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.
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 alpha-spectrin was analyzed by immunoblotting.


RESULTS
Elevated IOP caused retinal degeneration and proteolysis of alpha-spectrin. Both i.p. and oral administration of SNJ-1945 inhibited proteolysis of alpha-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 alpha-spectrin due to calpains in rat, monkey, and human samples. SNJ-1945 similarly inhibited proteolysis in all species.


CONCLUSIONS
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.
Oka
T
T
Kobe Creative Center, Senju Pharmaceutical Co., Ltd., 1-5-4 Murotani, Nishiku, Kobe, Hyogo 651-2241, Japan.
Walkup
R D
RD
Tamada
Y
Y
Nakajima
E
E
Tochigi
A
A
Shearer
T R
TR
Azuma
M
M
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002154:Calpain / Q000235:genetics / Q000378:metabolism; D002219:Carbamates / Q000008:administration & dosage; D004195:Disease Models, Animal; D004333:Drug Administration Routes; D006023:Glycoproteins / Q000008:administration & dosage*; D000882:Haplorhini; D006801:Humans; D007429:Intraocular Pressure / Q000187:drug effects; D009798:Ocular Hypertension / Q000150:complications / Q000188:drug therapy / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012162:Retinal Degeneration / Q000188:drug therapy* / Q000209:etiology / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors
C515164:((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester; D002135:Calcium-Binding Proteins; D002219:Carbamates; D006023:Glycoproteins; D012333:RNA, Messenger; C039304:calpain inhibitors; C033668:calpastatin; D002154:Calpain