Patent Application: US-201113006984-A

Abstract:
the invention concerns in one embodiment a method for treating glaucoma or elevated iop in a patient comprising administering to the patient an effective amount of a composition comprising an agent that inhibits pai - 1 expression or pai - 1 activity . another embodiment of the present invention is a method of treating a pai - 1 - associated ocular disorder in a subject in need , comprising administering to the patient an effective amount of a composition comprising an agent that inhibits pai - 1 activity or expression .

Description:
certain embodiments of the present invention are methods for targeting the effects of pai - 1 in ocular disorders such as glaucoma by interfering with pai - 1 activity relative to t - pa and u - pa and / or pai - 1 expression as shown in the following scheme , where tgfβ2 ( or other stimuli ) promotes pai - 1 gene transcription , followed by an increase in pai - 1 protein expression and increased levels of active pai - 1 . active pai - 1 inhibits conversion of plasminogen into plasmin by t - pa and / or u - pa . the subsequent decrease in plasmin levels reduces fibrinolytic capacity and increases extracellular matrix ( ecm ) accumulation . ecm accumulation increases outflow resistance and , ultimately , increases iop . embodiments of the present invention recognize that inhibition of pai - 1 expression and / or interfering with pai - 1 activity relative to t - pa and / or u - pa is a useful glaucoma therapy . various compounds that inhibit pai - 1 expression or activity are known in the art . u . s . patent application ser . no . 11 / 611 , 312 ( fleenor et al ., filed dec . 15 , 2006 and published as u . s . patent publication no . 2008 / 0107644 ) and u . s . pat . no . 7 , 351 , 407 ( fleenor et al , issued apr . 1 , 2008 ) disclose compounds that may be useful as compounds that inhibit pai - 1 expression or activity , and are hereby incorporated by reference in their entirety . the pai - 1 inhibitors of the present invention include , but are not limited to pai - 039 ( tiplaxtinin ) ( crandall et al ., arterioscler thrombosis vascular biology journal , vol . 26 ( 10 ): 2209 - 2215 , 2006 ); pai - 749 ( diaplasinin ) ( gardell et al ., molecular pharmacology , vol . 72 ( 4 ): 897 - 906 , 2007 ); zk4044 ( liang et al ., thrombosis research , vol . 115 ( 4 ): 341 - 350 , 2005 ); way - 140312 ( crandall et al ., journal thrombosis haemostasis , vol . 2 ( 8 ): 1422 - 1428 , 2004 ); hp - 129 ( fendosal ) ( gils et al ., thrombosis haemostasis , vol . 88 ( 1 ): 137 - 143 , 2002 ); t - 686 ( murakami et al ., japanese journal of pharmacology , vol . 75 ( 3 ): 291 - 294 , 1997 ); paz - 417 ( aleplasinin ) ( zhao et al ., cell research , vol . 18 : 803 - 804 , 2008 ); t - 2639 ( miyazaki et al ., biorganic & amp ; medicinal chemistry letters , vol . 18 : 6419 - 6422 , 2008 ); s - 35225 ( rupin et al ., thrombosis research , vol . 122 : 265 - 270 , 2008 ; sk - 216 & amp ; sk - 116 ( mutoh et al ., carcinogenesis , vol . 29 ( 4 ): 824 - 829 , 2008 ); and 2 -[ 2 - methoxy - 6 -[[[ 3 -( trifluoromethyl )- 4 -[ 4 -[ 3 -( trifluoromethyl ) phenyl ]- 1 - piperazinyl ] phenyl ] amino ] methyl ] phenoxy ]- 5 - nitrobenzoic acid (“ compound 39 ”; ( ye et al ., bioorganic & amp ; medicinal chemistry letters , vol . 14 ( 3 ): 761 - 765 , 2004 ). other small molecules such as piperazine and menthol derivatives ( ye et al ., bioorganic & amp ; medicinal chemistry letters , vol . 14 ( 3 ): 761 - 765 , 2004 ; ye et al ., biorganic & amp ; medicinal chemistry letters , vol . 13 ( 19 ): 3361 - 3365 , 2003 ), pai - 1 antibodies ( verbeke et al ., journal of thrombosis and haemostasis , vol . 2 ( 2 ): 289 - 297 , 2004 ; van giezen et al ., thrombosis and haemostasis , vol . 77 ( 5 ): 964 - 969 , 1997 ; and abrahamsson et al ., thrombosis and haemostasis , vol . 75 ( 1 ): 118 - 126 , 1996 ), and protein agents such as paionin - 4 ( mathiasen et al ., molecular pharmacology , vol . 74 ( 3 ): 641 - 653 , 2008 ) may also be used as compounds that inhibit pai - 1 expression or activity in certain embodiments of the present invention . other embodiments may use agents such as sb202190 , hp - 129 , u0126 , sp600125 , bisindolylmaleimide i , rottlerin , sb431542 and sis3 . statin agents such as lovastatin , simvastatin , pravastatin , fluvastatin , atorvastatin may be used as agents in yet other embodiments . preferred compounds that inhibit pai - 1 expression or activity are tiplaxtinin , diaplasinin , compound 39 and t - 2639 . the compounds that inhibit pai - 1 expression or activity of the present invention can be incorporated into various types of ophthalmic formulations for delivery . the compounds may be delivered directly to the eye ( for example : topical ocular drops or ointments ; slow release devices such as pharmaceutical drug delivery sponges implanted in the cul - de - sac or implanted adjacent to the sclera or within the eye ; periocular , conjunctival , sub - tenons , intracameral , intravitreal , or intracanalicular injections ) or systemically ( for example : orally , intravenous , subcutaneous or intramuscular injections ; parenteral , dermal or nasal delivery ) using techniques well known by those of ordinary skill in the art . it is further contemplated that the pai - 1 expression or activity inhibitors of the invention may be formulated in intraocular inserts or implantable devices . the pai - 1 expression or activity inhibitors disclosed herein are preferably incorporated into topical ophthalmic formulations for delivery to the eye . the compounds may be combined with ophthalmologically acceptable preservatives , surfactants , viscosity enhancers , penetration enhancers , buffers , sodium chloride , and water to form an aqueous , sterile ophthalmic suspension or solution . ophthalmic solution formulations may be prepared by dissolving a compound in a physiologically acceptable isotonic aqueous buffer . further , the ophthalmic solution may include an ophthalmologically acceptable surfactant to assist in dissolving the compound . furthermore , the ophthalmic solution may contain an agent to increase viscosity such as hydroxymethylcellulose , hydroxyethylcellulose , hydroxypropylmethylcellulose , methylcellulose , polyvinylpyrrolidone , or the like , to improve the retention of the formulation in the conjunctival sac . gelling agents can also be used , including , but not limited to , gellan and xanthan gum . in order to prepare sterile ophthalmic ointment formulations , the active ingredient is combined with a preservative in an appropriate vehicle such as mineral oil , liquid lanolin , or white petrolatum . sterile ophthalmic gel formulations may be prepared by suspending the compound in a hydrophilic base prepared from the combination of , for example , carbopol - 974 , or the like , according to the published formulations for analogous ophthalmic preparations ; preservatives and tonicity agents can be incorporated . pai - 1 expression or activity inhibitors are preferably formulated as topical ophthalmic suspensions or solutions , with a ph of about 4 to 8 . the compounds are contained in the topical suspensions or solutions in amounts sufficient to lower iop in patients experiencing elevated iop and / or maintaining normal iop levels in glaucoma patients . such amounts are referred to herein as “ an amount effective to control iop ,” or more simply “ an effective amount .” the compounds will normally be contained in these formulations in an amount 0 . 01 to 5 percent by weight / volume (“ w / v %”), but preferably in an amount of 0 . 25 to 2 w / v %. thus , for topical presentation 1 to 2 drops of these formulations would be delivered to the surface of the eye 1 to 4 times per day , according to the discretion of a skilled clinician . the pai - 1 expression or activity inhibitors may also be used in combination with other elevated iop or glaucoma treatment agents , such as , but not limited to , rho kinase inhibitors , β - blockers , prostaglandin analogs , carbonic anhydrase inhibitors , α 2 agonists , miotics , serotonergic agonists and neuroprotectants . as used herein , “ pai - 1 expression or activity inhibitor ” encompasses such inhibitors as well as their pharmaceutically - acceptable salts . a pharmaceutically acceptable salt of a pai - 1 expression or activity inhibitor is a salt that retains pai - 1 expression or activity inhibitory activity and is acceptable by the human body . salts may be acid or base salts since agents herein may have amino or carboxy substituents . a salt may be formed with an acid such as acetic acid , benzoic acid , cinnamic acid , citric acid , ethanesulfonic acid , fumaric acid , glycolic acid , hydrobromic acid , hydrochloric acid , maleic acid , malonic acid , mandelic acid , methanesulfonic acid , nitric acid , oxalic acid , phosphoric acid , propionic acid , pyruvic acid , salicylic acid , succinic acid , sulfuric acid , tartaric acid , p - toluenesulfonic acid , trifluoroacetic acid , and the like . a salt may be formed with a base such as a primary , secondary , or tertiary amine , aluminum , ammonium , calcium , copper , iron , lithium , magnesium , manganese , potassium , sodium , zinc , and the like . the following examples are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . pai - 1 expression or activity inhibitors can be selected using binding assays or functional assays that can also be used to determine their biological activity . such assays can be developed by those of skill in the art using previously described methods . other useful assays for selecting pai - 1 expression or activity inhibitors are presented in examples 2 - 5 . the ability of certain pai - 1 expression or activity inhibitors to safely lower iop may be evaluated by means of in vivo assays . in one such assay using cynomolgus monkeys , iop is determined with an alcon pneumatonometer after light corneal anesthesia with 0 . 