AU717966B2 - Method of treating colonic adenomas - Google Patents

Description

tr-V S F Ref: 406743D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

0 *000 0 0 0000 000.

00 0 *a.

0* Name and Address of Applicant: Merck Co., Inc.

126 East Lincoln Avenue Rahway New Jersey 07065 UNITED STATES OF AMERICA Merck Frosst Canada Co.

16711 Trans-Canada Highway Kirkland Quebec H9H 3L1

CANADA

0000 0 *00009 0 Actual Inventor(s): Address for Service: Invention Title: Stacia Kargman, Jilly Evans and Thomas 3. Simon.

Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Method of Treating Colonic Adenomas The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845 Method of Treating Colonic Adenomas Background of the Invention This application is directed to a method of treating or preventing colonic adenomas in a mammalian patient, the method comprising administering to the patient a compound that is a potent inhibitor of PGHS-2 in an amount that is effective for treating or preventing colonic adenomas.

Typically the inhibitor is a non-steroidal anti-inflammatory agent ("NSAID"). This application also discloses a method of preventing or retarding the transformation of colonic adenomas to colonic adenocarcinomas by administration of a non-toxic therapeutically effective, specific PGHS-2 inhibitor.

The enzyme prostaglandin G/H synthase. (PGHS) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins (Watkins, W. Peterson, M. and Fletcher, J. R. ed.

Prostaglandins in Clinical Practice. New York: Raven, 1989 and Dewitt, D. L. Prostaglandin endoperoxide synthase: regulation and enzyme expression. Biochim. Biophys. Acta, 1083; 1121-134, 1991). These prostanoids are potent biological mediators with diverse normal physiological effects and are also implicated in a variety of pathological conditions including inflammation and neoplastic transformation (Watkins, W. Peterson, M. and Fletcher, J. R. ed. Prostaglandins in Clinical Practice. New York: Raven, 1989 and Dewitt, D. L. Prostaglandin endoperoxide synthase: regulation and enzyme expression. Biochim. Biophys. Acta, 1083; 121-134, 1991 and Xie, Robertson, D. L., and Simmons, D. L. Mitogen-inducible prostaglandin G/H synthase: a new target for nonsteroidal antiinflammatory drugs. Drug Dev. Res., 25; 249-265, 1992). Two isoforms of PGHS have been identified 20 (Loll, P. J. and Garavito, R. M. The isoforms of cyclooxygenase: structure and function. Expert Opin.

Invest. Drugs, 3; 1171-1180, 1994). PGHS-1 is constitutively expressed in most tissues and has been proposed to generate prostaglandins for normal physiological functions. The second isoform, PGHS- 2, is characterised by a rapid induction by a variety of stimuli, including mitogens, hormones, cytokines and growth factors (Loll, P. J. and Garavito, R. M. The isoforms of cyclooxygenase: structure and function. Expert Opin. Invest. Drugs, 3; 1171-1180, 1994 and Battistini, Botting, R., and Bakhle, Y. S. COX-1 and COX-2: Toward the development of more selective NSAIDS. Drug News Perspectives, 7; 501-512, 1994). In conditions such as inflammation, PGHS-2-derived

.S..S

prostaglandins may be the predominant effectors (Masferrer, J. Zweifel, B. Manning, P. T., Hauser, S. Leahy, K. Smith, Isakson, P. and Seibert, K. Selective inhibition of 30 inducible cyclooxygenase 2 in vivo is anti-inflammatory and non-ulcerogenic. Proc. Natl. Acad. Sci.

USA, 91; 3228-3232, 1994). Both PGHS-1 and PGHS-2 have been shown to be the target of nonsteroidal anti-inflammatory drugs (NSAIDS) (Battistini, Botting, and Bakhle, Y. S. COX-] and COX-2: Toward the development of more selective NSAIDS. Drug News Perspectives, 7; 501-5 12, 1994 and O'Neill, G. Mancini, J. Kargman, Yergey, Kwan, M. Falgueyret, J. Abramovitz, Kennedy, B. Ouellet, Cromlish, Culp, Evans, J. Ford-Hutchinson, A.

W. and Vickers, P. J. Overexpression of human prostaglandin G/H synthase-l and -2 by recombinant [R:\L1BAAj07725.doc:tab vaccinia virus: inhibition by nonsteroidal anti-inflammatory drugs and biosynthesis of 20 hydroxyeicosatetraenoic acid. Mol. Pharmacol., 45; 245-254, 1994 and DeWitt, D. Meade, E. A., and Smith, W. L. PGH synthase isoenzyme selectivity: the potential for safer nonsteroidal antiinflammatory drugs. Am. J. Med. (Suppl.), 95; 405-445, 1993). See also WO 94/14977, published July 7, 1994, which discloses a method of evaluating the potency of PGHS-2 inhibiting agents as well as the selectivity for PGHS-2 over PGHS-1.

