Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins

Definitions

• This invention relates to a method of treating cyclooxygenase mediated diseases in patients with ulcers.
• a method of treating cyclooxygenase mediated disease while promoting the healing of certain lesions including gastric ulcers comprising the co- administration of certain prostaglandins and a selective cyclooxygenase-
• Non-steroidal, anti inflammatory drugs exert most of their anti inflammatory, analgesic and antipyretic activity and inhibit hormone-induced uterine contractions and certain types of cancer
• cyclooxygenase-2 has been cloned, sequenced and characterized from chicken, murine and human sources. This enzyme is distinct from the cyclooxygenase- 1 which has now also been cloned, sequenced and characterized from sheep, murine and human sources.
• prostaglandins have both physiological and pathological roles, we have concluded that the constitutive enzyme, cyclooxygenase- 1, is responsible, in large part, for endogenous basal release of prostaglandins and hence is important in
• cyclooxygenase-2 is mainly responsible for the pathological effects of prostaglandins where rapid induction of the enzyme would occur in response to such agents as inflammatory agents, hormones, growth factors, and cytokines.
• a selective inhibitor of cyclooxygenase-2 will have similar anti inflammatory, antipyretic and analgesic properties to a conventional non-steroidal anti inflammatory drug, and in addition would inhibit hormone-induced uterine contractions and have potential anti-cancer effects, but will have a diminished ability to induce some of the mechanism-based side effects.
• such a compound should have a reduced potential for gastrointestinal toxicity, a reduced potential for renal side effects, a reduced effect on bleeding times and possibly a lessened ability to induce asthma attacks in aspirin-sensitive asthmatic subjects.
• US 5,015,481, issued May 14, 1991 discloses the use of defined combinations of NSAID's and prostaglandins for the prevention of NSAID induced ulcers.
• WO 91/16896, published November 14, 1991 discloses defined combinations of NSAID's and prostaglandins to treat mild to moderate pain.
• WO 91/16895, published November 14, 1991 discloses a pharmaceutical composition including a core of an NSAID selected from diclofenac and piroxacam which core is surrounded by a mantle coating of a prostaglandin, wherein an intermediate coating can be present between the NSAID core and the prostaglandin mantle coating.
• US 5,232,704 discloses a sustained release dosage form of prostaglandin which in combination which is said to be useful for the prevention of NSAID induced ulcers.
• NSAID induced gastric ulcers are caused by the cyclooxygenase- 1 activity found inmost NSAID's. Accordingly, the treatment of cyclooxygenase-2 mediated diseases by administration of an NSAID that selectively inhibits cyclooxygenase-2 in substantial preference to cyclooxygenase- 1 eliminates the advantage of co-administering prostaglandin with the NSAID for purposes of preventing NSAID induced ulcers.
• cyclooxygenase-2 also plays an important positive role in gastric mucosal protection and in promoting the healing of certain lesions including gastric ulcers.
• the applicants disclose a method of treating cyclooxygenase mediated disease while promoting the healing of certain lesions including gastric ulcers comprising the co- administration of certain prostaglandins and a selective cycooxygenase-2 inhibitor, or the co-administration of an anti-ulcer agent and a selective cyclooxygenase-2 inhibitor as defined below.
• a method of treating cyclooxygenase mediated disease while promoting the healing of certain lesions including gastric ulcers and protecting the gastric mucosa comprising the co-administration of certain prostaglandin and a selective cyclooxygenase-2 inhibitor, or the co-administration of an anti-ulcer agent and a selective cyclooxygenase- 2 inhibitor as defined below.
• the invention encompasses a method of treating cyclooxygenase mediated disease while promoting the healing of certain lesions including gastric ulcers comprising the co-administration of a prostaglandins and a selective cycooxygenase-2 inhibitor, or the co- administration of an anti-ulcer agent and a selective cyclooxygenase-2 inhibitor as defined below.
