A Novel 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), Reduces Murine Retinal Cell Death In Vitro and In Vivo

Abstract

We examined whether ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), a new orally available calpain inhibitor, might reduce retinal cell death in vivo and/or in vitro. Retinal cell damage was induced in vivo in mice by intravitreal injection of N-methyl-D-aspartate (NMDA), and SNJ-1945 was intraperitoneally or orally administered twice. NMDA-induced calpain activity (measured as the cleaved products of ␣-spectrin) and its substrate, p35 (a neuron-specific activator for cyclin-dependent kinase 5), in the retina were examined by immunoblotting. In RGC-5 (a rat retinal ganglion cell line) cell culture, cell damage was induced by a 4-h oxygenglucose deprivation (OGD) treatment followed by an 18-h reoxygenation period. In mouse retinas, SNJ-1945 (30 or 100 mg/kg i.p., 100 or 200 mg/kg p.o.) significantly inhibited the cell loss in the ganglion cell layer (GCL) and the thinning of the inner plexiform layer induced by NMDA. Furthermore, the number of positive cells for terminal deoxynucleotidyl transferase dUTP nick-end labeling was significantly reduced in the GCL and the inner nuclear layer of retinas treated with SNJ-1945 compared with vehicle-treated retinas 24 h after NMDA injection. Levels of cleaved ␣-spectrin products increased and p35 decreased 6 h after NMDA injection or later, and their effects were attenuated by SNJ-1945. In vitro, SNJ-1945 (10 and 100 M) inhibited the OGD stress-induced reduction in cell viability. In conclusion, SNJ-1945 may afford valuable neuroprotection against retinal diseases, because it was effective against retinal damage both in vitro and in vivo. Our results also indicate that calpain activation and subsequent p35 degradation may be involved in the mechanisms underlying retinal cell death.Retinal ganglion cell (RGC) death is a common feature of many ophthalmic disorders, including glaucoma, optic neuropathies, and various retinovascular diseases (diabetic retinopathy and retinal vein occlusions). Retinal ganglion cells are extremely sensitive to the effects of both glutamate and its analog N-methyl-D-aspartate (NMDA), both of which produce a dosedependent cell loss in vivo and in vitro. In addition, glutamate toxicity has been implicated in the pathophysiology of glaucoma (Dreyer, 1998). Activation of Ca 2ϩ -permeable NMDA receptors and the subsequent neuronal Ca 2ϩ overloading have been shown to mediate glutamate-induced neuron death (Choi, 1994). Induction of the Ca 2ϩ -activated neutral cysteine protease -calpain by micromolar concentrations of Ca 2ϩ occurs early in excitotoxic neuron death (Siman et al., 1989;Faddis et al., 1997). Similar to the caspases, -calpain cleaves a variety of cytoskeletal proteins, enzymes, and transcription factors and could also interfere with the proteolytic activity of the caspases (Croall and DeMartino, 1991).

Calpain inhibition stabilizes the platelet proteome and reactivity in diabetes

“…Calpain inhibition may also underlie platelet activation in other conditions, because many of the proteins identified as calpain substrates in diabetic humans and mice (including ILK) were also reported to be altered in platelets from patients with acute coronary syndromes. 41,42 Although in the present study, we found no significant effects on platelet number or function in otherwise healthy mice after 12 days of treatment, other studies indicate that the long-term treatment with A-705253 is well tolerated and can protect against retinal damage 43 and penile nitrergic nerve and dysfunction in diabetic mice. 44 Other calpain inhibitors attenuate angiotensin II-induced abdominal aortic aneurysms and atherosclerosis in LDL receptordeficient mice 45 and angiotensin II-induced endothelial dysfunction in rats and mice.…”

Effect of Nafamostat on <i>N</i>-Methyl-<small>D</small>-aspartate-Induced Retinal Neuronal and Capillary Degeneration in Rats

“…6,7) In addition, upregulation of pro-inflammatory cytokines and inflammatory adhesion molecules, 8,9) increase in matrix metalloproteinase (MMP) activity, [10][11][12] and recruitment of leukocytes into the retina 8) are also involved. These events precede the occurrence of retinal ganglion cell apoptosis, and preventive interventions have been shown to ameliorate NMDA-induced retinal ganglion cell death.…”

Calpain Inhibitor Protects Cells against Light-Induced Retinal Degeneration

“…Accumulation of hyperphosphorylated protein causes neurofibrillary tangles and neuronal cell death. In retina, p35 was proteolyzed to p25 by several factors, such as hypoxia, ocular hypertension, photoreceptor cell death, and ganglion cell death (Tamada et al, 2005;Oka et al, 2006Oka et al, , 2007Shimazawa et al, 2010). In the present study, p35 was decreased 48 h after light exposure, and SNJ-1945 treatment (200 mg/kg p.o.…”

“…SNJ-1945 has more favorable retinal pharmacokinetics, such as good retinal penetration, high oral bioavailability, and long half-life (Shirasaki et al, 2006). We have reported previously that SNJ-1945 has a protective effect against cerebral ischemia-induced neuronal cell death (Koumura et al, 2008) and N-methyl-D-aspartate-induced retinal cell death (Shimazawa et al, 2010). It is noteworthy that SNJ-1945 showed protective effects by oral administration in these models.…”