Classifications

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  • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
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  • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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  • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
  • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
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  • A61K31/60—Salicylic acid; Derivatives thereof
  • A61K31/603—Salicylic acid; Derivatives thereof having further aromatic rings, e.g. diflunisal
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  • A61K31/612—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid
  • A61K31/616—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid by carboxylic acids, e.g. acetylsalicylic acid
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Definitions

• This invention relates to treating or preventing certain diseases or conditions using therapeutically active compositions.
• this invention relates to methods using certain prostaglandin EP 4 agonist components to treat or prevent certain diseases or conditions of the gastrointestinal tract, including, without limitation, those conditions having an effect on the esophagus, stomach, small intestine, and/or large intestine.
• Prostaglandins potentiate members of a subfamily of the G-protem-coupled receptor (GPCR) family of cell surface receptors.
• GPCR G-protem-coupled receptor
• Members of the GPCR family of receptors are characterized by being folded and configured in such a manner as to cross the cell membrane seven times; thus, these receptors are also sometimes referred to as seven-transmembrane receptors .
• receptors are termed DP1-2, EP1-4, FP, IP, and TP, with the nomenclature corresponding to the receptor that binds and is potentiated by the corresponding prostaglandin (e.g., EP 4 receptors bind to and are potentiated by prostaglandin E 4 or PGE 4 ) .
• Prostaglandins thus act on a variety of cells such as vascular smooth muscle cells causing constriction or dilation, on platelets causing aggregation or disaggregation and on spinal neurons causing pain.
• Prostaglandins have a wide variety of actions, including, but not limited to muscular constriction and the mediation of inflammation. Other effects include calcium movement, hormone regulation and cell growth control.
• Thromboxane is created m platelets and causes vascular constriction and platelet aggregation.
• Prostacyclin is synthesized by cells in the blood vessel walls and is antagonistic to thromboxane.
• Prostaglandins can be described with reference to their structural similarity to prostanoic acid, and have the following structural formula and numbering scheme:
• prostaglandins are known, depending on the structure of and substituents carried on the alicyclic ring of the prostanoic acid skeleton. Further classification of prostaglandins is based on the number of unsaturated bonds in the side chain indicated by numerical subscripts after the generic type of prostaglandin [e.g. prostaglandin Ei (PGEi), prostaglandin E2 (PGE2)], and on the configuration of the substituents on the alicyclic ring indicated by ⁇ or ⁇ [e.g. prostaglandin F2 ⁇ (PGF2 ⁇ ) ] .
• PGEi prostaglandin Ei
• PGE2 prostaglandin E2
• prostaglandins In addition to the naturally occurring prostaglandins, various non-naturally occurring analogs, derivatives, and other compounds having some structural similarity to the prostaglandins have also been discovered to have activity at the prostaglandin receptors in general, and the EP 4 receptor in particular.
• a compound of particular interest that has prostaglandin EP 4 receptor agonist activity has the chemical name (Z) -7- ⁇ (IR, 4S, 5R) -5- [ (E) -5- (3-chloro- benzo [b] thiophene-2-yl) -3-hydroxy-pent-l-enyl] -A- hydroxy-3, 3-dimethyl-2-oxo-cyclopentyl ⁇ -hept-5-enoic acid, and is disclosed in U.S. Patent Publication No. 2005/0164992, hereby incorporated by reference herein.
• Prostaglandin EP 4 receptor selective agonists are believed to have several medical uses. For example, U.S. Patent No.
• 6,552,067 B2 expressly incorporated by reference herein, teaches the use of prostaglandin EP 4 receptor selective agonists for the treatment of "methods of treating conditions which present with low bone mass, particularly osteoporosis, frailty, an osteoporotic fracture, a bone defect, childhood idiopathic bone loss, alveolar bone loss, mandibular bone loss, bone fracture, osteotomy, bone loss associated with periodontitis, or prosthetic ingrowth in a mammal . "
• U.S. Patent No. 6,586,468 Bl expressly incorporated by reference herein, teaches that certain prostaglandin EP 4 receptor selective agonists "are useful for the prophylaxis and/or treatment of immune diseases (autoimmune diseases (amyotrophic lateral sclerosis (ALS), multiple sclerosis, Sjoegren's syndrome, arthritis, rheumatoid arthritis, systemic lupus erythematosus, etc.), post-transplantation graft rejection, etc.), asthma, abnormal bone formation, neurocyte death, pulmopathy, hepatopathy, acute hepatitis, nephritis, renal insufficiency, hypertension, myocardial ischemia, systemic inflammatory syndrome, pain induced by ambustion, sepsis, hemophagocytosis syndrome, macrophage activation syndrome, Still's diseases, Kawasaki diseases, burns, systemic granuloma, ulcerative colititis, Crohn's diseases, hypercytokin
• Patent Publication No. 2005/0164992 incorporated by reference above and commonly owned by the current assignee discloses the use of certain prostaglandin EP 4 receptor agonists for the treatment of inflammatory bowel disease (IBD), characterized by inflammation in the large or small intestines and is manifest in symptoms such as diarrhea, pain, and weight loss.
• IBD inflammatory bowel disease
• Nonsteroidal anti-inflammatory drugs are thought to be associated with the risk of developing IBD, and Kabashima and colleagues have disclosed that "EP 4 works to keep mucosal integrity, to suppress the innate immunity, and to downregulate the proliferation and activation of CD4+ T cells.
