Preliminary studies conducted by the principal investigator indicate that intravitreal injection of the decapeptide hormone, luteinizing hormone releasing hormone (LHRH), into test eyes of rabbits produces a marked lowering of intraocular pressure (IOP) while other ocular properties appear unchanged. Intravitreal injection of structural analogs of LHRH known to possess little or no activity in the glycoprotein hormone releasing assays for LHRH did not lower IOP suggesting that LHRH is producing a true biological effect with structural specificity required. The long term goal of this project is to obtain a superagonist of LHRH which effectively treats glaucoma.
The specific aim of this proposal is to demonstrate that LHRH or an LHRH-like peptide is a modulator of IOP by (1) ascertaining that high affinity receptors for an analog of the hormone are found in ciliary processes and/or ciliary epithelia and (2) isolating LHRH or LHRH-like material from extracts of the same tissue or cells or from aqueous humor. Radioreceptor assays will be performed using I125-[D-Ala6-desGly10]-LHRH ethylamide, as that analog is less likely to be inactivated and binds more readily to high affinity sites than LHRH itself. Incubation mixtures will include plasma membrane proteins, a fixed amount of radiolabeled analog and varying amounts of the same unlabeled analog. Bound hormone will be separated from free hormone by passage through glass fiber filters and corrections will be made for non-specific binding. LHRH and LHRH-peptides will be isolated from biological sources by passing acid extracts of tissue or acidified aqueous humor through cartridges of c18-silica. LHRH-like peptides will bind to the cartridges. Contaminants will be removed by washing the cartridges and the peptides will be eluted with a mixture of aqueous acid and acetonitrile. Final separation and quantitation will be performed by high performance liquid chromatography with sensitive fluorometric and immunological detection. Attempts will be made to achieve the long range goal by preparing and testing in rabbits superagonists or modified derivatives of LHRH. The latter might serve as prodrugs, if the chemical modifications are removable enzymatically in situ.
