Abstract:
Compounds of Formula 1, where the symbols are as defined in the specification, are useful as ocular hypotensive agents but do not exert their activity through the FP prostaglandin receptor.                            
     In particular the compound PGF 2α  1-ethanolamide, having the formula                            
     was discovered to be present in mammalian tissue as a naturally occurring substance, was synthesized in a substantially pure form and was found to be effective for lowering intraocular pressure in the mammalian eye, but not acting through the FP receptor through which many ocular hypotensive prostaglandins act.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to ocular hypotensive lipids. More particularly the present invention relates to PGF 2α  1-ethanolamide and related compounds, and to pharmaceutical compositions containing these compounds, as well as methods for using the compounds to lower intraocular pressure in a mammal. 
     2. Brief Description of the Prior Art 
     The prior art is well aware of numerous ocular hypotensive agents which are used to treat various ocular hypertensive conditions, including primary and secondary glaucoma which represent a serious human health problem. Drugs used for treating ocular hypertension (glaucoma) include β-adrenoreceptor antagonists, and various prostaglandins. There is a substantial volume of scientific and patent literature pertaining to prostaglandins and to their use in the treatment of ocular hypertension. See for example Bito L. Z.  Biological Protection with Prostaglandins  Cohen, M. M., ed., Boca Raton. Fla., CRC Press Inc., 1985, pp. 231-252; and Bito, L. Z.,  Applied Pharmacology in the Medical Treatment of Glaucomas  Drance, S. M. and Neufeld, A. H. eds., New York, Grune &amp; Stratton, 1984, pp. 477-505. 
     U.S. Pat. No. 5,288,754 includes further citations to specific prior art directed to prostaglandins and related derivatives which are active as agents for reducing intraocular pressure in a mammal. U.S. Pat. No. 5,288,754 itself, describes “Polar C-1 Esters of Prostaglandins”, including C-1 amides and C-1 substituted amides of the carboxylic acid compound known as PGF 2α . Additional prostaglandin derivatives related to PGF 2α  which have emerged in prior art research conducted in the research facilities of the corporate assignee of the present application and which show strong intraocular hypotensive activity, are shown below by formula and are identified by arbitrary numbers as “prior art compound no. 1” and “prior art compound no. 2”. Prior art compound no. 1 is described in U.S. Pat. Nos. 5,352,708; 5,607,978 and 5,688,819, and prior art compound no. 2 is described in U.S. Pat. No. 5,545,665.                           
     The vast majority of ocular hypotensive agents which have a prostaglandin (or closely) related structure act through known “prostglandin” receptors. Particularly, the compound PGF 2α  is known to exert its ocular hypotensive action through the receptor known as FP. By “FP receptor” is meant a human prostaglandin receptor as disclosed in Abramovitz et al.,  J. Biol. Chem.  269:2632 (1994), hereby incorporated by reference herein. The structure of PGF 2α , including the numbering customarily used in the nomenclature of prostaglandin and related compounds, is shown below.                           
     The present invention is directed to a class of compounds related to PGF 2α  1-ethanolamide, which suprisingly have been discovered, together with the prior art compound nos. 1 and 2, as agents having strong ocular hypotensive activity, but which do no exert their ocular hypotensive effects through the FP receptor, nor through any hitherto recognized prostaglandin receptor, such as DP, EP, EP 2 , EP 3 , EP 4 , FP, IP and TP. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to compounds of Formula 1 wherein the dashed lines represent absence of a bond, or a bond with the proviso that there are no two adjacent double bonds in the formula; 
     the wavy line attachments represent either alpha (α, down) or beta (β, up) configuration, where the wavy lines are attached to a double bond they represent either Z (cis) or E (trans) configuration; 
     the hatched lines indicate alpha (α) configuration and solid triangles indicate beta (β) configuration; 
     m is an integer having the values of 0 to 5; 
     n is an integer having the values 1-6, with the proviso that the compound represented by the formula is not PGF 2α  1-ethanolamide; 
     q and r each independently are integers having the value of 0 to 6; 
     X is CH 2 , O or S with the proviso that when X is O or S then the dashed line adjacent to X represents absence of a bond; 
     R is CH 3 , phenyl, furyl, thienyl, cycloalkyl of 3 to 8 carbons, or phenyl furyl or thienyl itself substituted with one or two substituents selected from the group consisting of F, Cl, Br, alkyl of 1 to 6 carbons, NO 2 , CN, COOH and COOalkyl where alkyl has 1 to 6 carbons; 
     R 1 , R 2 , R 3 , and R 4  each independently represent H, a straight or branch-chained alkanoyl group having 1 to 6 carbons, benzoyl or lower alkyl of 1 to 6 carbons; 
     R 5  is H or straight or branch-chained alkyl group having 1 to 6 carbons, and 
     R 6  is H or straight or branch-chained alkyl of 1 to 4 carbons or a pharmaceutically acceptable salt of said compound, and said compounds being active to lower intraocular pressure in the eye of a mammal but do not exert their ocular hypotensive activity through the FP prostaglandin receptor, nor through any hitherto recognized prostaglandin receptor.                           
     The present invention is also directed to isolated substantially pure PGF 2α  1-ethanolamide, which has surprisingly been discovered to be present in certain biological systems as a naturally occurring substance. Further, the present invention is directed to pharmaceutical compositions containing as an active ingredient PGF 2α  1-ethanolamide and/or one or more of the compounds in accordance with Formula 1, and to the process of treating a mammal, including a human, in need of such treatment with an effective amount of a pharmaceutical composition containing as active ingredient PGF 2α  1-ethanolamide and/or one or more of the compounds in accordance with Formula 1, for the purpose of lowering intraocular pressure. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a graph showing the effect of graded doses of PGF 2α  1-ethanolamide on feline iris sphincter smooth muscle. Points are mean values±SEM; n=4. 
     FIG. 2 is a graph showing the inhibitory effect of graded doses of PGF 2α  1-ethanolamide on field stimulated contraction of the isolated guinea pig vas deferens. Points are mean values±SEM; n=4. 
     FIG. 3 is a graph showing the effect of graded doses of PGF 2α  1-ethanolamide on the isolated chick ileum. Points are mean values±SEM; n=4. 
     FIG. 4 is a graph showing the effect of graded doses of PGF 2α  1-ethanolamide on the isolated guinea pig ileum. Points are mean values±SEM; n=4. 
     FIG. 5 is a graph showing the effect of graded doses of PGF 2α  1-ethanolamide on isolated rat aortic smooth muscle. Points are mean values±SEM; n=4. 
     FIG. 6 is a graph showing the increases in intracellular Ca 2+  concentration [Ca 2+ ] i  produced by graded doses of PGF 2α  (▪) and PGF 2α  1-ethanolamide () in CRL 1497 cells. Points are mean values±SEM; n=4. 
     FIG. 7 is a graph showing the vasorelaxation effects of Prostaglandin F 2α  1-ethanolamide () and Prostaglandin F 2α  (▪) on the histamine precontracted rabbit jugular vein segment. Points are mean values±SEM; n=6 for PGF 2α  1-ethanolamide, n=7 for PGF 2α . 
     FIG. 8 is a graph showing the increases in intracellular Ca 2+  concentration [Ca 2+ ] i  produced by graded doses of PGF 2α  (▪) and PGF 2α  1-ethanolamide () in HEK-293 cells stably transfected with the recombinant feline FP receptor. Points are mean values±SEM; n=3. 