• gastroesophageal reflux disease acute and chronic gastritis (including, but not limited to Heliobacter pylori-induced gastritis, atrophic gastritis, pernicious anemia, stress-related mucosal damage, alcohol gastropathy, postoperative alkaline gastritis, and eosinophilic gastroenteritis)
• gastritis including, but not limited to Heliobacter pylori-induced gastritis, atrophic gastritis, pernicious anemia, stress-related mucosal damage, alcohol gastropathy, postoperative alkaline gastritis, and eosinophilic gastroenteritis
• duodenitis diverticulitis
• Zolliger-Ellison syndrome chmotherepy-induced gastrointestinal toxicity
• radiation-induced gastrointestinal toxicity and wounds or damage to the gastrointestinal tract caused by injury or surgery.
• the present invention relates to methods and compositions for treating or preventing one or more diseases or conditions of the gastrointestinal tract of for example, the mammalian body.
• the present invention relates to methods and compositions for treating gastrointestinal conditions including gastroesophageal reflux disease, acute and chronic gastritis (including, but not limited to Heliobacter pylor ⁇ -induced gastritis, atrophic gastritis, pernicious anemia, stress-related mucosal damage, alcohol gastropathy, postoperative alkaline gastritis, and eosinophilic gastroenteritis), duodenitis, diverticulitis, Zolliger-Ellison syndrome, chemotherapy-induced gastrointestinal toxicity, radiation-induced gastrointestinal toxicity, and wounds or damage to the gastrointestinal tract caused by injury or surgery.
• gastroesophageal reflux disease including, but not limited to Heliobacter pylor ⁇ -induced gastritis, atrophic gastritis, pernicious anemia, stress-related mucosal damage, alcohol gastro
• Treating or preventing such disease (s) or condition (s) provides one or more substantial advantages, for example, enhances or maintains the health of the individual, for example, human or animal, afflicted with or prone to affliction with such disease(s) or condition (s) .
• the present invention is drawn to methods comprise administering a therapeutically effective amount of a therapeutically effective composition
• a therapeutically effective composition comprising a prostaglandin EP 4 agonist having a structure as follows (and salts and esters thereof, as well as mixtures of these) :
• the invention is drawn to pharmacologically effective compositions comprising a prostaglandin EP 4 agonist having a structure as follows (and salts and esters thereof, as well as mixtures of these) :
• the therapeutically effective composition is administered to a human.
• the prostaglandin EP 4 agonist component may be administered, for example, orally administered, to the gastrointestinal tract of a mammal, for example, a human .
• Fig. IA shows a Western blot of a polyacrylamide electrophoresis gel of an IEC-18 cell lysate having bands for the Akt protein, in which an antibody selective for phophorylated Akt has been secondarily used as a probe of Akt species.
• Lanes include samples from: cells treated with vehicle (0.1% DMSO) alone, cells treated with 10 ⁇ M celecoxib, cells treated with
• Fig IB shows a Western blot of a polyacrylamide electrophoresis gel of an IEC-18 cell lysate having bands for the Akt protein, in which an antibody selective for phophorylated Akt has been secondarily used as a probe of Akt species.
• Lanes include samples from: vehicle-treated cells, cells treated with indomethacm (10 ⁇ M) alone, and cells treated with indomethacm and 10 nM Compound 7.
• Fig. 2 shows flow cytometry (FACS) scans of IEC- 18 cells treated with vehicle alone (Fig. 2A) , cells treated with both doxorubicin (005 ⁇ g/ml) and celecoxib (10 ⁇ M) (Fig. 2B), and cells treated with both doxorubicin and celecoxib and 10 nM Compound 7 (Fig. 2C) .
• FACS flow cytometry
• Fig. 2D is a bar graph showing the relative degrees of apoptosis in these samples.
• Fig. 3A is a bar graph showing ulcer sizes in a mouse ulcer model, wherein non-control mice were given indomethacm .
• Fig. 3B is a bar graph showing white blood cells, lymphocytes, neutrophils and monocyte cell counts in a mouse ulcer model, wherein non-control mice were given indomethacm .
• Fig. 3C is a bar graph showing red blood cell, hemoglobin (HGB) and hematocrit levels in a mouse ulcer model, wherein non-control mice were given indomethacm .
• Fig. 4A is a bar graph showing ulcer sizes in a mouse ulcer model, wherein control mice were given vehicle and non-control mice were given Compound 7.
• Fig. 4B is a bar graph showing neutrophil and necrosis levels in granulation tissue in a mouse ulcer model, wherein control mice were given vehicle and non-control mice were given Compound 7.
• Figure 4C shows photographs comparing gross and microscopic morphologies of ulcer tissue in a mouse ulcer model, wherein control mice were given vehicle and non-control mice were given Compound 7.
• Fig. 5A is a bar graph showing red blood cell, hemoglobin (HGB) and hematocrit levels in a mouse ulcer model, wherein control mice were given indomethacm (3 mg/kg/d) and non-control mice were given indomethacm (3 mg/kg/d) and Compound 7 (0.1 mg/kg/d) .