Elevated levels of prostaglandins have been demonstrated in various cancers including lung and colon carcinomas (McLemore, T Hubbard, W. Litterst, C. Liu, M. Miller, S., McMahon, N. Eggleston, J. and Boyd, M. R. Profiles of prostaglandin biosynthesis in normal lung and tumour tissue from lung cancer patients. Cancer Res., 48; 3140-3147, 1988 and Rigas, B., Goldman, I. and Levine, L. Altered eicosanoid levels in human colon cancer. J. Lab. Clin. Med., 122; 518-523, 1993). In particular, prostaglandin levels have been shown to be elevated in benign adenomatous polyps and further increased in cancerous colon tissue, as compared to histologically normal mucosa. Since prostanoids have been shown to be immunosuppressive, they may play a role 1i in tumour development (Eamest, D. Hixson, L. and Alberts, D. S. Piroxicam and other cyclooxygenase inhibitors: potential for cancer chemoprevention. J. Cell. Biochem., 161 (Suppl.); 156- 166,1992).

Since the late 1970's, investigators have considered the possibility that aspirin and related nonsteroidal anti-inflammatory drugs (NSAIDS) might be beneficial to the treatment of certain cancers, 20 including colon cancer.

The first epidemiological study suggesting that aspirin might reduce the risk of colorectal cancer Se" came in 1988 in a retrospective, exploratory analysis from Melbourne, Australia (Cancer, Res., 48: 4399-4404, 1988). The study found a 40 percent lower risk of incident colon cancer among persons who regularly used aspirin compared to those who used no aspirin. More recently the data of Heath, 25 et al, suggests the possible benefit of NSAIDS for prevention of colorectal neoplasms (Heath, C. W., Jr., Thun, M. Greenberg, E. Levin, and Marnett, L. J. Nonsteroidal anti-inflammatory drugs and human cancer. Cancer, 74; 2885-2888, 1994). However, despite this and subsequent studies, Sthere has been no hard evidence linking the use of and the prevention of colon cancer, nor has there been any hard evidence demonstrating a pathological link between colorectal cancer and PGHS or 30 the therapeutic value of inhibiting PGHS by administration of aspirin. For example, arthritis patients (many of whom take aspirin) may simply be less prone to cancer of the colon.

In this application we disclose studies in which we have analysed the expression of human PGHS-I and PGHS-2 protein in 25 paired normal and autologous colon tumours, 4 premalignant colon polyps, 5 control colon tissues (from non-cancer patients) and 3 matched normal and cancerous human breast tissues. Among the observations from this study are that PGHS-1 protein is reduced in colon tumour tissue as compared to histologically normal colonic mucosa, and that PGHS-2 is [R:\LIBAA]07725.doc:tab detected in the majority of colon tumour samples while being virtually undetectable in normal tissues, polyps and breast cancer samples. The increased levels of prostaglandins in tumour tissue of the colon is derived from the inducible PGHS-2 isoform. These studies support our belief that the transformation of a colonic adenoma to a colonic adenocarcinoma is mediated by the dramatic and surprising over production of PGHS-2 in the adenoma. Accordingly, we have surprisingly found a method of retarding or preventing the transformation of a colonic adenoma to an colonic adenocarcinoma comprising the administration to a patient with a history of FAP (Familial adenomatous Polyposis) or a patient with one or more colonic adenomas a non-toxic therapeutically effective amount of NSAID; said amount effective to inhibit the PGHS-2 in said adenoma.

1o Brief Description of the Figures Fig. 1 Representative immunoblot analysis of PGHS-1 and PGHS-2 protein expression in normal human colonic: mucosa and autologous tumour tissue.

Fig. 2 Quantitation of change in PGHS-1 and PGHS-2 20 protein expression in matched tumour as compared to autologous normal mucosa.

Fig. 3 Immunoblot analyses demonstrating PGHS-I and PGHS-2 expression in polyps in ApcA716 knockout mice ranging in size from 0.3-5mm in diameter.

Summary of the Invention This invention is directed to a method of treating or preventing colonic adenomas in a mammalian patient, the method comprising administering to a patient in need of such treatment or 20 prevention, a compound that is a potent inhibitor of PGHS-2 in an amount that is effective for treating or preventing colonic adenomas.

Detailed Description of the Figures Figure. 1. Representative immunoblot analysis of PGHS-1 and PGHS-2 protein expression in normal human colonic mucosa and autologous tumour tissue. Immunoblot analysis using an anti- 25 PGHS-1 antiserum (panel A) and immunoblot analysis of the same samples as those shown in panel S A using anti-PGHS-2 antiserum (panel Purified PGHS standards and microsomal protein samples per lane) were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-PGHS antisera with detection by enhanced chemiluminescence. Numbers 1-4 indicate samples from 4 representative patients of 25 patients examined. The densitometric values for patients 1-4 in S 30 this figure correspond to patients 17-20 shown in Fig. 2. N and T denote matched normal colonic mucosa and tumour tissue for each patient, respectively. Purified PGHS-1 and PGHS-2 standards are shown at the left. The positions of molecular weight markers are indicated.

