-inflammatory agent in
  the rat carrageenan paw edema (NaproxengtHCT
  3012gtNMI-1182) and air pouch model
  (NaproxenNMI-1182gtHCT 3012).
• NMI-1182 exhibits GI tolerance comparable to HCT
  3012 (NMI-1182HCT 3012gtgtNaproxen).
• 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

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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

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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.

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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
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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
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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
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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
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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.

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Summary Biological Properties of NMI-1182 and
HCT 3012
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Schematic of NMI-1182 and HCT 3012
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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

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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 RD Gordon Letts