Elsevier

Neuroscience

Volume 141, Issue 4, 2006, Pages 2139-2145
Neuroscience

Sensory system
Amelioration of retinal degeneration and proteolysis in acute ocular hypertensive rats by calpain inhibitor ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester

https://doi.org/10.1016/j.neuroscience.2006.05.060Get rights and content

Abstract

Background

Our recent study suggested involvement of calpain-induced proteolysis in retinal degeneration and dysfunction in acute ocular hypertensive rats. The purpose of the present study was to determine if an orally available form of calpain inhibitor, ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), ameliorated retinal degeneration induced by acute hypertension in rats. To help extrapolate the effect of SNJ-1945 from the rat model to the human glaucomatous patient, in vitro inhibition of calpain-induced proteolysis by SNJ-1945 in monkey and human retinal proteins was compared with proteolysis in rat proteins.

Methods

Intraocular pressure (IOP) in rats was elevated to 110 mm Hg for 50 min. SNJ-1945 was administrated i.p. or orally before ocular hypertension. Retinal degeneration was evaluated by hematoxylin and eosin (H&E) staining and cell counting. Transcripts for calpains and calpastatin in rat, monkey, and human retinas were measured by quantitative RT-PCR. Calpain activities were determined by casein zymography. Soluble retinal proteins from rat, monkey, and humans were incubated with calcium to activate calpains, with or without SNJ-1945. Proteolysis of calpain substrate α-spectrin was analyzed by immunoblotting.

Results

Elevated IOP caused retinal degeneration and proteolysis of α-spectrin. Both i.p. and oral administration of SNJ-1945 inhibited proteolysis of α-spectrin and ameliorated retinal degeneration. Transcript levels for calpain 1 and calpastatin were similar in rat, monkey, and human retinas. Calpain 2 transcript levels were higher in rats compared with monkey and human. Appreciable caseinolytic activities due to calpains were observed in monkey and human retinas. Incubation of retinal soluble proteins with calcium led to proteolysis of α-spectrin due to calpains in rat, monkey, and human samples. SNJ-1945 similarly inhibited proteolysis in all species.

Conclusion

Our results suggested that orally available calpain inhibitor SNJ-1945 might be a possible candidate drug for testing in preventing progression of glaucomatous retinal degeneration.

Section snippets

Experimental animals and eyes from human and monkey

Sprague–Dawley rats at 11–12 weeks of age were obtained from Charles River (Yokohama, Japan) and Harlan (Indianapolis, IN, USA). Rhesus monkey (Macaca mulatta) eyes ranging in age from 7 to 15 years were obtained from the Oregon National Primate Research Center (Beaverton, OR, USA) from procedures unrelated to the present studies. Experimental animals were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with the Guiding Principles in

Effect of SNJ-1945 in acute ocular hypertensive rats

H&E staining of retinal sections from rats revealed degeneration after ocular hypertension. The thickness of inner plexiform layer (IPL) and the inner nuclear layer (INL) decreased 7 days after ocular hypertension as compared with sham-operated animals (Fig. 1A). A single i.p. (Fig. 1A) or oral (data not shown) administration of SNJ-1945 helped protect against decreased retinal thickness. The number of cells in GCL was also counted, since glaucoma is associated with the degeneration of RGCs (

Discussion

The major finding of the present study was that a new calpain inhibitor SNJ-1945 with enhanced oral availability ameliorated retinal degeneration in rats suffering from acute ocular hypertension. These are the first data showing that a calpain inhibitor ameliorated retinal degeneration in acute hypertensive rats. The dipeptidyl aldehyde inhibitor SJA6017, N-(4-fluorophenylsulfonyl)-l-valyl-l-leucinal, first showed effectiveness against cataract formation in cultured lenses (Fukiage et al., 1997

Acknowledgments

Dr. Shearer has a significant financial interest (research contract and consulting fee) in Senju Pharmaceutical Co., Ltd. and Dr. Azuma is an employee of Senju Pharmaceutical Co., Ltd., a company that may have a commercial interest in the results of this research and technology. This potential conflict was reviewed and a management plan approved by the OHSU Conflict of Interest in Research Committee was implemented. We thank Noriko Nada for technical assistance.

References (33)

  • A.K. Sharma et al.

    Calcium-induced calpain mediates apoptosis via caspase-3 in a mouse photoreceptor cell line

    J Biol Chem

    (2004)
  • Y. Shirasaki et al.

    Exploration of orally available calpain inhibitorspeptidyl α-ketoamides containing an amphiphile at P3 site

    Bioorg Med Chem

    (2005)
  • Y. Tamada et al.

    Involvement of calpain in hypoxia-induced damage in rat retina in vitro

    Comp Biochem Physiol

    (2002)
  • Y. Tamada et al.

    Proteolysis of neuronal cytoskeletal proteins by calpain contributes to rat retinal cell death induced by hypoxia

    Brain Res

    (2005)
  • A. Agar et al.

    Pressure related apoptosis in neuronal cell lines

    J Neurosci Res

    (2000)
  • A. Agar et al.

    In-vitro pressure induced apoptosis in retinal ganglion cell line shows graded response to simulated acute, chronic glaucoma and normal pressures

    Invest Ophthalmol Vis Sci Abstr

    (2002)
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