Patent Application: US-44532099-A

Abstract:
a method of lowering intraocular pressure employs an upregulating agent that induces increased prostaglandin synthesis in the eye . the method of treatment entails administering to the eye of a mammal in need thereof a prostaglandin upregulating agent to increase endogenous prostaglandin synthesis and thereby effect a reduction in intraocular pressure . in a preferred embodiment the upregulating agent is il - 1 .

Description:
the present invention involves a method of treating the eyes of a mammal suffering from a glaucomatous condition , wherein the intraocular pressure ( iop ) of the eye is or is likely to become elevated above its “ normal ” state . the present method can be employed to reduce , or ameliorate elevated iop and to prevent or impede increases in normal iop , thereby controlling or slowing the progression of the disease . a method of the present invention comprises administering to an eye of the mammal a therapeutically effective amount of a prostaglandin upregulating agent . as used herein , the term “ upregulating agent ” means any agent which , directly or indirectly , induces increased synthesis of a prostaglandin in the eye to effect a reduction of intraocular pressure . the method of the present invention may be practiced with any agent which directly or indirectly upregulates prostaglandin synthesis in the eye . preferred among such upregulating agents are il - 1 beta ( genzyme , mass ., usa ), tgf - beta 1 or 2 ( oncogene research products , cambridge , mass ., usa ), levamisole ( flavine international , inc ., closter , n . j ., usa ), muramyl dipeptide ( mdp ) ( glycotech corporation , rockville , md ., usa ) ( see also u . s . pat . no . 4 , 235 , 771 , the disclosure of which is incorporated herein by reference ), and muramyl tripeptide ( mtp ). other ingredients which may be desirable to use in the ophthalmic preparations of the present invention include preservatives , co - solvents , and viscosity building agents . ophthalmic products are typically packaged in multidose form . preservatives are thus required to prevent microbial contamination during use . suitable preservatives include : benzalkonium chloride , thimerosal , chlorobutanol , methyl paraben , propyl paraben , phenylethyl alcohol , edetate disodium , sorbic acid , onamer m , or other agents known to those skilled in the art . such preservatives are typically employed at a level between about 0 . 001 % and about 1 . 0 % by weight . some upregulating agents of the present invention may have limited solubility in water and therefore may require a surfactant or other appropriate co - solvent in the composition . such co - solvents include : polysorbate 20 , 60 and 80 ; pluronic f - 68 , f - 84 and p - 103 ; cremophore ® el ( polyoxyl 35 castor oil ) cyclodextrin ; or other agents known to those skilled in the art . such co - solvents are typically employed at a level between about 0 . 01 % and about 2 % by weight . viscosity greater than that of simple aqueous solutions may be desirable to increase ocular absorption of the active compound , to decrease variability in dispensing the formulations , to decrease physical separation of components of a suspension or emulsion of formulation and / or otherwise to improve the ophthalmic formulation . such viscosity building agents include , for example , polyvinyl alcohol , polyvinyl pyrrolidone , methyl cellulose , hydroxy propyl methyl cellulose , hydroxyethyl cellulose , carboxymethyl cellulose , hydroxy propyl cellulose , chondroitin sulfate and salts thereof , hyaluronic acid and salts thereof , and other agents known to those skilled in the art . such agents are typically employed at a level between about 0 . 