• Figure 5B is a bar graph showing white blood cell
• WBC lymphocyte
• NEU neutrophil
• a prostaglandin EP 4 agonist is broadly defined as a compound that the person of ordinary skill in the art reasonably believes agonizes a prostaglandin EP 4 receptor according to any one or more of numerous assays for determination of the EP 4 activity that are well known to such persons.
• one such assay system is known as the RSAT assay technology disclosed in part in Brann, U.S. Patent No. 5,707,798, hereby incorporated herein by reference.
• This assay system is commonly used to assay the effects (rather than simply the binding) of putative modulators of members of the G-protein coupled receptor (GPCR) superfamily of cell surface receptors at a given cloned receptor.
• GPCR G-protein coupled receptor
• Other assays that may be used to determine the activity of a modulator of the prostaglandin EP4 receptor include employing cells expressing and displaying recombinant EP4 receptor in an assay of intracellular calcium signaling.
• a fluorescent dye such as the Fluo-4 ® dye
• the subsequent release of Ca ++ from intracellular depots is detected as an increase in fluorescence at the emission maximum of Fluo-4 ® (between 510 and 570 ran) .
• Prostaglandin EP 4 agonist activity can therefore be measured by a change in intracellular calcium concentration using a device such as the FLIPR ® apparatus .
• Prodrugs of the prostaglandin EP 4 agonists disclosed and used in U.S. Patent Publication No. 2005/0164992, incorporated by reference in its entirety as part of this specification above and commonly owned by the current assignee, are contemplated to be within the scope of the methods and compositions of present invention as they may apply to the particular EP 4 receptor agonists disclosed herein.
• EP4 agonist compounds disclosed herein may be administered as therapeutic agents as the biologically active compound or as one or more prodrug, for example and without limitation, those derived from or containing alkene, alkyne and ester linkages; an ester, ether, or amide of a carbohydrate; an ester, ether, or amide of an amino acid; a glucoside ester, ether, or amide; a glucuronide ester, ether, or amide; a cyclodextrin ester, ether, or amide; or a dextran ester, ether, or amide, and mixtures thereof.
• oral dosage forms may include solid forms and semi-solid forms (such as tablets, hard and soft capsules, including liquid or gel filled capsules or tablets) , and aqueous and non-aqueous liquid forms, including but not limited to, oil-m-water and water- in-o ⁇ .1 emulsions, liquid suspensions, solutions, syrups and the like, are known in the art.
• Specific formulations of oral dosage forms include, without limitation, 1) contacting the drug with compatible excipients, for example, conventional excipients, including, without limitation, oils, such as hydrogenated castor oil other vegetable oils, and the like and mixtures thereof; cellulosic derivatives and starch derivatives, such as alkyl celluloses, hydroxyl alkyl celluloses, alkali metal starch carboxylates, e.g., sodium starch glycolate, and the like and mixtures thereof; and sugars and sugar derivatives and the like and mixtures thereof; so that the drug is released m the upper or lower gastrointestinal tract, for example, esophagus, stomach, duodenum and colon.
• compatible excipients for example, conventional excipients, including, without limitation, oils, such as hydrogenated castor oil other vegetable oils, and the like and mixtures thereof; cellulosic derivatives and starch derivatives, such as alkyl celluloses, hydroxyl alkyl celluloses, alkali metal starch carboxy
• a prodrug with compatible excipients, for example the conventional excipients noted in 1) above, with the prodrug being selected so that the drug is released in the upper gastrointestinal tract and/or lower gastrointestinal tract, as desired, 3) coating the drug and/or prodrug with, or encapsulating or impregnating the drug and/or prodrug into, a polymer such as a alkyl cellulose or derivative thereof and/or gelatin designed for delivery to the upper and or lower gastrointestinal tract, 4) formulations for time released delivery of the drug and/or prodrug, 5) use of a bioadhesive system, such as a transdermal patch and the like.
• compatible excipients for example the conventional excipients noted in 1) above, with the prodrug being selected so that the drug is released in the upper gastrointestinal tract and/or lower gastrointestinal tract, as desired
• a polymer such as a alkyl cellulose or derivative thereof and/or gelatin designed for delivery to the upper and or lower gastrointestinal tract
• compositions or dosage forms may additionally comprise other pharmaceutically acceptable excipients, such as tonicity components, buffer components, electrolyte components, thickeners, fillers, diluents, flavoring agents, coloring agents, antioxidants, preservatives (such as antibacterial or antifungal agents) , acids and/or bases to adjust pH, and the like and mixtures thereof.
• each such additive if present, may typically comprise about 0.0001% or less or about 0.01% or less to about 10% or more by weight of the 5 composition.
• additives may include those additives which are conventional and/or well known for use in similar pharmaceutical compositions.
• suitable thickening agents include any of those known in the art, as for example iO pharmaceutically acceptable polymers and/or inorganic thickeners.
• Such agents include, but are not limited to, polyacrylate homo- and co-polymers; celluloses and cellulose derivatives; polyvinyl pyrrolidones; polyvinyl resins; silicates; and the like and mixtures i5 thereof.
• an azo-based prodrug may be employed to provide the drug in the lower gastrointestinal tract.
• Lower intestinal microflora are believed to be capable of reductive
• Bacteria of the lower gastrointestinal tract also have enzymes which can digest glycosides, glucuronides, cyclodextrins, dextrans, and other carbohydrates, and ester prodrugs
• Carbohydrate polymers including, without limitation, amylase, arabinogalactan, chitosan,
• chondroiton sulfate, dextran, guar gum, pectin, xylm, and the like and mixtures thereof can be used to coat a drug and/or prodrug, or a drug and/or prodrug may be impregnated or encapsulated in the polymer.