     FIG. 9 is a graph showing the total inositol phosphate accumulation produced by graded doses of 17-phenyl prostaglandin F 2α  (▴) and prostaglandin F 2α  1-ethanolamide () in HEK-293 cells stably transfected with the recombinant feline FP receptor. Values are mean±SEM. N=3. 
     FIG. 10 is a graph showing the total inositol phosphate accumulation produced by graded doses of 17-phenyl prostaglandin F 2α  (▴) and prostaglandin F 2α  1-ethanolamide () in HEK-293 cells stably transfected with the recombinant human FP receptor. Values are mean±SEM. n=3. 
     FIG. 11 is a graph showing the radioligand binding competition studies vs. 5 nM  3 H-17-phenyl PGF 2α  using HEK-293 cells stably transfected with the recombinant feline FP receptor. Competition afforded by unlabeled PGF 2α  1-ethanolamide () and unlabelled 17-phenyl PGF 2α  (▴) is depicted. Points are mean values±SEM; n=3 of experiments performed in triplicate. 
     FIG. 12 is a graph showing the radioligand binding competition studies vs. 5 nM  3 H-17-phenyl PGF 2α  using HEK-293 cells stably transfected with the recombinant human FP receptor. Competition afforded by unlabeled PGF 2α  1-ethanolamide () and unlabelled 17-phenyl PGF 2α  (▴) is depicted. Points are mean values±SEM; n=3 of experiments performed in triplicate. 
     FIG. 13 is a graph showing the effect of 0.1% PGF 2α  1-ethanolamide () on dog intraocular pressure. Contralateral eyes received vehicle (∘) as a control. Points are mean values±SEM; n=6. 
     FIG. 14 is a graph showing the effect of 0.1% PGF 2α  1-ethanolamide () on dog pupil diameter. Contralateral eyes received vehicle (∘) as a control. Points are mean values±SEM; n=6. 
     FIG. 15 is a graph showing the effect of PGF 2α  1-ethanolamide () on dog ocular surface hyperemia. Contralateral eyes received vehicle (∘) as a control. Points are mean values±SEM; n=6. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Surprisingly it was discovered in connection with the present invention that PGF 2α  1-ethanolamide is a naturally occurring substance that may be formed from anandamide, a natural cannabinomimetic, by recombinant COX-2 and PGF synthase. It is known that COX-2 (cyclo-oxygenase-2) is a prostaglandin endoperoxide synthase which is inducible and not constitutively present in tissues (see Meade et al. (1993) J. Biol. Chem. 268: 6610-6614.) Since COX-2 would not be expected to be present in non-stimulated tissues, the finding in connection with the present invention that substantial quantities of PGF 2α  ethanolamide were formed following anandamide administration to naive mice is unexpected. Moreover, surprisingly PGF 2α  ethanolamide was also detected in mice that did not receive anandamide, suggesting that PGF 2α  1-ethanolamide is constitutive and may behave as an endogenously synthesized hormone. 
     In accordance with the invention PGF 2α  1-ethanolamide was synthesized to provide this substance in an isolated and substantially pure form, and to enable the formulation of pharmaceutical compositions containing this substance. The synthesis of PGF 2α  ethanolamide also enables the synthesis of all compounds within the scope of Formula 1 with only such modifications in the synthetic process which are readily available to the practicing organic chemist, and particularly to the chemist practicing in the field of prostaglandin and related chemistry. Thus, the present invention encompasses the compounds of Formula 1.                           
     Preferred compounds within the scope of the invention are shown in Formula 1a, and particularly preferred are compounds within the scope of Formula 1a where the dashed lines between carbons 10 and 11, 8 and 12 and 17 and 18 represent absence of a bond, and between carbons 5 and 6 represent a bond. Further, as far as the substitution of the hydroxyl functions of the compounds of Formula 1a or of Formula 1 are concerned (R 1  through R 4  groups), compounds are presently preferred where one or more of the hydroxyl functions is not esterified nor alkylated (one or more of R 1  through R 4  is H), or when esterified the esterifying group is alkanoyl having 1 through 6 carbons (formyl, acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, or any branched chain alkanoyl of 3 to 6 carbons). As far as the substituent designated R 5  in Formula 1a or in Formula 1 is concerned, compounds are preferred where R 5  is H, or lower alkyl of 1 to 3 carbons, more preferably H. R 6  is preferably H or lower alkyl of 1 to 3 carbons, more preferably H, and n is preferably 2. 
     Referring still to Formula 1a and Formula 1, the configuration of the OR 1 , OR 2  and OR 3  groups is preferably alpha (C-9, C-11 and C-15 α), and the configuration of the 5-6 double bond is preferably Z (cis). 
     The most preferred compounds in accordance with the present invention are PGF 2α  1-ethanolamide and its acylated or alkylated derivatives, shown in Formula 2, where R 1  through R 4  are defined as in connection with Formula 1, but the acylated derivatives are more preferred than the alkylated ones.                           
     The synthesis of the compounds of the invention is illustrated by the synthesis of PGF 2α  1-ethanolamide shown in Reaction Scheme 1, and also disclosed below in experimental detail. Referring now to the reaction scheme, the commercially available triethanolamine (tromethamine) salt of the carboxylic acid PGF 2α  is treated with acid to provide the free carboxylic acid. The free carboxylic acid PGF 2α  is converted to the methyl ester by treatment with a methylating agent (preferably methyl iodide) in the presence of an acid acceptor (preferably 1,8-diazabicyclo[5.4.0]undec-7-ene) to provide PGF 2α  methyl ester. PGF 2α  methyl ester is thereafter treated with ethanolamine to provide PGF 2α  1-ethanolamide. Thus, the entire “amide”moiety is introduced into the molecule in the reaction between PGF 2α  methyl ester and a hydroxyalkylamine derivative of Formula 3, where R 5  and R 6  and n are defined as in connection with Formula 1, and where Formula 3 represents ethanolamine when R 5 , R 6  are H and n is 2. The hydroxylalkylamines of Formula 3 are available from the chemical literature and are well within the skill of the practicing organic chemist. Compounds where in Formula 1 one or more of the dashed lines represent a bond, and/or where the configuration of the hydroxyl groups, or the configuration of the substituents about one or more double bonds is different than in PGF 2α  1-ethanolamide, can be obtained by reactions analogous to the ones shown in Reaction Scheme 1, however starting with the prostaglandin derivative C-1 carboxylic acid of the appropriate corresponding structure and stereochemistry. These starting compounds are available in accordance with the chemical literature and are within the skill of the practicing organic chemist. Compounds where in Formula 1 one or more of the R 1  through R 4  groups is other than H, that is, compounds where one or more of the C-9, C-11, C-15 and the hydroxyl group of the hydroxyalkyl moiety in the amide portion are esterified, or alkylated can be obtained by esterification or alkylation reactions from the “free hydroxyl” compounds. Generally speaking, the modifications of the α and ω side chains of the compounds of Formula 1 as defined in connection with that formula, are within the skill of the practicing synthetic organic chemist in accordance with or in analogy to the disclosures in U.S. Pat. Nos. 5,545,665; 5,834,498 and 5,352,708 the specifications of which are expressly incorporated herein by reference.                           
     Biological Activity 
     The compounds of the invention are unique in that they are potent ocular hypotensive agents which nevertheless do not act through the FP receptor. This is demonstrated in a number of assay procedures which are well accepted in the art for establishing intraocular hypotensive activity as well as for determining the receptor through which the compounds act. The intraocular hypotensive activity of PGF 2α  1-ethanolamine and its virtual inertness in a number of assays which probe for activity of the FP or other known prostaglandin receptors are particularly striking and surprising when the biological profile of this compound is compared with the “parent” compound PGF 2α . 