• a compound shall be defined as a selective cyclooxygenase-2 inhibitor if the ratio of it's IC50 for the inhibition of cyclooxygenase- 1 divided by it's IC50 for the inhibition of cyclooxygenase-2, as measured as described in this specification or a comparable method is 200 or greater; preferably 1000 or greater.
• selective cyclooxygenase-2 inhibitors includes, but is not limited to compounds of Formula I
• X-Y-Z- is selected from the group consisting of:
• Rl is selected from the group consisting of
• R2 is selected from the group consisting of (a) Cl-6alkyl,
• heteroaryl is a monocyclic aromatic ring of 5 atoms, said ring having one hetero atom which is S, O, or N, and optionally 1, 2, or 3 additionally N atoms; or the heteroaryl is a monocyclic ring of 6 atoms, said ring having one hetero atom which is N, and optionally 1, 2, 3, or 4 additional N atoms; said substituents are selected from the group consisting of
• halo including fluoro, chloro, bromo and iodo
• R3, R4, R5 and R5' are each independently selected from the group consisting of
• Additional selective cyclooxygenase-2 inhibitors within the scope of claimed method include: as well as compounds disclosed in WO 94/13635, published June 23, 1994; US 5,344,911, issued September 6, 1994; and WO 94/15932, published July 21 1994, all of which are hereby incorporated by reference. Additional selective cyclooxygenase-2 inhibitors within the scope of claimed method include Diclofenac, those disclosed in USSN 08/330, filed October 27, 1994; USSN 08/361,268, filed December 21, 1994 and USSN 08/443,620, filed May 18, 1995, all of which are incorporated by method.
• alkyl is defined to include linear, branched, and cyclic structures, with Cl-6alkyl including methyl, ethyl, propyl, 2-propyl, s- and t-butyl, butyl, pentyl, hexyl, 1,1- dimethylethyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
• Cl-6alkoxy is intended to include alkoxy groups of from 1 to 6 carbon atoms of a straight, branched, or cyclic configuration.
• lower alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy, and the like.
• Cl-6alkylthio is intended to include alkylthio groups of from 1 to 6 carbon atoms of a straight, branched or cyclic configuration.
• lower alkylthio groups include methylthio, propylthio, isopropylthio, cycloheptylthio, etc.
• the propylthio group signifies -SCH2CH2CH3.
• the prostaglandin suitable sed method includes the compounds of Formula II
• R is hydrogen or Ci-6alkyl
• Rl is hydrogen, vinyl or Cl-4alkyl and the wavy line represents R or S stereochemistry
• R2, R3, and R4 are hydrogen or Cl-4alkyl or R2 and R3 together with carbon b form a cycloalkenyl having 4 to 6 carbon atoms or R3 and R4 together with carbons a and b form a cycloalkenyl having 4 to 6 carbons and wherein the a-b bond can be saturated or unsaturated.
• prostaglandins include misoprostol, ⁇ methyl Hoc, 16-dihydroxy-16-methyl-9-oxo ⁇ rost 13E-en-l-oate; enisoprost and methyl-7- [2B- [6-( 1 -cyclopenten- 1 -yl)-4-hydroxy-4-methyl- 1 E, 5E- hexadienyl]-3 ⁇ -hydroxy-5-oxo IR, l ⁇ -cyclo ⁇ entyl]-4Z-heptenoate.
• Prostaglandins within the scope of the invention also include arbaprostil, enprostil, rioprostol, nocloprost, mexiprostil, ornoprostol, dimoxaprost, tiprostanide, and rosaprostol.
• applicants method includes the use of the prostaglandin misoprostol with Diclofenac.
• the dashed line indicates the grouping being behind the plane of the paper and the solid, blackened triangular shape indicates that the group is in front of the plane of the paper.
• prostaglandins useful in the composition of the invention herein can be prepared by known reaction schemes such as by the methods taught in U.S. Pat. Nos. 3,965,143; 4,271,314; and 4,683,328 and in an article by P.W. Collins and J.W. Djurie, Chem. Rev. 1993, £2., 1533-1564..