• the polymers After oral administration, the polymers remain stable in the upper gastrointestinal tract, but are digested by the microflora of the lower gastrointestinal tract thus releasing the drug for therapeutic effect.
• Polymers that are sensitive to pH may also be used since the lower gastrointestinal tract has a higher pH than the upper gastrointestinal tract.
• Such polymers are commercially available.
• Rohm Pharmaceuticals, Darmstadt, Germany markets pH dependent methacrylate based polymers and copolymers sold under the trademark Eudragit®, which have varying solubilities over different pH ranges based upon the number of free carboxylate groups in the polymer.
• Time release systems, bioadhesive systems, and other delivery systems may also be employed.
• Coadministration of prostaglandin EP 4 agonists with one or more other drugs, such as drugs other than EP4 agonists, either in a single composition or in separate dosage forms, is also contemplated.
• Anti-inflammatory drugs such as non-selective COX inhibitors and selective COX-2 inhibitors including without limitation, aspirin and aspirin derivatives, acetomenifen, lbuprofen, ketorolac, napoxen, piroxicam, nabumetone, diclofenac, diflunisal, etodolac, fenoprofen, ketoprofen, mefenamic acid, meloxicam, nabumetone, oxaprozm, sulindac, and tolmetin and the like and mixtures thereof; indoles, such as indomethacin and the like; diarylpyrazoles, such as celecoxib and the like; pyrrolo pyrroles; other agents that inhibit prostaglan
• Such other drug or drugs are administered in amounts effective to provide the desired therapeutic effect or effects.
• TP, IP and DP receptors are prepared by cloning the respective coding sequences into the eukaryotic expression vector pCEP 4 (Invitrogen) .
• the pCEP 4 vector contains an Epstein-Barr virus (EBV) origin of replication, which permits episomal replication in primate cell lines expressing EBV nuclear antigen (EBNA-I) . It also contains a hygromycin resistance gene that is used for eukaryotic selection.
• the cells employed for stable transfection are human embryonic kidney cells (HEK-293) that are transfected with and express the EBNA-I protein. These HEK-293-EBNA cells (Invitrogen) are grown in medium containing Geneticin (G418) to maintain expression of the EBNA-I protein.
• HEK-293 cells are grown in DMEM with 10% fetal bovine serum (FBS), 250 ⁇ g ml 1 G418 (Life Technologies) and 200 ⁇ g ml "1 gentamicm or penicillin/streptomycin. Selection of stable transfectants is achieved with 200 ⁇ g ml 1 hygromycin, the optimal concentration being determined by previous hygromycin kill curve studies. For transfection, the cells are grown to 50-60% confluency on 10 cm plates. The plasmid pCEP 4 incorporating cDNA inserts for the respective human prostanoid receptor (20 ⁇ g) is added to 500 ⁇ l of 250 mM CaCl 2 .
• FBS fetal bovine serum
• 250 ⁇ g ml 1 G418 Life Technologies
• 200 ⁇ g ml "1 gentamicm or penicillin/streptomycin Selection of stable transfectants is achieved with 200 ⁇ g ml 1 hygromycin, the optimal concentration being determined by previous h
• HEPES buffered saline x 2 (2 x HBS, 280 mM NaCl, 20 mM HEPES acid, 1.5 mM Na 2 HPO 4 , pH 7.05 - 7.12) is then added dropwise to a total of 500 ⁇ l, with continuous vortexmg at room temperature. After 30 mm, 9 ml DMEM are added to the mixture.
• DNA/DMEM/calcium phosphate mixture is then added to the cells, which is previously rinsed with 10 ml PBS.
• the cells are then incubated for 5 hr at 37 0 C in humidified 95% air/5% CO 2 .
• the calcium phosphate solution is then removed and the cells are treated with 10% glycerol in DMEM for 2 min.
• the glycerol solution is then replaced by DMEM with 10% FBS.
• the cells are incubated overnight and the medium is replaced by DMEM/10% FBS containing 250 ⁇ g ml "1 G418 and penicillin/streptomycin.
• hygromycin B is added to a final concentration of 200 ⁇ g ml "1 .
• hygromycin B resistant clones are individually selected and transferred to a separate well on a 24 well plate. At confluence each clone is transferred to one well of a 6 well plate, and then expanded in a 10 cm dish. Cells are maintained under continuous hygromycin selection until use.
• Radioligand binding studies are a useful initial step in screening for prospective modulators of the prostaglandin EP receptor; however, binding studies alone can give no information concerning whether a binding ligand is an agonist, inverse agonist, or antagonist of the receptor.
• Radioligand binding studies on plasma membrane fractions prepared from cells are performed as follows. Cells washed with TME buffer are scraped from the bottom of the flasks and homogenized for 30 sec using a Bnnkman PT 10/35 polytron. TME buffer is added as necessary to achieve a 40 ml volume in the centrifuge tubes. TME is comprised of 50 mM TRIS base, 10 mM MgCl 2 , 1 mM EDTA; pH 7.4 is achieved by adding 1 N HCl.
