title: Evaluation of a novel calpain inhibitor as a treatment for cataract.
abstract: OBJECTIVE
  The aim of this study is to evaluate the therapeutic potential of a newly synthesized calpain inhibitor, CAT0059, using a naturally occurring in vivo sheep cataract model.
  
  
  METHODS
  The selectivity of CAT0059 was investigated by an in vitro protease assay. The efficacy of CAT0059 in preventing proteolysis of lens cytoskeletal proteins by calpain 2 was investigated using a lens-based cell-free method. The cytotoxicity and stability of CAT0059 in physiological conditions were examined using cultured sheep lenses. Protein binding of CAT0059 by ocular proteins was assessed and quantified by a modified high-performance liquid chromatography assay. CAT0059 was formulated in an eye drop solution and as an eye ointment. These were applied in vivo daily to one eye of the cataract lambs, over a 67- and 97-day trial period, respectively. The progression of cataracts in the treated and untreated eyes was assessed by an independent veterinary ophthalmologist using a slit-lamp microscope.
  
  
  RESULTS
  In vitro assays revealed that CAT0059 was selective for cysteine proteases and also protected lens cytoskeletal proteins from degradation. CAT0059 was stable in physiological conditions and non-toxic to the lens. Only 15% of CAT0059 is bound to proteins in the aqueous humour but >90% bound to lens homogenate. The 67-day CAT0059 eye drop treatment was not effective in slowing the rate of cataract development. However, application of CAT0059 in an eye ointment initially slowed cataract development compared with the untreated eye. This effect was temporary.
  
  
  CONCLUSIONS
  In vitro assays confirmed CAT0059 to be a potent calpain inhibitor. The two in vivo trials addressed the ability of CAT0059 to reach the lens and established its limitations as a therapeutic molecule for cataract treatment.
authors.affiliation: Department of Wine, Food and Molecular Biosciences, Lincoln University, 7647 Canterbury, New Zealand.
authors.forename: Hannah Y Y
authors.initials: HY
authors.lastname: Lee
authors.forename: James D
authors.initials: JD
authors.lastname: Morton
authors.forename: Lucinda J G
authors.initials: LJ
authors.lastname: Robertson
authors.forename: Joshua D
authors.initials: JD
authors.lastname: McDermott
authors.forename: Roy
authors.initials: R
authors.lastname: Bickerstaffe
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Abell
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Jones
authors.forename: Janna M
authors.initials: JM
authors.lastname: Mehrtens
authors.forename: James M
authors.initials: JM
authors.lastname: Coxon
mesh_terms: D000818:Animals; D002154:Calpain / Q000378:metabolism*; D002386:Cataract / Q000188:drug therapy* / Q000201:enzymology / Q000662:veterinary; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004151:Dipeptides / Q000627:therapeutic use*; D004195:Disease Models, Animal; D006023:Glycoproteins / Q000008:administration & dosage / Q000737:chemistry / Q000627:therapeutic use* / Q000633:toxicity; D006801:Humans; D008958:Models, Molecular; D009824:Ointments; D009883:Ophthalmic Solutions; D011758:Pyrroles / Q000627:therapeutic use*; D012756:Sheep; D012757:Sheep Diseases / Q000188:drug therapy
chemical_list: C541390:5-formyl-pyrrole-2-carboxylic acid (1-(1-formyl-3-methyl-butylcarbamoyl)-2-methyl-propyl)amide; D004151:Dipeptides; D006023:Glycoproteins; D009824:Ointments; D009883:Ophthalmic Solutions; D011758:Pyrroles; C039304:calpain inhibitors; D002154:Calpain; D003546:Cysteine Endopeptidases