• the individual isomers can be obtained by chromatographic separation.
• the prostaglandin is preferably an orally available prostaglandin.
• the prostaglandin is misoprostol, ( ⁇ ) methyl 11 alpha, 16 dihydroxy 16 methyl-9-oxoprostl3E-en-l-oate
• the misoprostol is present in an amount of about 100 to 200 meg (micrograms).
• the anti-ulcer agent shall be defined to include cimetidine, famotidine, omeprazole, ranitidine and the like.
• the co- administration of a selective cyclooxygenase-2 inhibitor with an additional active agent includes situations wherein both active agents are provided in a single dosage orm as well as situations wherein the active agents are provided in separate dosage forms.
• an additional active agent such as a prostaglandin or anti-ulcer agent
• both active agents are provided in a single dosage orm as well as situations wherein the active agents are provided in separate dosage forms.
• Some of the compounds described herein contain one or more asymmetric centers and may thus give rise to diastereomers and optical isomers.
• the present invention is meant to comprehend such possible diastereomers as well as their racemic and resolved, enantiomerically pure forms and pharmaceutically acceptable salts thereof.
• compositions of the present invention comprise a compound of Formula I as an active ingredient or a pharmaceutically acceptable salt, thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.
• pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts.
• Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N_- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropy lamine, tromethamine, and the like.
• basic ion exchange resins such as arg
• the Compound of Formula I is useful for the relief of pain, fever and inflammation of a variety of conditions including rheumatic fever, symptoms associated with influenza or other viral infections, common cold, low back and neck pain, dysmenorrhea, headache, toothache, sprains and strains, myositis, neuralgia, synovitis, arthritis, including rheumatoid arthritis degenerative joint diseases (osteoarthritis), gout and ankylosing spondylitis, bursitis, burns, injuries, following surgical and dental procedures.
• a compound may inhibit cellular neoplastic transformations and metastic tumor growth and hence can be used in the treatment of cancer.
• Compounds of formula I may also be useful for the treatment of dementia including pre-senile and senile dementia, and in particular, dementia associated with Alzheimer Disease (ie Alzheimer's dementia).
• Compounds of formula I will also inhibit prostanoid- induced smooth muscle contraction by preventing the synthesis of contractile prostanoids and hence may be of use in the treatment of dysmenorrhea, premature labor and asthma.
• non-steroidal antiinflammatory drugs such as in patients with peptic ulcers, gastritis, regional enteritis, ulcerative colitis, diverticulitis or with a recurrent history of gastrointestinal lesions; GI bleeding, coagulation disorders including anemia such as hypoprothrombinemia, haemophilia or other bleeding problems (including those relating to reduced or impaired platelet function); kidney disease (eg impaired renal function); those prior to surgery or taking anticoagulants; and those susceptable to NSAID induced asthma.
• NSAID'S non-steroidal antiinflammatory drugs
• compositions for treating cyclooxygenase-2 mediated diseases as defined above comprising a non-toxic therapeutically effective amount of the compound of Formula I as defined above and one or more ingredients such as another pain reliever including acetominophen or phenacetin; a potentiator including caffeine; an H2- antagonist, aluminum or magnesium hydroxide, simethicone, a decongestant including phenylephrine, phenylpropanolamine, pseudophedrine, oxymetazoline, ephinephrine, naphazoline, xylometazoline, propylhexedrine, or levo-desoxyephedrine; an antiitussive including codeine, hydrocodone, caramiphen, carbetapentane, or
• the invention encompasses a method of treating cyclooxygenase mediated diseases comprising: administration to a patient in need of such treatment a non-toxic therapeutically effect amount of the compound of Formula I, optionally co-administered with one or more of such ingredients as listed immediately above.
• compositions for treating cyclooxygenase-2 mediated diseases as defined may include one or more ingredients as listed above as well as a compound of formula II.
• compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
• Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
• excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc.
• the tablets may be uncoated or they may be coated by known techniques to delay disintegration and abso ⁇ tion in the gastrointestinal tract and thereby provide a sustained action over a longer period.
• a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the technique described in the U.S. Patent 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.
• Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
• an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
• an oil medium for example peanut oil, liquid paraffin, or olive oil.
• Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions.
• excipients are suspending agents, for example sodium carboxymethyl-cellulose, methylcellulose, hydroxy- propylmethycellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene- oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene
• the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n- propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
• preservatives for example ethyl, or n- propyl, p-hydroxybenzoate
• coloring agents for example ethyl, or n- propyl, p-hydroxybenzoate
• coloring agents for example ethyl, or n- propyl, p-hydroxybenzoate
• flavoring agents such as sucrose, saccharin or aspartame.
• sweetening agents such as sucrose, saccharin or aspartame.
• Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin.
• the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
• Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
• a dispersing or wetting agent e.g., glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerin, glycerin, glycerin, glycerin, glycerin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol
• the pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsions.
• the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
• Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
• the emulsions may also contain sweetening and flavouring agents.
• Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
• the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
• Suitable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid find use in the preparation of injectables.
• Compounds of formula I may also be administered in the form of a suppositories for rectal administration of the drug.
• These compositions can be prepared by mixing the drug with a suitable non ⁇ irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
• a suitable non ⁇ irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
• Such materials are cocoa butter and polyethylene glycols.
• creams, ointments, jellies, solutions or suspensions, etc., containing the compound of Formula I are employed. (For pu ⁇ oses of this application, topical application shall include mouth washes and gargles.)
• Specific cyclooxygenase-2 inhibitor dosage levels of the order of from about 0.01 mg to about 140 mg/kg of body weight per day are useful in the treatment of the above-indicated conditions, or alternatively about 0.5 mg to about 7 g per patient per day.
• inflammation may be effectively treated by the administration of from about 0.01 to 50 mg of the compound per kilogram of body weight per day, or alternatively about 0.5 mg to about 3.5 g per patient per day.
• typical dosages by be as much as 25 to 1600 ⁇ g per day; more typically 200 to 800 ⁇ g per day (eg 200, 400, 600 or 800 ⁇ g per day.
• Single dosage forms may typically contain 5, 25, 50, 100, 200, 250, 400 or 500 ⁇ g per tablet.
• the misoprostol is present in the dosage form (eg tablet) in an amount of about 100 to 200 ⁇ g. See, for example, a Physicans Desk Reference (PDR).
• PDR Physicans Desk Reference
• the dosages may typically range from 10 to 800 mg per day or more, with single dosages containing 10, 20, 30 ,100, 200, 400 or 800 mg of active agent.
• the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
• a formulation intended for the oral administration of humans may contain from 0.5 mg to 5 g of specific cyclooxygenase-2 inhibitor compounded with a prostaglandin of formula II and an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition.
• Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg of specific cyclooxygenase-2 inhibitor.
• the compound of Formula I can be tested using the following assays to determine their cyclooxygenase-2 inhibiting activity.
• osteosarcoma cells are cultured in 1 mL of media in 24-well multidishes (Nunclon) until confluent (1-2 x 10 cells/well).
• U-937 cells are grown in spinner flasks and resuspended to a final density of 1.5 x 10 cells/mL in 24-well multidishes (Nunclon).
• 1 ⁇ L of a DMSO solution of test compound or DMSO vehicle is added, and samples gently mixed. All assays are performed in triplicate.
• CHO Chinese hamster ovary
• CHO[hCOX-l] cells from suspension cultures and CHO[hCOX-2] cells prepared by trypsinization of adherent cultures are harvested by centrifugation (300 x g, 10 min) and washed once in HBSS containing 15 mM HEPES, pH 7.4, and resuspended in HBSS, 15 mM HEPES, pH 7.4, at a cell concentration of 1.5 x 10 cells/ml.