     Description of the biological assays, and results in the assays is provided below. 
     Methods 
     Isolated Tissue Studies 
     Smooth muscle tension of isolated tissues was measured isometrically with force displacement transducers (Grass FT-03) and recorded on Grass Polygraphs (Models 7G and 79E). The organ baths contained Krebs solution maintained at 37° C. and gassed with 95% 0 2 /5% CO 2  to give a pH of 7.4. The Krebs solution had the following composition (mM): NaCl, 118.0; KCl, 4.7; KH 2 PO 4 , 1.2; CaCl 2 , 1.9; MgSO 4 , 1.18; NaHCO 3 , 25.0; glucose, 11.7; indomethacin, 0.001. 
     (a) Cat Iris 
     Adult domestic cats were sacrificed by intravenous overdose of sodium pentobarbital (Anthony, Arcadia, Calif.). The eyes were enucleated immediately and placed on ice. The iris sphincter muscle was mounted vertically under 50 to 100 mg tension in a jacketed 10 ml organ bath. A 60 min stabilization period was allowed before commencing each experiment. Activity was determined as contractile responses. Compounds were added cumulatively to the organ bath and at least 30 min was allowed for recovery, after complete wash-out, and return to baseline tension. The response to 10 −7 M PGF 2α  was determined at the beginning and end of each experiment and between dose-response curves as a reference. No more than two compounds were tested in each tissue. 
     (b) Rat Aorta 
     Adult Sprague Dawley rats weighing 180-220 grams were anesthetized by CO 2  inhalation and then decapitated and exsanguinated. The thoracic aorta was removed and cleaned of any adhering tissue. The lumen was flushed to remove any blood. The aorta was dissected into three small segments of 5-8 mm length. Each segment was mounted under 2 g of tension in a jacketed 10 ml organ bath with the aid of two wire hooks placed through the lumen of the vessel. This arrangement allowed for measurement of contraction forces developed by the circular smooth muscle. The tissues were allowed to equilibrate for 1 hour before compounds were added cumulatively to the organ baths. The response to 10 −7  M U-46619 (a thromboxane mimetic) was determined at the beginning and end of each experiment as a reference. A 30-45 minute recovery period was allowed after complete wash-out of drug. Only one test compound was examined in each tissue. 
     (c) Chick Ileum 
     The chick ileum is an EP 3  receptor preparation. Portions of the chick ileum of 1.5 cm length were suspended under 1 g of tension. After a one hour equilibration period, a standard dose-response curve for PGE 2  was obtained in a non-cumulative fashion with 30 minute wash-out periods between individual doses. Subsequently, test compound was added non-cumulatively. A maximal dose of PGE 2  (10 −6  M) was finally given to serve as a second reference standard. Contractile activity at each concentration was then calculated as a % of the maximal PGE 2  response reached at a 10 −6  M concentration. 
     (d) Guinea Pig Ileum 
     The guinea pig ileum is an EP 1  receptor preparation. An approximately 1.5 cm segment of guinea pig ileum was suspended in a jacketed organ bath under 1 g tension. Following a 1 hr equilibration period, a wash-out dose response curve for PGE 2  was obtained. 30 min wash-out periods were allowed between doses. A dose response curve was then generated for PGF 2α  1-ethanolamide in an identical manner. A final administration of PGE 2  10 −6  M was given as a reference. Data were expressed as % maximal of the PGE 2  response. 
     (e) Guinea Pig Vas Deferens 
     Activity at the prostaglandin-sensitive EP 3  receptor subtype was also determined by the ability of prostanoids to inhibit the smooth muscle twitch response stimulated by an electrical current in the isolated guinea pig vas deferens. 1.5 cm portions of the guinea pig vas deferens were suspended under 1 g initial tension and allowed to equilibrate for at least 30 minutes. Tissues were then subjected to stimulation every 30 seconds by 20 volt electrical pulses. After stabilization of the twitch response, a standard dose response curve to PGE 2  was performed in a cumulative fashion. Test compounds were then evaluated by performing a cumulative dose response curve. Tissues were washed-out and allowed to equilibrate for 1 hr between compounds. PGE 2  was once again evaluated at the conclusion of the experiment as a standard reference. Activity was calculated as the percent inhibition of the height of the muscle twitch response. 
     (f) Rabbit Jugular Vein 
     New Zealand albino rabbit of either sex, weighing 2-4 kg, were injected with 1000 U heparin into the marginal ear vein and then sacrificed by CO 2  gas. The external jugular veins were cleared of fat and adherent connective tissue and removed. The veins were transected and each ring of 3-4 mm length was suspended between two metal hooks in a jacketed organ bath. The tissues were equilibrated for 1 hr under 0.75 g tension, which was readjusted as the tissues relaxed. Single doses of histamine, 10 −5  M then 2-3×10 −6  M, with washing after each dose, were given to contract the tissue and establish responsiveness. The TP-receptor antagonist SQ29548, (Ogletree et al. (1985) J. Pharmacol. Exp. Ther. 234 435-441) at (10 −6  M), was applied for 5 min and then histamine 2-3×10 −6  M was added to elicit the contractile response. After 30 min of pretreatment with the histamine, the relaxant response was tested by adding cumulative doses of the test compounds with 10 −8  M to 10 −7  M PGE 2  at the end of each dose-response curve as a reference. A recovery period of 30-50 min was allowed after wash-out of the tissues. Relaxant activity was calculated as % of the control tone elicited by histamine. 
     (g) Human Platelets 
     Prostanoid activity at the DP-, TP- and IP-receptor types was determined by an ability to cause aggregation (TP-receptor activity) or to inhibit ADP-induced aggregation of human platelets in vitro (DP- and IP-receptor activity). Fresh whole blood was obtained from consenting healthy human volunteers and mixed with acid citrate-dextrose (ACD). The blood was centrifuged at 1000 rpm for 15-20 minutes to obtain platelet rich plasma (PRP). 4.5 μl of prostanoid or vehicle was added to 450 μl of PRP and incubated for 2 minutes at 37° C. in a Payton aggregometer and observed for any aggregatory activity. 2×10 −5  M ADP (final concentration) was then added to induce full aggregation. Inhibition of aggregation was calculated as the percentage difference between aggregation evoked by 2×10 −5  M ADP in the absence and presence of drug. Aggregatory activity was calculated as the percent aggregation induced by the prostanoid relative to the aggregation induced by 2×10 −5  M ADP. Standard aggregatory responses to 2×10 −5  M ADP alone were performed at the beginning and end of each experiment. 
     (h) CRL 1497 Cells: Ca 2+  Signal. 
     Human CRL 1497 cells were plated in culture flasks and fed Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) containing 10% fetal calf serum, 2 mM 1-glutamine, and 0.05 mg/ml gentacin (all purchased from Gibco, Grand Island, N.Y.). Cell cultures were maintained in a humidified atmosphere of 95% air, 5% CO 2  and grown to confluency. Cells were removed from the culture flasks by approximately 1 min treatment with trypsin 0.05%/0.53 mM EDTA in Hanks Balanced Salt Solution (HBSS, Gibco, Grand Island, N.Y.) at 37° C. Proteolytic activity was arrested by adding 5 ml of 10% fetal bovine serum in DMEM. The cells were consecutively washed in Hank&#39;s BSS and medium containing 140 mM NaCl, 50 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES: TRIS, 5 mM glucose, 5 mM Na pyruvate, 0.1% bovine serum albumin at pH 7.4: Centrifugation for the washes was performed for 15 minutes at 200 g at room temperature. Cells were counted, resuspended in the above medium and incubated with 2×10 −6  M Fura 2/acetoxymethyl ester in a shaking water bath for 30 minutes at 37° C. The cells were subsequently washed in medium as above and resuspended at a concentration of 2×10 6  cells ml −1 . Aliquots of 0.5 ml cell suspension were then added to autocap microtubes to provide 10 6  cells per experimental determination of intracellular free Ca 2+  concentration ([Ca 2+ ] i ). 