• the cell homogenate is centrifuged at 19,000 rpm for 20-25 mm at 4°C using a Beckman Ti-60 or T ⁇ -70 rotor. The pellet is then resuspended in TME buffer to provide a final protein concentration of 1 mg/ml, as determined by Bio-Rad assay. Radioligand binding assays are performed in a 100 ⁇ l or 200 ⁇ l volume.
• Ci/mmol is determined in duplicate and in at least 3 separate experiments. Incubations are for 60 mm at
• the criteria for inclusion are >50% specific binding and between 500 and 1000 displaceable counts or better.
• HEK-293 (EBNA) cells stably expressing one type or subtype of recombinant human prostaglandin receptors (prostaglandin receptors expressed: hDP/Gqs5; hEP 1 ; hEP 2 /Gqs5; hEP 3ft /Gqi5; hEP 4 /Gqs5; hFP; hIP; hTP) , are cultured in 100 mm culture dishes in high-glucose DMEM medium containing 10% fetal bovine serum, 2 mM 1- glutamme, 250 ⁇ g/ml geneticm (G418) and 200 ⁇ g/ml hygromycm B as selection markers, and 100 units/ml penicillin G, 100 ⁇ g/ml streptomycin and 0.25 ⁇ g/ml amphotericin B.
• Transfectant cells are seeded at a density of 5 xlO 4 cells per well in Biocoat*.
• Cells are then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 ⁇ iM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems) .
• Cells are excited with an Argon laser at 488 nm, and emission measured through a 510-570 nm bandwidth emission filter (FLIPR. TM., Molecular Devices, Sunnyvale, Calif.). Drug solution is added in a 50 ⁇ l volume to each well to give the desired final concentration. The peak increase in fluorescence intensity is recorded for each well.
• FLIPR. TM. Molecular Devices, Sunnyvale, Calif.
• HBSS-HEPES buffer On each plate, four wells each serve as negative (HBSS-HEPES buffer) and positive controls (standard agonists: BW245C (hDP) ; PGE 2 (hEPi ; hEP 2 /Gqs5; hEP 3A /Gqi5; hEP 4 /Gqs5) ; PGF 2 ⁇ ; (hFP); carbacyclm (hIP) ; U-46619 (M 1 P) , depending on receptor) .
• the peak fluorescence change in each drug-containing well is then expressed relative to the controls.
• HTS high-throughput
• CoRe concentration-response
• cAMP assays may employ the detection of changes in the concentrations of intracellular second messengers other than Ca++, such as cyclic AMP (cAMP) .
• cAMP assay methods are very well known. Such methods may include, for example, the cotransfection of nucleic acids encoding the receptor, such as the prostaglandin EP 4 receptor aderenergic receptor, with a cAMP-dependent chloramphenicol acetyl transferase (CAT) reporter plasmid into human JEG-3 choriocarcinoma cells, and challenging the cells with modulators of the receptor.
• CAT chloramphenicol acetyl transferase
• human JEG-3 cells (American Type Culture Collection, Rockville, Md.) are cultured in Delbecco' s Modified Eagle's Medium (DMEM) containing 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 micrograms/ml streptomycin. Cells are plated in 10 cm dishes 1-2 days before transfection. Cells are then transfected with 10 micrograms of a CAT reporter such as plasmid TESBgIIICRE (+) ⁇ NHSE (provided by P.
• DMEM Delbecco' s Modified Eagle's Medium
• FCS fetal calf serum
• FCS fetal calf serum
• P fetal calf serum
• the dosage of the prostaglandin EP 4 agonist component employed in accordance with the present invention varies over a relatively wide range and depends on factors well known in the medicinal arts including, but not limited to: the weight of the individual to whom the agonist component is administered, the general health status/condition of such individual, the disease/condition sought to be treated/prevented by such administration, the severity of such disease/condition in such individual, the potency of the specific EP 4 receptor agonist component being administered, the sensitivity of such individual to the specific agonist component being administered, the mode of administration, the age of such individual, the sex of such individual, the pregnancy status of such individual, the other ongoing drug therapies being administered to such individual and the like factors.
• the amount of prostaglandin EP 4 agonist component employed on a daily basis for each human or animal may be in a range of about 0.1 mg to about 30 mg or about 50 mg or about 100 mg or about 150 mg or about 200 mg or more. In one embodiment, such daily amount may be in a range of about 5 mg to about 150 mg or about 200 mg or more.
• the prostaglandin EP 4 agonist component may be administered in one or more doses daily, for example, once daily, twice daily, three times daily or more frequently. In one embodiment, once daily dosage is useful.
• the prostaglandin EP 4 agonist component is administered for a period of time sufficient to obtain the desired therapeutic effect or effects; that is, relief of the symptoms of the gastrointestinal disorder for which the drug is administered.
• the duration of treatment may be, for example, in a range of about 1 day or about 3 days or about 1 week or about 2 weeks to about 4 weeks or about 8 weeks or about 12 weeks or about 20 weeks or longer. In one useful embodiment, the duration of treatment is in a range of about 2 weeks to about 12 weeks.
• a series of four (4) tablet compositions are produced using a prostaglandin EP 4 agonist and three (3) different prostaglandin EP 4 agonist prodrugs.
• Each of the tablet compositions is prepared as follows.
• a mixture of ingredients is prepared and blended until the mixture is uniform.