Figure. 2. Quantitation of change in PGHS-1 and PGHS-2 protein expression in matched tumour tissue as compared to autologous normal mucosa. Aliquots of PGHS standards and of microsomal proteins from 25 patients (50p.g per lane) were separated by SDS-PAGE, transferred to s nitrocellulose, and immunoblotted with anti-PGHS-1 or anti-PGHS-2 antisera, with detection by [R:\LIBAA]07725.doc:tab enhanced chemiluminescence. The amounts of PGHS protein were determined by densitometry. The optical density reading from the scanned autoradiograph for known quantities of purified PGHS-1 and PGHS-2 standards were used to quantitate approximate amounts of PGHS-I and PGHS-2 protein in microsomal samples from normal mucosa and tumour samples. Values represent the change in expression in ng from normal mucosa to tumour tissue for each patient.

Figure 3 An immunoblot analyses demonstrating PGHS-1 and PGHS-2 expression in polyps ranging in size from 0.3-5mm in diameter. (Lanes 1-5 correspond to samples derived from polyps of approximately 1-5mm in diameter, respectively.) PGHS-1 protein was demonstrated in all intestinal and colonic control and polyps samples. PGHS-2 immunoreactivity was not detected in control intestine or colon samples. Although low levels of PGHS-2 protein were detected in larger intestinal polyps (3-5mm in diameter) (lanes 3,4 and higher levels of PGHS-2 immunoreactive species were detected in all colonic polyp samples ranging in size from 1-5mm in diameter (lanes 1-5) with very high levels of PGHS-2 protein being demonstrated in polyps of 2-3mm in diameter (lanes 2 and 3).

Purified sheep PGHS-1 and PGHS-2 protein (5ng, 10ng, and 20ng) were electrophoresed as standards and are labelled accordingly.

Detailed Description of the Invention There is disclosed herein a method of treating or preventing colonic adenomas in a mammalian patient, the method comprising administering to a patient in need of such treatment or prevention, a compound that is a potent inhibitor of PGHS-2 in an amount that is effective for treating or preventing S 20 colonic adenomas.

For purposes of this specification, such NSAIDS includes, but is not limited to aspirin, ibuprofen, indomethacin, sulindac, dolobid, diclofenac, naproxen, piroxicam, etodolac, ketoprofen, flurbiprofen, meloxicam, flosulide and nabumetone.

As is appreciated by those of skill in the art, a milestone event in the transformation of a colonic 25 adenoma to a colonic adenocarcinoma is the acquired capacity of adenoma cells to invade the basement membrane of the sub-mucosal tissue of the colon.

In one genus of this embodiment, this invention is directed to the use of NSAIDS which are potent PGHS-2 inhibiting agents. For purposes of this specification an NSAID is potent if it possess an ICso for the inhibition of PGHS-2 of 1lM or less as measured by the cell or microsomal assay disclosed herein.

In one subgenus of this genus the invention is directed to the use of NSAIDS which are specific inhibitors of PGHS-2. For purposes of this specification NSAIDS which are specific inhibitors of PGHS-2 is defined as those which possess a specificity for inhibiting PGHS-2 over PGHS-1 of at least 100 fold as measured by the ratio of ICso for PGHS-2 over ICso for PGHS-1 evaluated by the cell or microsmal assay disclosed hereinunder. Such compounds include, but are not limited to those [R:LIBAA]07725.doc:tab disclosed in WO 94/13635, WO 95/00501, WO 94/15932, US 5 344 991, EP 0418 845, US 5 380 738, and US 5 393 790, all of which are hereby incorporated by reference.

In a second genus the invention is directed to a method of treating or preventing colonic adenomas in a mammalian patent, the method comprising the administration to a patient of a nontoxic therapeutically effective amount of compound of formula I

H

3 C' N

S

C B 0I wherein A, B and C are each independently selected from the group consisting of: hydrogen F, Cl, Br or I, methyl or ethyl, CF3 vinyl, OCH3 or OCF3, SCH3 or SCF 3 CN, N 3 with the proviso that at least one of A, B and C must be hydrogen.

Within genus there is a sub-genus directed to the use of compounds of formula I wherein A and B are each independently selected from the group consisting of hydrogen, F, CI, Br or I, (c) methyl or ethyl, CF3, vinyl, SCH 3 or SCF3, CN, N 3 and C is hydrogen.

Within the class is the sub-class directed to the use of compounds of the formula

H

HC A 0.

B*\

0 15 wherein B is hydrogen, F, Cl, Br or I, methyl or ethyl, CF 3 vinyl, -OCF 3 or SCH3.

C..HIllustrating this aspect of the invention is the use of a compound selected from OS O O O H Hs H' Ha 3 Cs N F H 3 C N' F H 3 C' N Cl 0 0 o /o /o H'CSN' H c H H S H HC" W Cl B rCNs A H 3 C XN A o o or a pharmaceutically acceptable salt thereof.