     Fluorescence was measured in a Perkin-Elmer LS-5 fluorescence spectrophotometer at excitation and emission wavelengths of 340 and 492 nm, respectively, with both slits at 10 nm. For each experimental determination 10 6  cells were washed (200×g for 5 min) and suspended in a 2 ml cuvette with buffer containing 120 mM NaCl, 6 mM KCl, 1 mM MgSO 4 , 1.5 mM CaCl 2 , 20 mM HEPES, 1 mg ml −1  glucose and 1 mg ml −1  Na pyruvate. Stirring was achieved by an overhead-mounted, paddle stirrer with the temperature maintained at 37° C. The cells were lysed with digitonin (10 μl of 100 mg ml −1  DMSO) to obtain f max . EGTA (100 mM) and sufficient 10N NaOH to adjust the pH to 8.5 were then successively added to obtain f min . 
     (i) Recombinant Cat and Human FP Receptor; Stable Transfectants 
     HEK-293 cells were grown in DMEM with 10% fetal bovine serum (FBS), 250 μg ml −1  G418 (Life Technologies) and 200 μg ml −1  gentamicin or penicillin/streptomycin. Selection of stable transfectants was achieved with 200 μg ml −1  hygromycin, the optimal concentration being determined by hygromycin kill curve studies. For transfection, the cells were grown to 50-60% confluency on 10 cm plates. The plasmid pCEP4 incorporating cDNA inserts for the human FP receptor (20 μg) was added to 500 μl of 250 mM CaCl 2 . HEPES buffered saline×2 (2×HBSS, 280 mM NaCl, 20 mM HEPES acid, 1.5 mM Na 2  HPO 4 , pH 7.05-7.12) was then added dropwise to a total of 500 μl, with continuous vortexing at room temperature. After 30 min, 9 ml DMEM were added to the mixture. The DNA/DMEM/calcium phosphate mixture was then added to the cells, which had been previously rinsed with 10 ml phosphate buffered saline (PBS). The cells were then incubated for 5 h. at 37° C. in humidified 95% air/5% CO 2 . The calcium phosphate solution was then removed and the cells were treated with 10% glycerol in DMEM for 2 min. The glycerol solution was then replaced by DMEM with 10% fetal bovine serum (FBS). The cells were incubated overnight and the medium was replaced by DMEM/10% FBS containing 250 μg ml −1  G418 and penicillin/streptomycin. The following day hygromycin B was added to a final concentration of 200 μg ml −1 . 
     Ten days after transfection, hygromycin B resistant clones were individually selected and transferred to a separate well on a 24 well plate. At confluence each clone was transferred to one well of a 6 well plate, and then expanded in a 10 cm dish. Cells were maintained under continuous hygromycin selection until use. The stable cat FP receptor transfectants were similarly prepared using lipofectamine. Again cells were grown to 50-60% confluency on 10 cm plates and the plasmid pCEP4, incorporating cDNA inserts for the feline FP receptor, was transfected using lipofectin. Hygromycin B selection was started 48 hr post-transfection. Eight days after transfection, hygromycin B resistant clones were selected and transferred to 24 well plates. Ca 2+  signaling studies on recombinant cat and human FP receptors were performed in an identical manner to that described for the CRL 1497 cell studies. 
     (j) Inositol Phosphate Formation 
     Receptor-mediated phosphoinositide (PI) hydrolysis was determined by measuring the accumulation of total inositol phosphates (IP) in cells preincubated with  3 H-myoinositol. Stable cell lines of HEK 293 cells expressing human or cat FP receptors were plated in 10 cm dishes (10 6  cells per dish in DMEM with 10% FBS). The following day, the cells were incubated with 18 μCi myo-[2  3 H] inositol (Amersham; 10-20 μCi mmol −1 ) in 6 ml of DMEM with 10% FBS for 24 hours at 37° C. The cells were then rinsed twice with phosphate buffered saline (PBS), incubated for 5 min with 1 ml of trypsin-EDTA, and suspended in 10 ml DMEM containing 25 mM HEPES buffer. Cells were pelleted at 1000 rpm and resuspended in DMEM/25 mM HEPES containing 10 mM LiCl for 10 minutes. Aliquots of 200 μl cell suspension were incubated with 50 μl drug for 30 minutes at 37° C. (duplicate determination), and agonist stimulation was terminated with a 750 μl mixture of chloroform:methanol:4N HCl (100:200:2), followed by addition of 250 μl of chloroform and 0.5 N HCl for extraction of inositol phosphates. A 750 μl volume of the aqueous layer was loaded on columns packed with 0.5 ml of Dowex AG 1-X8 anion-exchange resin (formate form, 100-200 mesh; BioRad) to separate radiolabeled components. The elution procedure consisted of three 3 ml washes with 5 mM inositol, then two elutions with 750 μl of 1.3 M ammonium formate with 0.1 M formic acid. The eluate was mixed with 10 ml Aquasol scintillation fluid (Packard Instrument Co.) and total [ 3 H] inositol phosphates was determined by a liquid scintillation counter. 
     (k) Radioligand Binding 
     Plasma membrane preparations of HEK 293 cells stably transfected with the human or feline FP receptor were obtained as follows. Cells washed with TME buffer were scraped from the bottom of the flasks and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added as necessary to achieve a 40 ml volume in the centrifuge tubes. TME is comprised of 100 mM TRIS base, 20 mM Mg Cl 2 , 2 m EDTA; physiological pH is achieved by adding 10 N HCl. The cell homogenate was centrifuged at 19,000 rpm for 20 min at 4-6° C. using a Beckman Ti-60 rotor. The pellet was then resuspended in TME buffer to provide a final protein concentration of 1 mg/ml, as determined by Biorad assay. Radioligand binding assays were performed in a 200 μl volume. 
     The binding of [ 3 H](N) 17-phenyl PGF 2α  (specific activity 85 Ci/mmol) was determined in duplicate and experiments were replicated three times. Incubations were for 60 min at 25° C. and were terminated by the addition of 4 ml of ice-cold 50 mM TRIS-HCl followed by rapid filtration through Whatman GF/B filters and three additional 4 ml washes in a cell harvester (Brandel). Competition studies were performed using a final concentration of 5 nM [ 3 H](N) 17-phenyl PGF 2α  and non-specific binding was determined with 10 −5  M unlabelled 17-phenyl PGF 2α . 
     (l) Intraocular Pressure (IOP) 
     Intraocular pressure studies in dogs involved pneumatonometry performed in conscious, Beagle dogs of both sexes (10-15 kg). The animals remained conscious throughout the study and were gently restrained by hand. Drugs were administered topically to one eye as a 25 μL volume drop, the other eye received 25 μL vehicle (0.1% polysorbate 80:10 mM TRIS) as a control. 0.1% proparacaine was used for corneal anesthesia during tonometry. Intraocular pressure was determined just before drug administration and at 2, 4 and 6 hr thereafter on each day of the 5 day study. Drug was administered immediately after the first IOP reading. 
     (m) Pupil Diameter 
     Dog pupil diameter was measured using an optistick (a mm ruler which included half-circle references of standard widths (mm) for reference. Gently restraining the dog by hand, pupil diameter was determined by matching a half-circle to the pupil in normal room light. In dogs with very dark pupils a specialized penlight was used, but only very briefly to avoid pupil constriction. Pupil diameter was measured at the same time as IOP and hyperemia. 
     (n) Ocular Surface Hyperemia 
     Ocular surface hyperemia was visually assessed and scored according to a system typically used clinically. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Hyperemia Score 
                 Assigned Value 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 &lt;1 trace 
                 0.5 
               