• the uniform mixture having a composition as listed in the table directly below, is then used in a conventional tablettmg machine to produce 100 mg tablets having such composition.
• the tablets may be packaged, for example, in high density polyethylene bottles, with appropriate silica gel packs, capped and labeled.
• the mixtures and tablets have the following compositions:
• Each of the tablets that is produced in Examples 1 to 4 includes about 10 mg of the agonist preparation or prodrug preparation, as the case may be, the total weight of each tablet being about 100 mg.
• a series of two (2) hard shell gelatin capsule compositions are produced using a prostaglandin EP 4 agonist and a prostaglandin EP 4 agonist prodrug. Each of these capsule compositions is prepared as follows.
• small sugar spheres are provided within a dust containment area.
• a second mixture including a binder/sealer, e.g., Opadry® clear, in a liquid carrier is sprayed onto the first sprayed spheres using a conventional fluid bed spraying system. This step results in agonist or prodrug loaded pellets with a sealing coat.
• pellets are coated with an aqueous mixture of triethyl citrate, talc and a methacrylic acid copolymer using a conventional fluid bed spraying system. This step results in agonist or prodrug loaded pellets with a sealing coat and an outer enteric coating. These pellets are encapsulated in natural transparent hard shell gelatin capsules.
• the filled capsules may be packaged, for example, in high density polyethylene bottles, with appropriate silica gel packs, capped and labeled.
• the pellets with the enteric coating have the following compositions.
• Prostaglandin EP 4 Agonist 1 (1> 35.5 -
• Each of the capsules that is produced in Examples 5 and 6 includes about 35.5 mg of the agonist or prodrug.
• a series of four (4) soft shell gelatin capsule compositions are produced using a prostaglandin EP 4 agonist and three (3) different prostaglandin EP 4 agonist prodrugs.
• Each of the capsule compositions is prepared as follows.
• a uniform soft shell gelatin mixture which comprises about 20-45% gelatin, about 15-30% water, about 17.5-35% of a plasticizer which in turn is comprised of glycerin or sorbitol or a mixture thereof and about 5-25% of a hydrogenated starch hydrolysate.
• the soft shell gelatin mixture is agitated with heat until a uniform melt results.
• a second mixture which is a uniform suspension or solution of the agonist or agonist prodrug is provided as the soft gelatin capsule fill material.
• the fill material is intended to be substantially water free (less than or equal to about 5-7% water) and includes the agonist or agonist prodrug and optional amounts of co-solvents, buffers, surfactants, thickeners, and the like.
• the fill material may be of solid, semi-solid, gel, or liquid form, so long as it is uniform and is compatible with soft gelatin encapsulation, i.e., it does not substantially degrade the soft gelatin shell.
• the fill material has a composition as listed in the table directly below and is preferably degassed prior to use in the next step.
• the soft shell gelatin preparation above is used to encapsulate 100 mg portions of the fill material employing standard encapsulation technology to produce one-piece, hermetically sealed soft gelatin capsules.
• the soft shell gelatin capsules may be packaged, for example, in high density polyethylene bottles, with appropriate silica gel packs, capped and labeled.
• the fill materials of the capsules have the following make-ups:
• conventional pharmaceutical excipients useful, for example, as co-solvents, buffers, preservatives, surfactants, thickeners, and the like, provided they are substantially water free (less than or equal to about 5-7% water) and are compatible with soft gelatin encapsulation, i.e., they do not substantially degrade the soft gelatin shell.
• examples include, but are not limited to, mixtures of vegetable oils, liquid polyalkylene glycols and the like.
• Each of the capsules that is produced in Examples 7 to 10 includes about 10 mg of the agonist or prodrug, as the case may be, the total weight of each of the capsules depending on the weight of the soft gelatin shell.
• Ten adult humans are diagnosed with gastroesophageal reflux disease. Each of these people orally takes a tablet or a capsule (produced as described in Examples 1 to 10) having a different one of Compositions 1 to 10 once daily for twelve weeks. At the end of this period of time, each of the humans reports substantial relief from the gastroesophageal reflux disease. The pain and/or other symptoms of the disease are reduced.
• the gastritis is either acute or chronic and includes types such as helicobacter pylori-induced gastritis, atrophic gastritis, pernicious anemia induced gastritis, stress related mucosal damage induced gastritis, alcohol gastropathy induced gastritis, postoperative alkaline gastritis and eosinophilic gastroenteritis.
• Each of the ten people orally takes a tablet or a capsule (produced as described m Examples 1 to 10) having a different one of Compositions 1 to 10 once daily for twelve weeks. At the end of this period of time, each of the humans reports substantial relief from the gastritis. The pain and/or other symptoms of the disease are reduced.
• EXAMPLES 81 TO 90 Ten adult humans have wounds from either injury to and/or surgery of the gastrointestinal tract. Each of these people orally takes a tablet or a capsule (produced as described in Examples 1 to 10) having a different one of Compositions 1 to 10 once daily for twelve weeks. At the end of this period of time, each of the humans reports substantial relief from the pain and/or other symptoms of the injury and/or surgery. In addition, the wounds from the injury/surgery have substantially completely healed. EXAMPLES 91-100
• the PI3k (phosphomositide-3-kinase) /Akt phosphorylation pathway is an important cellular signal transduction pathway for the regulation of cell growth, migration, differentiation, and apoptosis.
