Retinal → 11-cis-Retinol

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A method for determining the effect of a compound on isomerase activity may be performed in vitro as described herein and in the art (Stecher et al., J Biol Chem 274:8577-85 (1999); see also Golczak et al., Proc. Natl. Acad. Sci. USA 102:8162-67 (2005)). Retinal pigment epithelium (RPE) microsome membranes isolated from an animal (such as bovine, porcine, human, for example) may serve as the source of the isomerase. The capability of the alkynyl phenyl-linked amine derivative compounds to inhibit isomerase may also be determined by an in vivo murine isomerase assay. Brief exposure of the eye to intense light (“photobleaching” of the visual pigment or simply “bleaching”) is known to photo-isomerize almost all 11-cis-retinal in the retina. The recovery of 11-cis-retinal after bleaching can be used to estimate the activity of isomerase in vivo (see, e.g., Maeda et al., J. Neurochem 85:944-956 (2003); Van Hooser et al., J Biol Chem 277:19173-82, 2002). Electroretinographic (ERG) recording may be performed as previously described (Haeseleer et al., Nat. Neurosci. 7:1079-87 (2004); Sugitomo et al., J. Toxicol. Sci. 22 Suppl 2:315-25 (1997); Keating et al., Documenta Opthalmologica 100:77-92 (2000)). See also Deigner et al., Science, 244: 968-971 (1989); Gollapalli et al., Biochim Biophys Acta. 1651: 93-101 (2003); Parish, et al., Proc. Natl. Acad. Sci. USA 95:14609-13 (1998); Radu, et al., Proc Natl Acad Sci USA 101: 5928-33 (2004)). In certain embodiments, compounds that are useful for treating a subject who has or who is at risk of developing any one of the ophthalmic and retinal diseases or disorders described herein have IC50 levels (compound concentration at which 50% of isomerase activity is inhibited) as measured in the isomerase assays described herein or known in the art that is less than about 1 μM; in other embodiments, the determined IC50 level is less than about 10 nM; in other embodiments, the determined IC50 level is less than about 50 nM; in certain other embodiments, the determined IC50 level is less than about 100 nM; in other certain embodiments, the determined IC50 level is less than about 10 μM; in other embodiments, the determined IC50 level is less than about 50 μM; in other certain embodiments, the determined IC50 level is less than about 100 μM or about 500 μM; in other embodiments, the determined IC50 level is between about 1 μM and 10 μM; in other embodiments, the determined IC50 level is between about 1 nM and 10 nM. When adminstered into a subject, one or more compounds of the present invention exhibits an ED50 value of about 5 mg/kg, 5 mg/kg or less as ascertained by inhibition of an isomerase reaction that results in production of 11-cis retinol. In some embodiments, the compounds of the present invention have ED50 values of about 1 mg/kg when administered into a subject. In other embodiments, the compounds of the present invention have ED50 values of about 0.1 mg/kg when administered into a subject. The ED50 values can be measured after about 2 hours, 4 hours, 6 hours, 8 hours or longer upon administering a subject compound or a pharmaceutical composition thereof.

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