1 % proparacaine . ( sharif et al ., journal ocular pharmacology & amp ; therapeutics , vol . 17 ( 4 ): 305 - 317 , 2001 ; may et al ., journal of pharmacology & amp ; experimental therapeutics , vol . 306 ( 1 ): 301 - 309 , 2003 ). eyes are rinsed with one or two drops of saline after each measurement . after a baseline iop measurement , test compound is instilled in one or two 30 μl aliquots to the selected eye . subsequent iop measurements are taken at 1 , 3 , and 6 hours . right eyes of all animals undergo laser trabeculoplasty to induce ocular hypertension . all left eyes are normal and thus have normal iop . in another assay using new zealand albino rabbits , iop is determined with a mentor classic 30 pneumatonometer after light corneal anesthesia with 0 . 1 % proparacaine . eyes are rinsed with one or two drops of saline after each measurement . after a baseline iop measurement , test compound is instilled in one 30 μl aliquot to one or both eye of each animal or compound to one eye and vehicle to the contralateral eye . subsequent iop measurements are taken at 0 . 5 , 1 , 2 , 3 , 4 , and 5 hours . human tm cells were isolated from post - mortem human donor tissue , characterized , and cultured as previously described . generation and characterization of the transformed ( gtm - 3 ) cell line was as previously described by pang et al . ( current eye research , vol . 13 ( 1 ): 51 - 63 , 1994 ). 24 - well plates of tm cell cultures were serum - deprived for 24 h followed by an additional 24 h ( or as indicated ) incubation with tgfβ2 in a serum - free medium . aliquots of supernatants from the treated cultures were quantified for secreted pai - 1 content by means of human pai - 1 elisa kit ( imubind ; american diagnostica inc ., greenwich , conn .). the elisa detects both latent and active pai - 1 , as well as pai - 1 complexes , with a minimum detectable limit of 50 pg / ml . fig1 is a graph showing that tgfβ2 increases the pai - 1 content in trabecular meshwork cell cultures ( gtm - 3 ). pai - 1 mediated effects may contribute to the previously observed tgfβ2 - mediated accumulation of extracellular matrix materials in various tissues , including tm tissues . fig2 demonstrates that such tgfβ2 - mediated pai - 1 increases are persistent in cell cultures treated with tgfβ2 . accordingly , tgfβ2 - treatment appears to result in both concentration - dependent and time - dependent accumulation of pai - 1 in tm cell supernatants . pai - 1 levels increase gradually in response to tgfβ2 , reaching a constant level at approximately 24 h post - treatment . transforming growth factor - beta ( tgfβ3 ) regulates the production of a wide variety of gene and protein products and , thus , multiple cellular processes . studies have shown that ex vivo treatment of human trabecular meshwork ( htm ) cells with the tgfβ2 isoform leads to changes in expression of plasminogen activator inhibitor - 1 ( pai - 1 ), an important mediator that likely contributes to ocular extracellular matrix ( ecm ) accumulation . a disproportionate accretion of ecm in the tm region may impart greater resistance to aqueous humor ( ah ) outflow and , consequently , increased intraocular pressure , such as seen in primary open angle glaucoma . additionally , levels of both tgfβ2 and pai - 1 are greater in ah collected from human poag eyes as compared to non - glaucomatous eyes . furthermore , ex vivo human anterior segments respond with decreases in outflow facility when perfused with tgfβ2 . in these studies , human tm cells were isolated , characterized , and cultured as previously described ( pang et al ., current eye research , vol . 13 ( 1 ): 51 - 63 , 1994 ). for these assays , plated cells were serum - deprived for 24 h followed by additional 24 h incubation with test agents in a serum - free medium . aliquots of supernatants from treated cultures were then removed for quantification of total pai - 1 content by means of a human pai - 1 elisa kit ( imubind ; american diagnostica inc ., greenwich , conn .). the elisa detects both latent and active pai - 1 , as well as pai - 1 complexes , with a minimum detectable limit of 50 pg / ml . active pai - 1 content in the cell supernatants was evaluated with an elisa kit ( molecular innovations , southfield , mich .) that quantifies binding of active pai - 1 to urokinase . latent and complexed pai - 1 does not bind urokinase and thus is not detected by the assay . the expected detection limit of the assay is approximately 0 . 045 u / ml ( where 1 unit equals approximately 1 . 34 ng active pai - 1 ). fig3 - 9 present the results of in vitro experiments conducted using the above protocols . the average basal pai - 1 secretion by gtm - 3 cells in the studies was 33 . 9 ± 1 . 5 ng / ml / 24 h ( n = 233 ). tgfβ2 increased pai - 1 content of gtm - 3 cell supernatants in a time and dose - dependent manner . a 24 h treatment with 5 ng / ml tgfβ2 elevated pai - 1 levels by 12 . 