               
                   
                 1 mild 
                 1 
               
               
                   
                 moderate 
                 2 
               
               
                   
                 severe 
                 3 
               
               
                   
                   
               
             
          
         
       
     
     Ocular surface hyperemia was evaluated at the same time points as intraocular pressure measurement. It should be noted that untreated dog eyes frequently have a pink/red tone. Thus, values of trace or even mild are not necessarily out of the normal range. 
     Results 
     The effects of Prostaglandin F 2α  1-ethanolamide on the feline iris sphincter are depicted in FIG.  1  and tabulated (Table 1). Prostaglandin F 2α  1-ethanolamide (PGF 2α  1-ethanolamide) produced dose dependent contraction of cat iris sphincter smooth muscle. An effective concentration (E.C.) 50  value of 58 nM was obtained. The data indicate a threshold concentration in the region of 10 nM. 
     Prostaglandin F 2α  1-ethanolamide was modestly effective in inhibiting field stimulation evoked contraction of the isolated guinea pig vas deferens preparation. The data are depicted in FIG.  2  and tabulated (Table 2). 
     FIG. 3 depicts the effect of PGF 2α  1-ethanolamide on the isolated chick ileum. Comparison of responses to the reference compound (prostaglandin E 2 , 10 −6  M) suggests that PGF 2α  1-ethanolamide may behave as a weak partial agonist. The data are listed in Table 3. PGF 2α  1-ethanolamide appeared less effective in the guinea pig ileum (FIG. 4; Table 4), which is reported to be an EP 1  receptor preparation. 
     The effects of graded doses of PGF 2α  1-ethanolamide on the isolated rat aorta are shown in FIG.  5  and Table 5. Residual activity occurred at high doses, 10 −6  and 10 −5  M. Since the rat aorta is a TP receptor preparation, activity at the TP receptor was further evaluated in terms of platelet aggregation. In addition, inhibition of ADP induced human platelet aggregation was determined to reveal any interaction at DP or IP receptors. No effect on platelets was apparent. The data are presented in Table 6; no graph is provided since absolutely no response occurred. 
     The Ca 2+  signal produced by graded doses of Prostaglandin F 2α  1-ethanolamide in human dermal fibroblasts (CRL 1497 cells) is compared to that produce by Prostaglandin F 2α  in FIG.  6 . The data are also tabulated (Table 7). The E.C. 50  values obtained were 16 nM for PGF 2α  and 1586 nM for PGF 2α  1-ethanolamide, indicating about a one hundred fold difference in potency at the human FP receptor. 
     Recent studies have shown that Prostaglandin F 2α  can produce endothelium dependent, nitric oxide (NO) mediated vasorelaxation (Chen et al.(1995) Br. J. Pharmacol. 116: 3035-3041) Thus, the effects of PGF 2α  1-ethanolamide were compared to those of PGF 2α  in producing endothelium dependent vasorelaxation of the histamine precontracted, isolated rabbit jugular vein segment. Although PGF 2α  potently produced marked vasorelaxation, PGF 2α  1-ethanolamide exhibited relatively little activity (FIG.  7 ). The data are also tabulated (Table 8). The following E.C. 50  values were obtained for vasorelaxation of the precontracted rabbit jugular vein segment: PGF 2α  1-ethanolamide=2000 nM, PGF 2α =5.3. 
     Since marked activity was observed for PGF 2α  1-ethanolamide in the cat iris sphincter preparation, its activity was compared to that of PGF 2α  at the recombinant feline FP receptor. FIG. 8 compares the activity of PGF 2α  1-ethanolamide and PGF 2α  in eliciting a Ca 2+  signal in HEK-293 cells stably transfected with the recombinant feline FP receptor. E.C. 50  values of 14 nM for PGF 2α  and 1458 for PGF 2α  1-ethanolamide were obtained, indicating an approximately 100 fold difference in potency. The data are also tabulated (Table 9). 
     The effects of PGF 2α  1-ethanolamide and 17-phenyl PGF 2α  on total inositol phosphate accumulation in HEK-293 cells stably transfected with the recombinant feline FP receptor are depicted in FIG.  9 . Again, a large separation in potency was apparent. E.C.50 values were 9 nM for 17-phenyl PGF 2α  and 524 nM for PGF 2α  1-ethanolamide. The data are tabulated (Table 10). 
     In addition, the effects of PGF 2α  1-ethanolamide and 17-phenyl PGF 2α  on total inositol phosphate accumulation in HEK-293 cells stably transfected with the recombinant human FP receptor were examined. E.C. 50  values of 8 nM for 17-phenyl PGF 2α  and 1003 nM were obtained. The data are presented in FIG.  10  and Table 11. Results were similar to those obtained for the recombinant feline FP receptor. 
     Consistent with the functional studies employing stably transfected FP receptor preparations, PGF 2α  1-ethanolamide had relatively modest binding affinity for the FP receptor. Thus, PGF 2α  1-ethanolamide (prior art compound no. 1) had only weak affinity for the feline receptor, whereas 17-phenyl PGF 2α  exhibited high affinity (FIG. 11; Table 12). The inhibitory concentration 50  (I.C. 50 ) for PGF 2α  1-ethanolamide obtained was 882 nM, compared to an I.C. 50  value of 43.5 nM for 17-phenyl PGF 2α . Similar results were obtained for the recombinant human FP receptor: PGF 2α  1-ethanolamide I.C. 50  =2015 nM, 17-phenyl PGF 2α  I.C. 50  =2.3 nM. These radioligand binding data are depicted in FIG.  12  and tabulated (Table 13). 
     The ocular effects of PGF 2α  1-ethanolamide were studied in dogs. PGF 2α  1-ethanolamide at a 0.1% dose produced a marked and highly significant decrease in intraocular pressure (FIG. 13; Table 14). The effect persisted over the entire 24 hr experimental period. PGF 2α  1-ethanolamide also caused marked miosis (FIG. 14; Table 15). Unlike the ocular hypotensive effect, the decrease in pupil diameter had resolved by the 24 hr time point. PGF 2α  1-ethanolamide caused mild ocular surface hyperemia, compared to trace for vehicle, throughout the 24 hr experimental period (FIG. 15; Table 16). 
     In the ensuing Tables, unless indicated otherwise, the concentration of the test or control compounds is indicated by the logarithmic exponent only. Thus and by way of example “−8” in the Tables means 10 −8  M concentration of the test or control substance. 
     