• Drugs to be tested are dissolved in DMSO to 66.7- fold the highest test drug concentration. Compounds are typically tested at 8 concentrations in duplicate using serial 3-fold serial dilutions in DMSO of the highest drug concentration.
• PGE2 levels of cells challenged with arachidonic acid versus the PGE2 levels in cells mock-challenged with ethanol vehicle. Inhibition of PGE2 synthesis by test compounds is calculated as a percentage of the activity in the presence of drug versus the activity in the positive control samples.
• U 937 cells are pelleted by centrifugation at 500 x g for 5 min and washed once with phosphate-buffered saline and repelleted.
• Cells are resuspended in homogenization buffer consisting of 0.1 M Tris-HCl, pH 7.4, 10 mM EDTA, 2 ⁇ g/ml leupeptin, 2 ⁇ g/ml soybean trypsin inhibitor, 2 ⁇ g/ml aprotinin and 1 mM phenyl methyl sulfonyl fluoride.
• the cell suspension is sonicated 4 times for 10 sec and is centrifuged at 10,000 x g for 10 min at 4° C.
• the supernatant is centrifuged at 100,000 x g for 1 hr at 4 ° C.
• the 100,000 x g microsomal pellet is resuspended in 0.1 M Tris-HCl, pH 7.4, 10 mM EDTA to approximately 7 mg protein/ml and stored at -80° C.
• Microsomal preparations are thawed immediately prior to use, subjected to a brief sonication, and then diluted to a protein concentration of 125 ⁇ g/ml in 0.1 M Tris-HCl buffer, pH 7.4 containing 10 mM EDTA, 0.5 mM phenol, 1 mM reduced glutathione and 1 ⁇ M hematin. Assays are performed in duplicate in a final volume of 250 ⁇ l. Initially, 5 ⁇ l of DMSO vehicle or drug in DMSO are added to 20 ⁇ l of 0.1 M Tris-HCl buffer, pH 7.4 containing 10 mM EDTA in wells of a 96-deepwell polypropylene titre plate.
• the enzyme activity is measured using a chromogenic assay based on the oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) during the reduction of PGG2 to PGH2 by COX-2 (Copeland et al. (1994) Proc. Natl. Acad. Sci. 91, 11202-11206).
• TMPD N,N,N',N'-tetramethyl-p-phenylenediamine
• Recombinant human COX-2 is purified from Sf9 cells as previously described (Percival et al (1994) Arch. Biochem. Biophys. 15, 111-118).
• the assay mixture (180 ⁇ L) contains 100 mM sodium phosphate, pH 6.5, 2 mM genapol X-100, 1 ⁇ M hematin, 1 mg/ml gelatin , 80-100 units of purified enzyme (One unit of enzyme is defined as the amount of enzyme required to produce an O.D. change of 0.00 l/min at 610 nm) and 4 ⁇ L of the test compound in DMSO.
• the mixture is pre-incubated at room temperature (22°C) for 15 minutes prior to initiation of the enzymatic reaction by the addition of 20 ⁇ L of a sonicated solution of 1 mM arachidonic acid (AA) and 1 mM TMPD in assay buffer (without enzyme or hematin).
• AA arachidonic acid
• TMPD TMPD in assay buffer (without enzyme or hematin).
• the enzymatic activity is measured by estimation of the initial velocity of TMPD oxidation over the first 36 sec of the reaction. A non-specific rate of oxidation is observed in the absence of enzyme (0.007 - 0.010 O.D. /min) and is subtracted before the calculation of the % inhibition.
• IC50 values are derived from 4- parameter least squares non-linear regression analysis of the log-dose vs % inhibition plot.
• Human whole blood provides a protein and cell-rich milieu appropriate for the study of biochemical efficacy of anti-inflammatory compounds such as selective COX-2 inhibitors.
• This assay can be used to evaluate the inhibitory effect of selective COX-2 inhibitors on PGE2 production.