[R:\LIBAA]07725.doc:tab In a third genus the invention is directed to a method of treating or preventing colonic adenomas in a mammalian patient, the method comprising the administration to a patient of a nontoxic therapeutically effective amount of compound of formula 11 r" II or a pharmaceutically acceptable salt thereof wherein: X-Y-Z-is selected from the group consisting of:

-CH

2

CH

2

CH

2

-C(O)CH

2

CH

2

-CH

2 CHC(O)-, -CR5(R 5 -C(O)-O-CR5(R 5 -CH2-NR 3 -CH2-, -CR 5

(R

5

')-NR

3

-CR

4

=CR

4

-S-CR

4

=_CR

4 CH=N-S-, -N=CR 4 (in) -O-CR 4

-N=CR

4

-N=CR

4 and -S-CR 4

C(O)-NR

3

-CR

5

(R

5

-NR

3 -CH=CH- provided R 1 is other than -S(O)2Me, -CH=CH-NR 3 provided RI is other than -S(O)2Me, when side b is a double bond, and sides a and c are single bonds; and X- Y-Z is selected from the group consisting of: =CH-O-CH=, and =CH-NR 3

=N-S-CH=,

when sides a and c are double bonds and side b is a single bond; R 1 is selected from the group consisting of S(O) 2

CH

3 S(O)2NH2, S(O) 2 NHC(O)CF3, S(O)(NH)CH 3

S(O)(NH)NH

2 S(O)(NH)NHC(O)CF3, (g)

P(O)(CH

3 )OH and P(O)(CH 3 )NH2, R 2 is selected from the group consisting of CI-6alkyl, C3- 7 cycloalkyl, mono-, di- or tri-substituted phenyl wherein the substituent is selected from the group consisting of hydrogen, halo, Cis6alkoxy, Ci-6alkylthio, CN, CF 3 Ci-6alkyl, (8) ~N3, -CO 2 H, (10) -C02-Cl~alkyl, (1I) -C(R 5

)(R

6 (12) -C(R 5

)(R

6 )-O-Ci4alkyl, and (13) -Clralkyl- C0 2

-R

5 mono-, di- or tni-substituted heteroaryl wherein the heteroaryl is a monocyclic aromatic 2o ring of 5 atoms, said ring having one hetero atom which is S, 0, or N, and optionally 1, 2, or 3 additional 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 or 3 additional N atoms; said substituents are selected from the group consisting of hydrogen, halo, including fluoro, chloro, bromo and iodo, Cis6alkyl, C1_ 6alkoxy, Ci-6alkylthio, CN, CF 3

N

3 -C(R5)(R 6 (10) -C(R 5

)(R

6 )-0-C14~alkyI; R 3 is selected from the group consisting of hydrogen, CF 3 CN, Ci-6alkyl, hydroxyCi-6alkyl, and -C(0)-Ci_6akyl, optionally substituted -C 1 -5 alkyl-Q, -Ci_3alky-0-C1_3alkyl-Q, -Cl- 3 alkyl-S-Cl-3alkyl-Q, -Cli 5 alkyl-0-Q, or -Cli5 alkyl-S-Q, wherein the substituent resides on the alkyl and the substituent is C 13 alkyl; R 4 and R 4 are each independently selected from the group consisting of hydrogen, CE 3 CN, C1_alkyI, and (h) optionally substituted -C 15 alkyl-Q, -0-C1.5 alkyl-Q, -S-C1_ 5 alkyl-Q, -Cl- 3 alkyl-0-C-3alkyl- Q, -Cl-3alkyl-S-Cl-3alkyl-Q, -01_5 alkyl-0-Q, -C 15 alkyl-S-Q, wherein the substituent resides on the alkyl and the substituent is C1-3alkyl, and R 5

R

5 and R 6

R

7 and R 8 are each independently selected from the group consisting of hydrogen, Ci-6alkyl, or R 5 and R 6 or R 7 and R 8 together with the carbon to which they are attached form a monocyclic saturated carbon ring of 3, 4, 5, 6 or 7 [RALIBAA]07725.dOC~tab atoms; Q is CO 2 H, C02-C14alkyl, tetrazolyl-5-yl, C(R 7

)(R

8 or C(R 7

)(R

8 )(O-C14alkyl); provided that when X-Y-Z is -S-CR 4

=CR

4 then R 4 and R 4 are other than CF3.