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Effect of graded doses of PGF 2α  1-ethanolamide on the isolated feline 
               
               
                 iris sphincter smooth muscle. Reference contraction was provided by 
               
               
                 10 −7  M PGF 2α . Values are mean ± SEM; n = 4 
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                   
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
                   
               
             
          
           
               
                 % Maximal 
                 2.7 ± 1.7 
                 77 ± 5.5 
                 119.2 ± 6.0 
                 126.0 ± 3.6 
               
               
                 contractile 
               
               
                 response mean 
               
               
                 χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Inhibitory effect of graded doses of PGF 2α  1-ethanolamide on field 
               
               
                 stimulated contraction of the isolated guinea pig vas deferens. Values are 
               
               
                 mean ± SEM; n= 4 
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                   
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
                   
               
             
          
           
               
                 % Maximal 
                 2.2 ± 1.3 
                 12.5 ± 6.2 
                 37.7 ± 8.4 
                 98.5 ± 1.0 
               
               
                 inhibition of 
               
               
                 responses mean 
               
               
                 χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Effect of graded doses of PGF 2α  1-ethanolamide on the isolated chick 
               
               
                 ileum. Reference contraction was provided by 10 −6  M PGE 2 . Values are 
               
               
                 mean ± SEM; n = 4 
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                   
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
                   
               
             
          
           
               
                 % Maximal 
                 0 ± 0 
                 15.0 ± 8.4 
                 50.7 ± 4.0 
                 66.5 ± 9.4 
               
               
                 contractile 
               
               
                 response mean 
               
               
                 χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Effect of graded doses of PGF 2α  1-ethanolamide on the isolated guinea 
               
               
                 pig ileum. Reference contraction was provided by PGE 2  10 −6  M. Values 
               
               
                 are mean ± SEM; n = 4 
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                   
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
                   
               
             
          
           
               
                 % Maximal 
                 0 ± 0 
                 0 ± 0 
                 4.0 ± 2.6 
                 9.7 ± 4.2 
               
               
                 response mean 
               
               
                 χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Effect of graded doses of PGF 2α  1-ethanolamide on the isolated rat aorta. 
               
               
                 Reference contraction was provided by 10 −7  M U-46619. Values are 
               
               
                 mean ± SEM; n = 4 
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                   
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
                   
               
             
          
           
               
                 % Maximal 
                 0 ± 0 
                 0 ± 0 
                 4.0 ± 2.6 
                 9.7 ± 4.2 
               
               
                 contractile 
               
               
                 response mean 
               
               
                 χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Effect of graded doses of prostaglandin F 2α  1-ethanolamide on platelet 
               
               
                 aggregation and inhibition of ADP induced aggregation. Reference 
               
               
                 responses are provided by 2 × 10 −5  M ADP. Values are mean ± SEM; 
               
               
                 n = 4 
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide 
               
               
                   
                 concentration, log [M] 
               
             
          
           
               
                   
                 −7 
                 −6 
                 −5 
               
               
                   
                   
               
             
          
           
               
                   
                 % Platelet 
                 0 ± 0 
                 0 ± 0 
                 0 ± 0 
               
               
                   
                 aggregation 
               
               
                   
                 % Inhibition 
                 0 ± 0 
                 0 ± 0 
                 0 ± 0 
               
               
                   
                 platelet 
               
               
                   
                 aggregation 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Increases in intracellular free Ca 2+  concentration [Ca 2+ ] i  produced by 
               
               
                 graded doses of prostaglandin F 2α  1-ethanolamide and prostaglandin F 2α  in 
               
               
                 CRL 1497 human dermal fibroblasts. Values are mean ± SEM; n = 4 
               
             
          
           
               
                   
                 −9 
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
                   
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                 Δ[Ca 2+ ] i   
                 — 
                  3.4 ± 1.5 
                  13.5 ± 3.9 
                 45.8 ± 11.5 
                 97.9 ± 10.8 
               
               
                 mean 
               
               
                 χ ± SEM 
               
             
          
           
               
                   
                 Prostaglandin F 2α  concentration, log [M] 
               
             
          
           
               
                 Δ[Ca 2+ ] i   
                 5.6 ± 2.4 
                 47.7 ± 9.4  
                 124.1 ± 6.5  
                 127.4 ± 7.6  
                 136.7 ± 11.2  
               
               
                 mean 
               
               
                 χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 Vasorelaxation of the pre-contracted rabbit jugular vein segment by 
               
               
                 prostaglandin F 2α  1-ethanolamide and prostaglandin F 2α . Reference vasorelaxation is 
               
               
                 provided by PGE 2  10 −7  M. Values are mean ± SEM; n = 6 for PGF 2α   
               
               
                 1-ethanolamide, n = 7 for PGF 2α . 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                   
                 −10 
                 −9 
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
               
               
                 % 
                 100 ± 0   
                 99.7 ± 0.3 
                 93.2 ± 2.2 
                 80.3 ± 3.2 
                 54.3 ± 10.2 
                 38.4 ± 11.3 
               
               
                 relaxation 
               
               
                 mean χ ± SEM 
               
               
                   
               
             
          
           
               
                   
                 Prostaglandin F 2α  concentration, log [M] 
               
             
          
           
               
                   
                 −11 
                 −10 
                 −9 
                 −8 
                 −7 
                 −6 
               
               
                   
               
               
                 % 
                  98 ± 1.1 
                 92.5 ± 2.3 
                 71.9 ± 7.2 
                 25.8 ± 7.8 
                 8.3 ± 3.0 
                 6.7 ± 2.7 
               
               
                 relaxation 
               
               
                 mean χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Increases in intracellular free Ca 2+  concentration [Ca 2+ ] i  produce by 
               
               
                 graded doses of prostaglandin F 2α  1-ethanolamide and prostaglandin F 2α  in HEK-293 
               
               
                 cells stably transfected with the recombinant feline FP receptor. 
               
               
                 Values are mean ± SEM; n = 3. 
               
             
          
           
               
                   
                 −10 
                 −9 
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
                   
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                 Δ[Ca 2+ ] i   
                 — 
                 — 
                 0 ± 0 
                 28.5 ± 3.1 
                 45.2 ± 6.4 
                 77.2 ± 14.7 
               
               
                 mean χ ± SEM 
               
             
          
           
               
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                 Δ[Ca 2+ ] i   
                 6.5 ± 5.0 
                 22.2 ± 6.4 
                 45.7 ± 5.3  
                 78.8 ± 8.3 
                 87.9 ± 9.0 
                 85.0 ± 9.6  
               
               
                 mean χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Total inositol phosphate accumulation produced by graded doses of 
               
               
                 prostaglandin F 2α  1-ethanolamide and 17-phenyl prostaglandin F 2α  in HEK-293 cells 
               
               
                 stably transfected with the recombinant feline FP receptor. Reference stimulation 
               
               
                 was provided by 17-phenyl PGF 2α  10 −6  M. Values are mean ± SEM; n = 3 for 
               
               
                 PGF 2α  1-ethanolamide and 4 for 17-phenyl PGF 2α  of experiments performed 
               
               
                 in duplicate. 
               