02 ± 0 . 03 fold . htm cell pai - 1 total protein levels are upregulated in vitro by factors ( tgfβ2 , tnfα , dexamethasone ) linked to increased intraocular pressure . active pai - 1 levels are also increased by tgfβ2 ( fig3 ). fig4 shows that tiplaxtinin reduces active pai - 1 levels in gtm - 3 cultures treated with tgfβ2 . tgfβ2 - stimulated pai - 1 levels were significantly ( p & lt ; 0 . 05 ) down - regulated by inhibitors of both canonical ( smad - mediated ) and non - canonical ( smad - independent ) signal transduction pathways . fig5 is a graph showing the effects of a pai - 1 synthesis inhibitor ( t - 2639 ) on the tgfβ2 - mediated increase of total pai - 1 protein levels in supernatants of treated gtm - 3 cell cultures . inhibitors of tgfβ2 - mediated canonical ( smad ) signaling pathways ( sb431542 and sis3 ) block the in vitro expression of total pai - 1 in human trabecular meshwork ( htm ) cell cultures ( fig6 and 7 ). inhibitors of tgfβ2 - mediated non - canonical ( smad - independent ) signaling pathways ( sb202190 , u0126 , sp600125 , bisindolylmaleimide i , and rottlerin ) also prevent in vitro expression of total pai - 1 in htm cell cultures . such signaling pathways identified to date include p38 mapk , mek1 / 2 , jnk , and pkc { tilde over ( δ )} ( fig8 ). treatment with statin agents also decreases in vitro expression of total pai - 1 in htm cell cultures . ( fig9 ). overall response varied from complete inhibition by agents such as sb431542 ( tgfβ type 1 receptor inhibitor ; 1 μm ) and rottlerin ( pkcδ inhibitor ; 10 μm ) to partial inhibition by sb202190 ( p38 mapk inhibitor ; 100 nm ), sp600125 ( c - jun n - terminal kinase inhibitor ; 1 μm ), and various statin agents . a study was conducted to evaluate the effect of the compounds of the present invention on extracellular matrix clearance . human tm cells are treated for 24 h in the presence or absence of tiplaxtinin , diaplasinin , and compound 39 . cell supernatant aliquots are then incubated for 2 h with irdye 800rs - labeled casein ( li - cor biosciences ), followed by detection of accumulated fluorescent degradation products with an odyssey infrared imaging system ( li - cor biosciences ). fig1 shows that tiplaxtinin , diaplasinin , and compound 39 elicited demonstrable increases over basal ( no treatment ) activity in supernatant aliquots from each of six different htm cell lines . accordingly , treatment with these compounds enhances the degradation of matrix protein by trabecular meshwork cells . to evaluate the in vivo effects of the compounds of the present invention , a mouse model was used . one eye each of balb / cj mice was injected intravitreally with either ad5 . cmv . hpai - 1 or ad . cmv . htgfβ2 226 / 228 . un - injected contralateral eyes served as controls . iop was measured in conscious animals at selected time points via rebound tonometer ( tonolab ®). test agents were administered via daily topical dosing ( bid ) during the time frames indicated on the graphs . fig1 presents two graphs of experimental results showing the effect of compounds ( tiplaxtinin and diaplasinin ) that inhibit the inhibitory activity of pai - 1 on t - pa and u - pa . the compounds almost completely reverse ad . tgfβ2 - induced increase in intraocular pressure in balb / c mice . iop reduction was achieved by both pre - and post - dosing of pai - 1 inhibitors , with respect to adv . tgfβ2 - injection . fig1 presents two graphs of experimental results showing the effect of two compounds ( tiplaxtinin and diaplasinin ) that prevent the inhibitory activity of pai - 1 on t - pa and u - pa . both agents reduced the ad . pai - 1 induced increase in intraocular pressure in balb / cj mice . the present invention and its embodiments have been described in detail . however , the scope of the present invention is not intended to be limited to the particular embodiments of any process , manufacture , composition of matter , compounds , means , methods , and / or steps described in the specification . various modifications , substitutions , and variations can be made to the disclosed material without departing from the spirit and / or essential characteristics of the present invention . accordingly , one of ordinary skill in the art will readily appreciate from the disclosure that later modifications , substitutions , and / or variations performing substantially the same function or achieving substantially the same result as embodiments described herein may be utilized according to such related embodiments of the present invention . thus , the following claims are intended to encompass within their scope modifications , substitutions , and variations to processes , manufactures , compositions of matter , compounds , means , methods , and / or steps disclosed herein . the following references are hereby incorporated by reference in their entirety : abrahamsson et al ., “ anti - 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