Within this genus is a sub-genus directed to the use of compound of formula III

R

5

R

Rio -0 R2

III

or a pharmaceutically acceptable salt thereof wherein: R 1 is selected from the group consisting of (a) S(0) 2

CH

3 S(0)2NH2, S(O) 2 NHC(O)CF3, S(O)(NH)CH 3

S(O)(NH)NH

2 (f) S(O)(NH)NHC(O)CF3 P(O)(CH 3 )OH and P(O)(CH 3 )NH2, R 2 is selected from the group consisting of C3-7 cycloalkyl, mono-, di- or tri-substituted phenyl wherein the substituent is selected from the group consisting of hydrogen, halo, Ci.alkoxy, Ci-.alkylthio, CN, to CF 3 C16alkyl, N 3 -CO2H, (10) -C02-Ci4alkyl, (11) -C(H)(R 6 (12) -C(H)(R 6 )-O-C-1 4alkyl, and (13) -C-6alkyl-CO2H; mono-, di- or tri-substituted heteroaryl wherein the 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 additional 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 or 3 additional N atoms; said substituents are selected from the group consisting of hydrogen, halo, including fluoro, chloro, bromo and iodo, Ci-alkyl, Ci-ealkoxy, C.e6alkylthio, CN, CF3, N3, -C(H)(R 6 (10) -C(H)(R 6 O-C-4alkyl; R 5 and R 5 are each methyl or ethyl, R 6 is selected from the group consisting of (a) hydrogen, C1-ealkyl.

Within this sub-genus is a class directed to the use of compound of formula III wherein R 1 is 20 selected from the group consisting of S(0) 2

CH

3 S(0) 2

NH

2 S(0) 2 NHC(0)CF3, (d)

S(O)NHCH

3 S(0)NHNH2, and S(0)NHNHC(O)CF3; R 2 is selected from the group consisting of (a) C-4alkyl, C37cycloalkyl, mono- or di-substituted phenyl wherein the substituent is selected from the group consisting of hydrogen, fluoro, chloro, and bromo, C14alkoxy, Cl4alkylthio, CN, CF3, C14alkyl, N 3 -CO2H, (10) -C 2 -Ci-3alkyl, (11) -C(H)(R 6 (12) -C(H)(R 6 25 C1.3alkyl.

Within this class is a sub-class directed the use of compound of formula III wherein R 2 is selected from the group consisting of cyclohexyl, and mono- or di-substituted phenyl, and wherein the substituents are selected from the group consisting of hydrogen, halo, Ci- 4alkoxy, C14alkylthio, CN, CF 3 C14alkyl, N3, and

R

5 and R5' are each independently selected from the group consisting of methyl or ethyl, R 6 is selected from the group consisting of hydrogen, methyl or ethyl.

Within this sub-class is a group directed to the use of compound of formula III wherein R 1 is selected from the group consisting of S(0) 2 CH3, S(0) 2 NH2, S(0)NHCH3, and (d) S(0)NHNH2; R 2 is selected from the group consisting of mono- or di-substituted phenyl wherein the [R:1IBAA]07725.doc:tab substituents are selected from the group consisting of hydrogen,(2) halo, selected from the group consisting of fluoro, chloro and bromo, C-.3alkoxy, Ci-salkythio, CN, and Cl-3alkyl.

Illustrating the invention is the use of compounds selected from 3-(3-fluorophenyl)-4-(4- 3-(3,4-difluorophenyl)-4-(4-(methylsulfonyl)phenyl)-2- (5H)-furanone, 3-(3,4-dichlorophenyl)-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone, 3-phenyl- 4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone and 5,5-dimethyl-3-(3-fluorophenyl)-4or a pharmaceutically acceptable salt thereof.

The pharmaceutical 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. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable nontoxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminium, 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-dibenzyl-ethylenediamine, diethylamine, 2diethylaminoethanol, 2-dimethylanilnoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N- 20 ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

Compounds of the present invention are inhibitors of cyclooxygenase-2 and are thereby useful in the treatment of cyclooxygenase-2 mediated diseases as enumerated above. This activity is illustrated by 25 their ability to selectively inhibit cyclooxygenase-2 over cyclooxygenase-1. Accordingly, in one assay, Sthe ability of the compounds of this invention to treat cyclooxygenase mediated diseases can be demonstrated by measuring the amount of prostaglandin E2 (PGE2) synthesised in the presence of arachidonic acid, cyclooxygenase-1 or cyclooxygenase-2 and a compound of formula I. The ICso values represent the concentration of inhibitor required to retum PGE2 synthesis to 50% of that obtained as compared to the uninhibited control.

For the treatment of any of these cyclooxygenase mediated diseases, compounds of formula I may be administered orally, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular,, intrastenal injection or infusion techniques. In addition to the treatment of warm [R:LIBAA]07725.doc:tab blooded animals such as mice, rats, horses, cattle sheep, dogs, cats, etc., the compound of the invention is effective in the treatment of humans.

As indicated above, pharmaceutical compositions for treating cyclooxygenase-2 mediated diseases as defined may optionally include one or more ingredients as listed above.

The pharmaceutical 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, flavouring agents, colouring 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. These 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, gelatine 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 absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

For example, 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 US 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 gelatine capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or S kaolin, or as soft gelatine capsules wherein the active ingredients is mixed with water or an oil 25 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. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethycellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a 30 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 sorbitan monooleate. The '0 ir2R aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p- [R:LIBAA]07725.doc:tab hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more 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 flavouring 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. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.