• platelets in whole blood contain a large amount of the COX- 1 enzyme. Immediately following blood clotting, platelets are activated through a thrombin- mediated mechanism.
• TxB2 thromboxane B2
• COX-1 thromboxane B2
• the degree of selectivity by the test compound can be determined by measuring the levels of PGE2 after LPS induction (COX-2) and TxB2 following blood clotting (COX-1) in the same assay.
• Fresh blood is collected in heparinized tubes by venipuncture from both male and female volunteers. The subjects have no apparent inflammatory conditions and have not taken any NSAIDs for at least 7 days prior to blood collection. Plasma is immediately obtained from a 2mL blood aliquot to use as blank (basal levels of PGE2). The remaining blood is incubated with LPS (100 ⁇ g/ml final concentration, Sigma Chem, #L-2630 from E. coli; diluted in 0.1% BSA (Phosphate buffered saline) for 5 minutes at room temperature. Five hundred ⁇ L aliquots of blood are incubated with either 2 ⁇ L of vehicle (DMSO) or 2 ⁇ L of a test compound at final concentrations varying from lOnM to
• Rationale The major side effect of conventional NSAIDs is their ability to produce gastric lesions in man. This action is believed to be caused by inhibition of Cox-1 in the gastrointestinal tract. Rats are particularly sensitive to the actions of NSAIDs. In fact, rat models have been used commonly in the past to evaluate the gastrointestinal side effects of current conventional NSAIDs. In the present assay, NSAID-induced gastrointestinal damage is observed by measuring fecal Cr excretion after systemic injection of Cr-labeled red blood cells. Fecal Cr excretion is a well-established and sensitive technique to detect gastrointestinal integrity in animals and man. Methods
• mice Male Sprague Dawley rats (150 - 200 g) are administered orally a test compound either once (acute dosing) or b.i.d. for 5 days (chronic dosing). Immediately after the administration of the last dose, the rats are injected via a tail vein with 0.5 mL of 5 Cr-labeled red blood cells from a donor rat. The animals are placed individually in metabolism cages with food and water ad lib. Feces are collected for a 48 h period and Cr fecal excretion is calculated as a percent of total injected dose. Cr-labeled red blood cells are prepared using the following procedures. Ten mL of blood is collected in heparinized tubes via the vena cava from a donor rat.
• Plasma is removed by centrifugation and replenished with equal volume of HBSS.
• the red blood cells are incubated with 400 Ci of sodium chromate for 30 min. at 37C. At the end of the incubation, the red blood cells are washed twice with 20 mL
• the red blood cells are finally reconstituted in 10 mL HBSS and 0.5 mL of the solution (about 20 Ci) is injected per rat.
• Protein-losing gastropathy (manifested as appearance of circulating cells and plasma proteins in the GI tract) is a significant and dose-limiting adverse response to standard non-steroidal anti ⁇ inflammatory drugs (NSAIDs). This can be quantitatively assessed by intravenous administration of C1CI3 solution. This isotopic ion can avidly bind to cell and serum globins and cell endoplasmic reticulum. Measurement of radioactivity appearing in feces collected for 24 h after administration of the isotope thus provides a sensitive and quantitative index of protein-losing gastropathy.
• NSAIDs non-steroidal anti ⁇ inflammatory drugs
• Groups of male squirrel monkeys (0.8 to 1.4 kg) are treated by gavage with either 1% methocell or 5% Tween 80 in H2 ⁇ vehicles, (3mL/kg b.i.d.) or test compounds at doses from 1 - 100 mg/kg b.i.d. for 5 days.
• Intravenous Cr (5Ci kg in 1 ml/kg phosphate buffer saline (PBS)) is administered 1 h after the last drug/vehicle dose, and feces collected for 24 h in a metabolism cage and assessed for excreted Cr by gamma-counting.
• Venous blood is sampled 1 h and 8 h after the last drug dose, and plasma concentrations of drug measured by RP- HPLC.

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