• the PI3k/Akt pathway is essential for the proliferation of intestinal epithelial cells both in vitro and m vivo. This pathway transduces proliferative signals between receptors and cell cycle machinery in intestinal epithelial cells. Sheng H., et al., GUT 52:1472-1478 (2003) .
• Akt also known as protein kinase B or PKB
• PKB protein kinase B
• PIP 3 phosphatidylmositol (3, 4, 5) -t ⁇ sphosphate
• PIP 3 phosphatidylmositol (3, 4, 5) -t ⁇ sphosphate
• PIP 3 is formed by phosphrylation of the di-phosphorylated phosphinositide Ptdins (4, 5) Pz by activated phosphomositide-3-kmase, known as PI3k. Once formed the PIP 3 can bind to Akt.
• Cycloxygenase is a family of enzymes (COX 1, COX 2) involved m prostaglandin synthesis. The mechanisms of COX 1 regulation are not entirely well understood. However, considerable work has shown that COX 2 synthesis is at least partly regulated by the PI3k (phosphomositide-3-kinase) /Akt phosphorylation pathway. Briefly, upon binding PIP 3 , Akt is partly activated and is able to be phosphorylated by PDKl (3- phosphmositide-dependent kmase-1) .
• Akt Upon phosphorylation, Akt is fully activated, and may in turn phosphorylate IKB kinase, which phosphorylates IKB to which is recruited the transcription factors p65 and p50.
• the phosphorlylated I ⁇ B/p65/p50 complex then enters the cell nucleus and stimulates the transcription of COX-2. See e.g., Little et al., J. VET. INTERN. MED. 21:367-377 (2007).
• the COX-2 inhibitor celecoxib is thought to inhibit the activity of PDKl, therefore also blocking phosphorylation of Akt.
• Rat intestinal epithelial cells are immortalized normal epithelial cells purchased from the American Type Culture Collection (ATCC) .
• IEC-18 cells were cultured in high glucose Delbecco' s Modified Eagle's Medium (DMEM) at 37°C under 5% CO 2 and incubated for 24 hours with either a) 5 ⁇ M of the COX-I inhibitor SC-560 (purchased from Sigma-Ald ⁇ ch, St. Louis, MO), 10 ⁇ M of the COX-2 inhibitor celecoxib, or 10 ⁇ M of the COX- 1/2 inhibitor indomethacin. Drugs were provided in a 0.1% dimethylsulfoxide (DMSO) vehicle.
• DMSO dimethylsulfoxide
• the PI3k/Akt pathway is monitored using the following general assay procedure. Following incubation cell lysates are collected with lysis buffer containing phosphatase and proteinase inhibitors. The supernanats are collected and protein concentrations are measured. Then SDS (sodium dodecyl sulfate) sample buffers are made to make sure final protein concentration is equal in each sample and boiled for 10 minutes at 100°C. Then the samples are loaded on to a 4%-12% pre-made SDS-PAGE (polyacrylamide gel electrophoresis) and the gel is developed under electrophoresis.
• SDS sodium dodecyl sulfate
• the gel is removed from the electrophoresis equipment and transferred to a nitrocellulose membrane and the membrane blocked with milk.
• the membrane is incubated with a primary antibody selective for either a) total Akt or b) phosphrylated Akt overnight at 4 0 C.
• a secondary antibody having a conjugated horseradish peroxidase (HRP) moiety is used to bind the constant regions of the primary antibodies.
• the membrane is washed under conditions favoring selective antibody binding.
• the HRP is detected using a enzymatically initiated chemilummescent substrate such as Super Signal® ELISA Pico Substrates, available from Thermo Fisher Scientific.
• each of these agents inhibited Akt phosphorylation.
• indomethacin inhibited phosphorylation of Akt to a greater extent that did SC-560 or celecoxib under these conditions.
• co-incubation of the cells for 24 hours with both 10 ⁇ M indomethacin and 1OnM of the prostaglandin EP4 agonist of Composition 7 maintained Akt phosphorylation at higher levels compared to those samples provided indomethacin in the absence of added EP4 agonist. Therefore, prostaglandin EP4 agonists such as Compound 7 may restore or strengthen Akt activity, either when used alone, or in the presence of COX inhibitors, such as non-steroidal antiinflammatory drugs (NSAIDS) .
• NSAIDS non-steroidal antiinflammatory drugs
• IEC-18 cells were cultured in high glucose DMEM containing 10% fetal bovine serum (FBS) , 1% penicillin, 1% streptomycin, and 50 unit insulin essentially as indicated in Example 101 for 48 hours in the presence of the 0.05 ⁇ g/ml chemotherapy agent doxorubicin to induce apoptosis.
• FBS fetal bovine serum
• doxorubicin apoptosis of doxorubicin-treated IEC-18 cells proceeds within 48 hours at such very low concentrations of doxorubicin.
• the cells collected by trypsin were subjected to flow cytometry analysis for identification of the percentage of cells in different stages of the cell cycle.
• Cultured cells were prepared for flow cytometry by trypsimzation and cent ⁇ fugation. The cells were then washed with cold phosphate buffered saline solution (PBS), then fixed with cold 70% ethanol overnight. The cells were then again washed with PBS and stained with propidium iodide, a nucleic acid intercalating dye, in the presence of RNase for 30 minutes at room temperature. The cells were then sorted using the buffer provided by Becton-Dickenson, the maker of the flow cytometry equipment.