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 naturallyoccurring 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 flavouring and colouring S agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous 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-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic monoor diglycerides. In addition, 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 Swill therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene [R:\LIBAA]07725.doc:tab glycols. For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compound of Formula I are employed. (For purposes of this application, topical application shall include mouth washes and gargles.) Dosage levels of the order of from about 0.01mg to about 140mg/kg of body weight per day are useful in the treatment of the above-indicated conditions, or alternatively about 0.5mg to about 7g per patient per day. For example, inflammation may be effectively treated by the administration of from about 0.01 to 50mg of the compound per kilogram of body weight per day, or alternatively about to about 3.5g per patient per day.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage firm will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 0.5mg to 5g of active agent compounded with 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 1mg to about 500mg of an active ingredient, typically 50mg, 100mg, 200mg, 300mg, 400mg, 500mg. Once a day dosage is anticipated.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

20 The examples below are intended to illustrate, but not limit the invention as disclosed herein.

Examples Patient Samples Colon cancer and matched normal mucosal tissues, adenomatous polyps, normal colonic 2 mucosa from non-cancer patients and matched normal and cancerous breast tissues were examined.

25 The mean age and range of matched normal and colon cancer samples were 63.3 and 41-93 years, respectively. Colon tissue specimens were obtained from a non-necrotic area of the tumour and from autologous normal mucosa from the same patient at a resection margin located at more than 5 cm S. from the tumour. Histologically, all colon tumours were adenocarcinomas and all patients had sporadic :colon cancer. One patient had a focus of adenocarcinoma within an adenomatous polyp. Colonic adenomas were from patients with familial adenomatous polyposis (FAP).

Preparation of microsomal membranes from colon tissues Frozen tissues were thawed in ice-cold homogenisation buffer [50mM potassium phosphate, pH7.1, containing 0.1M NaCI, 2mM EDTA, 0.4mM phenytmethylsulfonyl fluoride, 60p.g/mL soybean trypsin inhibitor, 2pg/mL leupeptin, 2p.g/mL aprotinin and 2gg/mL pepstatin, all from Sigma Chemical Co., St. Louis,, MO). Tissues were disrupted twice on ice using a tissue tearer (Biospec Products, 1 Bartlesville, OK) and homogenised by sonication at 4 0 C using a Cole Parmer 4710 series ultrasonic [R:\LIBAA]07725.doc:tab homogeniser (Cole Parmer Instrument Co., Chicago, IL). Cellular debris was removed by centrifugation at 1000 x g for 15min at 4 0 C and the resultant supenatants were subjected to centrifugation at 100 000 x g for 60min at 4 0 C. Membrane fractions were resuspended in homogenisation buffer and sonicated in order to obtain a homogenous membrane suspension. Protein concentrations were determined for each sample using a protein assay kit (Bio-Rad, Mississauga, Ontario, Canada).

Antisera Full-length sheep seminal vesicle PGHS-1 and placental PGHS-2 purified proteins were purchased from Cayman (Ann Arbor, MI) and used to generate rabbit polyclonal antibodies. New to Zealand White female rabbits were injected with 1mL of Freund's complete adjuvant containing 200pig of purified PGHS-1 or PGHS-2. Two weeks after the primary injection, rabbits were boosted with 100ig of purified PGHS-1 or PGHS-2 in 0.5mL of Freund's incomplete adjuvant. The anti-PGHS antisera recognise the homologous human PGHS isoforms with approximately one thousand-fold selectivity for the appropriate isoform. Under the conditions used in this study, the anti-PGHS antibodies demonstrated no significant cross-reactivity with the alternate PGHS isoform. For each experiment, two concentrations of both PGHS-1 and PGHS-2 proteins standards were loaded on each gel to assess selectivity of the antibodies.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis Membrane fractions were mixed with sodium dodecyl sulfate (SDS) sample buffer (20mM Tris- 20 HCI, pH6.8, containing 0.4% SDS, 4% glycerol, 0.24M p-mercaptoethanol, and bromphenol blue), boiled for 5min and analysed by SDS-polyacrylamide gel electrophoresis according to the method of Laemmli (Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227; 680-685, 1970). Proteins were electrophoretically transferred S to nitrocellulose membranes as described previously (Towbin, Staehelin, T. and Gordon, J.

25 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and Ssome applications. Proc. Natl. Acad. Sci. USA, 75; 4350-4354, 1979). Primary antibodies to PGHS-1 and PGHS-2 were used at a final dilution of 1:5000 and 1:7500, respectively. The secondary horseradish peroxidase-linked donkey anti-rabbit IgG antibody (Amersham Life Sciences, Oakville, Ontario, Canada) was used at a dilution of 1:3000. Immunodetection was performed using enhanced chemiluminescence according to the manufacturer's instructions (Amersham). Autoradiographs were scanned using a computing densitometer (Molecular Dynamics, Sunnyvale, CA) and the volume of optical density corresponding to the purified PGHS isoform was used to calculate quantity (ng) of PGHS protein in histologically normal colonic and tumour tissue.