01 % and about 2 % by weight . induction of prostglandin synthesis in human corneal fibroblasts by il - 1 alpha primary human corneal fibroblasts were grown in culture from freshly denuded corneal stromal tissue from a 55 - year old male donor . cells ( passage 4 ) were seeded into 12 - well plates and grown to 80 % confluency . selected cells were then exposed to interleukin - 1 alpha ( 10 ng / ml final ) in ham &# 39 ; s f - 10 nutrient mixture ( hyclone corporation ) containing 10 % fetal bovine serum by medium replacement in the wells . control cells received medium devoid of this inflammatory cytokine . cells were then incubated in a 37 ° c ., 5 % co 2 , humidified incubator for 24 hours . conditioned medium was then removed , centrifuged to remove any cellular debris ( 3 minutes at 2600 rpm and 4 ° c . ), and analyzed for pge 2 and pgf 2α , levels in the medium using specific enzyme immunoassay kits ( cayman scientific ). cells in each well were counted , and prostaglandin levels normalized to the cell count for the respective wells . the results of this study , detailed in table 1 , clearly indicate that the presence of il - 1 alpha stimulates a dramatic elevation of the levels of both pge 2 (& gt ; 47 fold ) and pgf 2α (& gt ; 3 fold ). these data support the premise of the present invention that cytokines like il - 1 alpha or others , including small synthetic molecules , which are known to act on cells through receptor mediated signal transduction , can lead to a significant elevation of extracellular levels of prostaglandins , especially pge 2 . primary human trabecular meshwork cells were grown in culture according to the method of weinreb , et al . [ 14 ] from an 18 - year old donor . cells ( passage 5 ) were seeded into 12 - well plates and grown until they just reached confluency , with culture medium replacement every 3 - 4 days . triplicate wells of cells were then exposed to interleukin - 1 beta ( 10 ng / ml final ), tgf - beta 1 ( 10 ng / ml final ) or both in 1 ml of ham &# 39 ; s f - 10 nutrient mixture ( gibco - brl ), containing 2 mm l - glutamine and 0 . 4 mg / ml bovine serum albumin ( sigma chemical co .). control cells received medium devoid of il - 1β and tgf - β1 . cells were then incubated in a 37 ° c ., 5 % co 2 , humidified incubator for 17 hours . 14 c - arachidonic acid ( 300 , 000 dpm ) was added to each well , and the cells were returned to the incubator for an additional 4 hours . a modified bligh & amp ; dyer lipid extraction [ can . j . biochem . physiol . 37 , 911 ( 1959 )] was then performed on the cells and supernatants . an aliquot of the extracted samples and fatty acid standards were spotted on a moderate hardness silica gel thin - layer chromatography plate ( alltech k5 , 20 × 20 cm , 250 μm thick ). the plate was developed in prostaglandin tlc solvent ( 11 : 5 : 2 : 10 ethyl acetate : 2 , 2 , 4 - trimethylpentane : acetic acid : distilled water ), air - dried , and standard bands were visualized with vaporization of iodine crystals . each lane on the plate was divided into segments based upon the position of the standards , and each segment was scraped off of the plate with a razor blade into a glass scintillation vial . opti - fluor scintillation cocktail ( packard ) was added to each vial , and the samples were counted on a beta scintillation counter . the amount of each fraction was calculated as a percentage of total counts per minute ( cpm ) from that sample . the results of this study , detailed in table 2 , show that both tgf - β1 and il - 1β increase pge 2 synthesis by cultured human trabecular meshwork cells . tgf - b1β increases pge 2 synthesis by 74 % over a non - treated control . moreover , il - 1β increases pge 2 synthesis by 440 % over the control . the combination of tgf - 1β and il - 1β unexpectedly yielded a total increase in pge 2 synthesis of 590 % over the control . the present invention has been described with reference to certain embodiments for purposes of clarity and understanding . it should be appreciated that various improvements and modifications can be practiced within the scope of the appended claims and equivalents . the pertinent disclosures of the following references are incorporated herein by reference . 1 . kimball , e . s ., schneider , c . r ., fisher , m . c . and clark , m . c ., levamisole causes differential cytokine expression by elicited mouse peritoneal macrophages . j leukoc biol ( 1992 ) 52 : 349 - 356 . 2 . kimball , e . s ., clark , m . c ., schneider , c . r . and persico , f . j ., enhancement of in vitro lipoplysaccharide - stimulated interleukin - 1 production by levamisole . clin immunol immunopathol ( 1991 ) 58 : 385 - 398 . 3 . medvedev , a . e ., fuks , b . b ., bovin , n . v . and zemliakov , a . e ., the immunomodulating activity of new muramyl dipeptide derivatives in vitro . biull eksp biol med ( 1992 ) 114 ( 12 ): 1838 - 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