             
          
           
               
                   
                 −9 
                 −8  
                 −7 
                 −6 
                 −5 
               
               
                   
                   
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                 % maximal 
                 — 
                 3.9 ± 3.2 
                 14.9 ± 4.7 
                 63.0 ± 16.4 
                 89.2 ± 4.8 
               
               
                 total inositol 
               
               
                 phosphate 
               
               
                 formation 
               
               
                 mean χ ± SEM 
               
             
          
           
               
                   
                 17-phenyl Prostaglandin F 2α  concentration, log [M ] 
               
             
          
           
               
                 % maximal 
                 17.8 ± 6.4 
                 56.8 ± 10.8 
                 89.7 ± 2.4 
                 99.9 ± 1.1  
                 — 
               
               
                 total inositol 
               
               
                 phosphate 
               
               
                 formation 
               
               
                 mean χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Total inositol phosphate accumulation produced by graded doses of 
               
               
                 prostaglandin F 2α  1-ethanolamide and 17-phenyl prostaglandin F 2α  in HEK-293 cells 
               
               
                 stably transfected with the recombinant human FP receptor. Reference stimulation 
               
               
                 was provided by 17-phenyl PGF 2α  10 −6  M. Values are mean ± SEM; n = 3 for 
               
               
                 PGF 2α  1-ethanolamide and 4 for 17-phenyl PGF 2α  of experiments performed 
               
               
                 in duplicate. 
               
             
          
           
               
                   
                 −9 
                 −8  
                 −7 
                 −6 
                 −5 
               
               
                   
                   
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                 % maximal 
                 — 
                 0.3 ± 0.6 
                 10.0 ± 1.6 
                 48.4 ± 3.3 
                 85.0 ± 1.9 
               
               
                 total inositol 
               
               
                 phosphate 
               
               
                 accumulation 
               
               
                 mean χ ± SEM 
               
             
          
           
               
                   
                 17-phenyl Prostaglandin F 2α  concentration, log [M ] 
               
             
          
           
               
                 % maximal 
                 14.7 ± 3.3 
                 57.8 ± 2.0  
                 92.3 ± 0.9 
                  100 ± 1.3 
                 — 
               
               
                 total inositol 
               
               
                 phosphate 
               
               
                 accumulation 
               
               
                 mean χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                 Radioligand binding competition studies (PGF 2α  1-ethanolamide and 
               
               
                 17-phenyl PGF 2α  vs. 5nM  3 H-17-phenyl PGF 2α ) on the recombinant feline FP 
               
               
                 receptor. Values are mean ± SEM; n = 3 of experiments performed in duplicate. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                   
                 −9 
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
               
               
                 % specific 
                 89.7 ± 3.2 
                 93.7 ± 3.8 
                 89.7 ± 4.9 
                 48.0 ± 2.9 
                 8.3 ± 4.8 
               
               
                 binding mean 
               
               
                 χ ± SEM 
               
               
                   
               
             
          
           
               
                   
                 17-phenyl Prostaglandin F 2α  concentration, log [M] 
               
             
          
           
               
                   
                 −10 
                 −9 
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
               
               
                 % specific 
                 92.7 ± 6.9 
                 80.7 ± 7.1 
                 43.7 ± 1.5 
                 17.0 ± 6.5 
                 10.3 ± 8.9  
                 0 ± 0 
               
               
                 binding mean 
               
               
                 χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 13 
               
               
                   
               
               
                 Radioligand binding competition studies (PGF 2α  1-ethanolamide and 
               
               
                 17-phenyl PGF 2α  vs. 5nM  3 H-17-phenyl PGF 2α ) on the recombinant human FP 
               
               
                 receptor. Values are mean ± SEM; n = 3 of experiments performed in duplicate. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Prostaglandin F 2α  1-ethanolamide concentration, log [M] 
               
             
          
           
               
                   
                 −9 
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
               
               
                 % specific 
                 94.7 ± 1.4 
                 103.3 ± 0.3 
                 95.9 ± 1.5 
                 63.7 ± 2.7 
                 19.7 ± 1.2 
               
               
                 binding mean 
               
               
                 χ ± SEM 
               
               
                   
               
             
          
           
               
                   
                 17-phenyl Prostaglandin F 2α  concentration, log [M] 
               
             
          
           
               
                   
                 −10 
                 −9 
                 −8 
                 −7 
                 −6 
                 −5 
               
               
                   
               
               
                 % specific 
                 98 ± 1.1 
                 65.0 ± 1.0 
                 24.0 ± 1.2 
                 5.3 ± 1.5 
                 1.7 ± 1.7 
                 0 ± 0 
               
               
                 binding mean 
               
               
                 χ ± SEM 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 Effect of 0.1% Prostaglandin F 2α  1-ethanolamide on dog intraocular 
               
               
                 pressure. Values are mean ± SEM; n = 6. **p &lt; 0.01 significant change from 
               
               
                 baseline intraocular pressure according to Student&#39;s paired t test. 
               
             
          
           
               
                   
                 Times of intraocular pressure measurement (HR) 
               
             
          
           
               
                   
                 0 
                 2 
                 4 
                 6 
                 24 
               
               
                   
                   
               
             
          
           
               
                 Intraocular 
                 Test eye 
                 19.1 ± 0.9 
                 16.2 ± 1.1** 
                 14.6 ± 0.9** 
                 14.0 ± 0.9** 
                 15.2 ± 0.7** 
               
               
                 pressure 
               
               
                 (mm Hg) 
               
               
                 mean 
               
               
                 χ ± SEM 
               
               
                   
                 Vehicle 
                 20.3 ± 0.7 
                 20.4 ± 1.0 
                 20.0 ± 1.0 
                 20.8 ± 0.6 
                 20.0 ± 0.9 
               
               
                   
                 control 
               
               
                   
                 eye 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                 Effect of 0.1% Prostaglandin F 2α  1-ethanolamide on dog pupil 
               
               
                 diameter. Values are mean ± SEM; n = 6. **p &lt; 0.01 significant change 
               
               
                 from baseline pupil diameter according to Student&#39;s paired t test. 
               
             
          
           
               
                   
                 Times of pupil diameter measurement (HR) 
               
             
          
           
               
                   
                 0 
                 2 
                 4 
                 6 
                 24 
               
               
                   
                   
               
             
          
           
               
                 Pupil 
                 Test eye 
                 6.7 ± 0.2 
                 1.0 ± 0.4** 
                 1.0 ± 0.3** 
                 1.5 ± 0.4** 
                 7.0 ± 0.3 
               
               
                 diameter 
               
               
                 (mm) 
               
               
                   
                 Vehicle 
                 6.3 ± 0.2 
                 6.3 ± 0.2 
                 6.7 ± 0.2 
                 6.3 ± 0.2 
                 6.2 0.2 
               
               
                   
                 control eye 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 16 
               
             
             
               
                   
               
               
                 Effect of 0.1% Prostaglandin F 2α  1-ethanolamide on dog ocular surface 
               
               
                 hyperemia. Values are mean ± SEM; n = 6. 
               