[R:\LIBAA]07725.doc:tab Statistical analysis The results of this study were analysed by a Wilcoxon signed-rank non-parametric test to determine significant differences between normal and tumour PGHS-1 and PGHS-2 levels (Freund, J.

E. Mathematical Statistics. Prentice Hall, Englewood Cliffs, New Jersey. 1992).

Expression of PGHS-1 in human colon: normal and tumour tissue Fig. 1 is a representative immunoblot demonstrating PGHS expression in 4 of the 25 patients examined in this study. The 72kD band, which is the reported molecular weight for PGHS-1 (Otto, J.

DeWitt, D. L. and Smith, W. L. N-glycosylation of prostaglandin endoperoxide synthases-1 and -2 and their orientations in the endoplasmic reticulum. J. Biol. Chem., 268; 18234-18242, 1993), comigrated with purified sheep seminal vesicle PGHS-1 standard (Fig. 1A) and with recombinant human PGHS-1 expressed in COS-7 cells (data not shown; The immunoblot results from all patients were quantitated by densitometric analysis and are shown in the bar graph in Fig 2. In 21 of patient, PGHS-1 levels were reduced in tumour tissue as compared to normal colon. The mean decrease of PGHS-1 in tumour as compared to normal tissue for all 25 patients examined was 170ng per mg nucrosomal protein. In comparison to the purified PGHS-1 standard, the range of concentrations of PGHS-1 in normal and tumour tissue were 0-760ng (median, 199.8) and 4-540ng (median, 51.1) per mg microsomal protein, respectively. The difference in PGHS-1 expression in normal versus tumour tissue was highly statistically significant as determined by nonparametric *6 analyses (Wilcoxon signed-rank test, p 0.0001).

20 Expression of PGHS-2 in human colon: normal and tumour tissue A representative immunoblot analysis of 4 of the 25 matched colon samples using a specific anti-PGHS-2 antibody is shown in Fig. 1B. Duplicate immunoblots were performed in order to assess the expression of PGHS-1 (Fig. 1A) and PGHS-2 (Fig. 1B) in samples derived from the same patient.

SPGHS-2 immunoreactivity was not detected in any of the 4 normal colon tissue samples. In contrast, S* 25 immunoreactive bands of 70-72kD, which comigrated with purified sheep placental PGHS-2 (Fig. 1 B) and human recombinant PGHS-2 expressed in COS-7 cells (data not shown; were detected in tumour tissue of 3 of the 4 patients shown here. In total, PGHS-2 immunoreactive protein was fo. detected in tumour tissue from 19 out of 25 patients examined (Fig. Overall, the mean PGHS-2 increase in colonic tumours for all 25 patients examined was approximately 73ng per mg microsomal protein. In comparison to the PGHS-2 standard, the range of concentrations of PGHS-2 immunoreactive protein in normal and tumour tissue was 0-49ng (median, 3.8ng) and 1.6-580 ng (median, 37.7ng), per mg microsomal protein, respectively. The difference in PGHS-2 expression in normal versus tumour tissue was highly statistically significant as determined by nonparametric analyses (Wilcoxon signed-rank test, p 0.0001).

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9. 9 4 9 9@ a Colonic tumours are classified by 4 Dukes' stages: A, tumour within the intestinal mucosa; B, tumour into muscularis mucosa; C, metastasis to lymph nodes and D, metastasis to other tissues. In this study, colonic tumours were from patients with all stages of Dukes' classification (Table 1).

Table 1 Patient Data Change in Protein Expression (ng) Patient# Medication Dukes Stage PGHS-2 PGHS-1 1 NA NA 6.20 -10.00 2 NA NA 1.70 .6.00 3 NA NA 2.80 .9.40 4 NA NA 1.20 .4.10 NONE B 0.10 ND 6 chlordiazepoxide hydrochloride/clidinium bromide, magaldrate C 3.20 .6.90 7 NSAID A 0.00 .1 7.30 8 levothyroxine sodium, lorazepam, omeprazole C 0.50 1.50 9 NONE C 0.90 0.50 NA C 4.70 -16.30 11 NONE B 0.90 -9.00 12 NA 0 -0.20 -14.40 13 NA B 4.30 -7.10 14 aspirin C 0.80 -5.60 deltacortisone, azathioprine, lovastatin B 0.00 5.70 16 enalapril maleate B 25.60 -6.60 17 NONE B .0.30 -1.50 18 NONE B 9.70 -11.20 19 aspirin C 13.70 -11.10 20 NONE B 8.30 -5.90 21 NONE C -0.10 -9.30 22 aspirin C 0.20 -3.00 23 none C 0.00 -12.30 24 diltiazem hydrochloride, omeprazole B 2.20 -17.00 25 flurbiprofen, aspirin, nifedipine. metoprolol tartrate B 0.10 -10.00 NA data not available ND not detectable Dukes Stages: A tumour within mucosa B tumour into muscularis mucosa to C metastasis to lymph nodes 0 metastasis to other tissues Table 1. Patient data. Information for each patient examined in this study is shown. When available, medications taken by patients are indicated. Dukes stage of disease is as follows: A tumour within mucosa; B tumour into muscularis mucosa; C metastasis to lymph nodes and D metastasis to other tissues. Amount of PGHS immunoreactive protein is expressed as relative change in expression from normal colonic mucosa to tumour tissue. NA: data not available and ND: immunoreactivity not detectable.