• PBS cold phosphate buffered saline solution
• Fig. 2A shows florescent flow cytometry analysis of IEC-18 cells in vehicle alone
• Fig. 2B shows flow cytometry analysis under the same conditions (except that the cells have previously been treated with 0.05 ⁇ g/ml doxorubicin (D) and 10 ⁇ M celecoxib (C)
• Fig. 2C shows the results when IEC-18 cells are incubated with 0.05 ⁇ g/ml doxorubicin (D), 10 ⁇ M celecoxib (C) and 10 nM Compound 7.
• Fig 2D shows the percent apoptosis of cells in 0.1% DMSO vehicle alone is approximately 2%.
• the percentage of cells undergoing apoptosis increases to about 13% upon treatment with doxorubicin and lO ⁇ M celecoxib.
• addition of 10 nM Compound 7 to the incubation mixture results in a reduction of apoptosis to approximately 5%, close to the baseline level.
• this experiment shows that Compound 7, a prostaglandin EP4 agonist, reduces the cytotoxic effect of NSAIDS and chemotherapeutic agents upon intestinal epithelial cells, thus providing a protective effect against injury to the gut caused by such agents.
• Gastric ulcers were produced in C57BL/6 mice by using 40% acetic acid as follows. Animals were anesthetized. The abdomen of each animal was surgically opened, and the stomach exposed. A 3 mm curette was placed onto the anterior surface of each stomach. A solution of 40% acetic acid was placed inside the curette. The curette prevents damage to surrounding tissue. The acetic acid treatment causes damage to blood vessels of the stomach, producing chemical death to the mucous layer.
• stomachs were processed for pathological analysis by dissection and section through the widest part of the ulcer. Sections were stained using hematoxylin & eosin.
• LYM lymphocytes
• Fig. 3B shows an x-axis legend of "WBC” (white blood cells) , LYM (lymphocytes; an increase in which is a standard indicator of chronic inflammation) , NEU (neutrophils) and MONO (monocytes ).
• WBC white blood cells
• LYM lymphocytes
• NEU neutrophils
• MONO monocytes
• the latter two cell types are associated with the acute (for example, bacte ⁇ ally- mediated) immune response.
• WBC therefore is a measurement of all white blood cells (including lymphocytes, neutrophils and monocytes) , with the particular cell types then "broken out” in the following bars. Indomethacm was found to exacerbate blood loss at ulcer sites (Fig. 3C) .
• mice were then treated with 10 nM Compound 7 or the vehicle under otherwise identical conditions.
• the vehicle was 4% DMSO in corn oil in a volume of 0.1 ml.
• the mice were sacrificed and the stomachs examined on either the 7 th day or the 11 th day following ulceration.
• the results indicate that mice treated with 10 nM Compound 7 resulted in significantly smaller ulcer sizes than mice treated with vehicle (Fig. 4A) .
• the areas of the ulcers in mice treated with Compound 7 were 60% and 32% of the control on days 7 and 11, respectively.
• Compound 7 had significantly less inflammatory cell infiltrations and necrotic tissue in granulation tissue compared to those from the control group, which may indicate a healthier condition for re- epithelization. (Fig. 4B) .
• Fig. 4C shows the representative gross and microscopic morphologies of the ulcer tissue in this experiment taken after sacrificing the mice treated with the vehicle on day 7 and the same vehicle containing Compound 7, respectively.
• the photographic results correlate with the pathology results and demonstrate that the prostaglandin EP4 agonist Compound 7 stimulates healing of ulcer and facilitate the removal of necrosis tissue.
• Fig. 5A shows that the number of red blood cells ("RBC") x 10 6 / ⁇ l of whole blood is significantly greater in mice administered Compound 7 ("treated mice") as compared to those given the vehicle alone (“control mice”) .
• RBC red blood cells
• HGB hemoglobin
• HCT hematocrit
• Fig. 5B shows that the treated group had significantly lessened inflammatory response than did the control group of mice.
• WBC and lymphocyte counts were significantly higher in the control group than in the treated group, while the level of neutrophils in the blood remains relatively constant.
• prostaglandin EP4 agonists such as Compound 7 are able to cause an acceleration in the healing of stomach ulcers. Additionally, such agents are able to prevent or moderate the cytotoxic effects of NSAIDS (for example, mdomethacm) in the gastrointestinal tract, particularly preventing or moderating the extent of apoptosis in intestinal and gastric epithelial cells.
• NSAIDS for example, mdomethacm
• the present invention also envisions a therapeutic composition
• the NSAID may be any NSAID, but in particular may be selected from the group consisting of aspirin, acetomenifen, mdomethacin, lbuprofen, ketorolac, napoxen, piroxicam, nabumetone, celecoxib, diclofenac, diflumsal, etodolac, fenoprofen, ketoprofen, mefenamic acid, meloxicam, nabumetone, oxaprozin, sulindac, and tolmetin.
• the present invention may comprise a method of treating a patient having a condition responsive to treatment by an NSAID comprising administering to said patient an NSAID and a prostaglandin EP 4 agonist component comprising a structure:
• the prostaglandin EP4 agonist component may comprise a prodrug of the compound corresponding to said structure.

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