             
          
           
               
                   
                 Times of ocular surface hyperemia measurement (HR) 
               
             
          
           
               
                   
                 0 
                 2 
                 4 
                 6 
                 24 
               
               
                   
                   
               
             
          
           
               
                 Ocular 
                 Test eye 
                 0.3 ± 0.1 
                 1.3 ± 0.2 
                 1.2 ± 0.2 
                 1.2 ± 0.2 
                 1.1 ± 0.2 
               
               
                 Surface 
               
               
                 Hyperemia 
               
               
                 Score 
               
               
                   
                 Vehicle 
                 0.2 ± 0.1 
                 0.5 ± 0 
                 0.5 ± 0 
                 0.5 ± 0 
                 0.4 ± 0.1 
               
               
                   
                 control 
               
               
                   
                 eye 
               
               
                   
               
             
          
         
       
     
     Pharmaceutical Compositions, Methods of Use 
     Pharmaceutical compositions may be prepared by combining a therapeutically effective amount of at least one compound according to the present invention, or a pharmaceutically acceptable salt thereof, as an active ingredient, with conventional ophthalmically acceptable pharmaceutical excipients, and by preparation of unit dosage forms suitable for topical ocular use. The therapeutically efficient amount typically is between about 0.0001 and about 5% (w/v), preferably about 0.001 to about 1.0% (w/v) in liquid formulations. 
     For ophthalmic application, preferably solutions are prepared using a physiological saline solution as a major vehicle. The pH of such ophthalmic solutions should preferably be maintained between 6.5 and 7.2 with an appropriate buffer system, a substantially neutral pH being preferred. The formulations may also contain conventional, pharmaceutically acceptable preservatives, stabilizers and surfactants. 
     Preferred preservatives that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate and phenylmercuric nitrate. A preferred surfactant is, for example, Tween 80. Likewise, various preferred vehicles may be used in the ophthalmic preparations of the present invention. These vehicles include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose and purified water. 
     Tonicity adjustors may be added as needed or convenient. They include, but are not limited to, salts, particularly sodium chloride, potassium chloride, mannitol and glycerin, or any other suitable opthalmically acceptable tonicity adjustor. 
     Various buffers and means for adjusting pH may be used so long as the resulting preparation is ophthalmically acceptable. Accordingly, buffers include acetate buffers, citrate buffers, phosphate buffers and borate buffers. Acids or bases may be used to adjust the pH of these formulations as needed. 
     In a similar vein, an ophthalmically acceptable antioxidant for use in the present invention includes, but is not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene. 
     Other excipient components which may be included in the ophthalmic preparations are chelating agents. The preferred chelating agent is edentate disodium, although other chelating agents may also be used in place or in conjunction with it. 
     The ingredients are usually used in the following amounts: 
     
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 ingredient 
                 amount (% w/v) 
               
               
                   
                   
               
             
             
               
                   
                 active ingredient 
                 about 0.001-5 
               
               
                   
                 preservative 
                 0-0.10 
               
               
                   
                 vehicle 
                 0-40 
               
               
                   
                 tonicity adjustor 
                 0-10 
               
               
                   
                 buffer 
                 0.01-10 
               
               
                   
                 pH adjustor 
                 Q.s. pH 4.5-7.5 
               
               
                   
                 antioxidant 
                 as needed 
               
               
                   
                 surfactant 
                 as needed 
               
               
                   
                 purified water 
                 as needed to make 100% 
               
               
                   
                   
               
             
          
         
       
     
     The actual dose of the active compounds of the present invention depends on the specific compound, and on the condition to be treated; the selection of the appropriate dose is well within the knowledge of the skilled artisan. 
     The ophthalmic formulations of the present invention are conveniently packaged in forms suitable for metered application, such as in containers equipped with a dropper, to facilitate application to the eye. Containers suitable for dropwise application are usually made of suitable inert, non-toxic plastic material, and generally contain between about 0.5 and about 15 ml solution. 
     Especially preservative-free solutions are often formulated in non-resealable containers containing up to about ten, preferably up to about five units doses, where a typical unit dose is from one to about 8 drops, preferably one to about 3 drops. The volume of one drop usually is about 20-35 μl. 
     DETAILED DESCRIPTION OF SYNTHETIC PROCEDURES 
     PGF 2α  (Free Carboxylic Acid) 
     To a stirred solution of 20 g PGF 2α  tromethamine salt (51.9 mmol) in 150 ml H 2 O was added concentrated HCl such that the pH of the solution was adjusted to 2. The solution was extracted with 3×200 ml chloroform and concentrated in vacuo. PGF 2α  free carboxylic acid appeared primarily as a solid layered between the aqueous and organic phases. The solid was collected from this layer and combined with solid obtained by filtration from the concentrated chloroform solution, and the combined solids were washed with 50% chloroform/methanol. 
     PGF 2α  methyl ester; methyl (5Z,8α, 9α, 11α,13E,15S)-9,11,15-trihydroxyprosta-5,13-dien-1-oate 
     To a stirred solution of PGF 2α  free carboxylic acid (2.528 g, 7.13 mmol) in 50 ml acetone was added 3.2 ml 1,8-diazabicyclo [5.4.0] undec-7-ene (21.39 mmol) and 1.33 ml iodomethane (21.39 mmol). The solution was stirred overnight, diluted with 150 ml ethyl aceate, washed with 2×50 ml 0.5 m LiOH, 1×50 ml brine and concentrated in vacuo to yield the title compound, pure methyl ester (purity checked by  1 H NMR). 
     H 1  NMR (CDCl 3 ): 5.56-5.34 (m, 4H), 4.01 (m, 1H), 3.87 (m, 1H), 3.87 (m, 1H), 3.63 (s, 3H), 3.18 (t, 2H, J=5.8 Hz), 2.31-2.02 (m, 8H), 1.69-1.25 (m, 12H), 0.849 (t, 3H, J=6.7 Hz). 
     PGF 2α  1-ethanolamide; (5Z,8α,9α,11α,13E,15S)-9,11,15-trihydroxy-N-(2-hydroxyethyL)prosta-5,13-dien-1-amide 
     To a stirred solution of PGF 2α  methyl ester (235.4 mg, 0.639 mmol) in 4.5 ml anhydrous methanol was added 771 μl ethanolamine (12.78 mmol). The tube containing the mixture was sealed and heated at 50° C. overnight. An aliquot was taken, concentrated in high vacuum with heat to remove excess ethanolamine and analyzed by  1 H NMR. The reaction was indicated to be 80% complete. The solution was concentrated under high vacuum at 65° C. for 1.5 hr, then at 60° C. overnight by Kugehlrohr distillation to remove residual ethanolamine.  1 H NMR and TLC in 5% methanol/ethylacetate indicated removal of the ethanolamine. (methyl ester R f =0.41, PGF 2α  1-ethanolamide R f =0.04). The crude material was flash chromatographed with 5% methanol/ethyl acetate to recover the remaining unreacted starting material, methyl ester in fractions 4-8 and 50%, and in methanol/ethyl acetate to yield the desired title compound in fractions 11-18. Further flash chromatography of fractions 11-18 with 15% methanol/ethyl acetate yielded the title compound free of starting material but still contaminated with with the C15 epimer in some fractions (fractions 7-10), and the desired product in pure state (in fractions 11-20). The  1 H+ 13 C NMR spectra of the title compound and its C 15 epimer are identical. Theoretical yield=254.0 mg, actual yield obtained=205.6 mg, 81%. 
     H 1  NMR (CD 3 OD): 5.4-5.2 (m, 4H), 3.98 (m, 1H), 3.89 (m, 1H), 3.72 (m, 1H), 3.47 (t, 2H, J=5.8 Hz,), 3.18 (t, 2H, J=5.8 Hz), 2.24-1.90 (m, 8H), 1.50-1.21 (m, 12H), 0.796 (t, 3H, J=6.5 Hz).