There was no association between stage and change in expression of PGHS-1 or PGHS-2 protein. This lack of association is consistent with the observation that PGE 2 levels in human colon Stumours do not appear to correlate with Dukes' stage (Rigas, Goldman, I. and Levine, L.

R:LIBAA7725.do:tab Altered eicosanoid levels in human colon cancer. J. Lab. Clin. Med., 122; 518-523, 1993).

Interestingly, PGHS-2 expression was either low or undetectable in 5 out of 6 patients currently using anti-inflammatory medications (Table 1).

Expression of PGHS-1 and -2 protein in normal colon, premalignant polyps and matched normal and breast cancer tissue The expression of PGHS-1 and -2 protein was analysed in a variety of other human tissues including S colon tissues from non-cancer patients, 4 premalignant polyps and 3 matched normal and cancerous breast tissues. Although PGHS-1 protein was observed in normal and cancer tissues, PGHS-2 protein was not detected in any of these samples.

to Discussion In this study, 19 out of 25 colon tumours examined expressed PGHS-2 protein whereas only 2 out of 25 normal colon tissue samples expressed this protein. Concomitant with the induced expression of PGHS-2, PGHS-I expression was reduced in 21 out of 25 of the tumour samples in comparison with normal adjacent colonic mucosa; however, PGHS-1 protein concentrations were similar in control colon and polyp samples.

To our knowledge, this is the first study examining the expression of PGHS-1 and -2 protein in human colon cancer. Previous research has shown increased eicosanoid levels, in particular, PGE2, in human colon cancer (Rigas, Goldman, I. and Levine, L. Altered eicosanoid levels in human colon cancer. J. Lab. Clin. Med., 122; 518-523, 1993, Bennett, Del Tacca, Stamford, and a 20 Zebro, T. Prostaglandins from tumours of human large bowel. Br. J. Cancer, 35; 881-884, 1977, Bennett, Civier, Hensby, C. Melhuish, P. and Stamford, I. F. Measurement of arachidonate and its metabolites extracted from human normal and malignant gastrointestinal tissues.

Gut, 28; 315-318, 1987). We conclude the increase in PGHS-2 enzyme that we observe in colonic tumour tissue results in elevated prostaglandin levels in these tumours. Moreover, we conclude that since prostaglandins may have multiple effects in the biology of cancer, including growth promotion and modulation of immune surveillance, elevated prostanoid levels within tumours will aid in tumour growth or development (Earnest, D. Hixson, L. and Alberts, D. S. Piroxicam and other cyclooxygenase inhibitors: potential for cancer chemoprevention. J. Cell. Biochem., 161 (Suppl.); 1S6i 166, 1992). Interestingly, PGHS-2 protein was not observed in the examination of breast cancer S 30 samples. Thus, PGHS-2 protein expression is a typical feature in the transformation of tissue from pro-malignant to malignant phases.

Eberhart and his coworkers demonstrated up-regulation of PGHS-2 mRNA in human colorectal adenomas and adenocarcinomas (Eberhart, C. Coffey, R. Radhika, Giardiello, F. M., Ferrenbach, Dubois, R. N. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology, 107; 1183-1188, 1994). While this is i an interesting finding, this finding but may not be indicative of actual enzyme expression due to the [R:LIBAA]07725.doc:tab complex post-transcriptional and -translational regulation of PGHS-2 mRNA. For example, Hoff et al.

(Hoff, DeWitt, Kaever, Resch, K. and Goppelt-Struebe, M. Differentiation-associated expression of prostaglandin G/H synthase in monocytic cells. FEBS Lett., 320; 38-42, 1993)., Lee et al (Lee, S. Soyoola, Chanmugam, Hart, Sun, Thong, Liou, Simmons, and Hwang, D. Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J. Biol. Chem., 267; 25934-25938, 1992) and our laboratories have shown substantial expression of PGHS-2 mRNA without concomitant expression of PGHS-2 protein.

Therefore, examination of PGHS protein expression is critical to estimate the concentration of PGHS- 2 enzyme.

Nothing in the art would suggest the absence of PGHS-2 protein in polyps from 4 patients with familial adenomatous polyposis, especially in light of evidence by several groups that sulindac (inhibiting both PGHS-1 and PGHS-2) results in polyp regression in patients with familial polyposis.

The human polyp samples were pools of small colon polyps ranging in size from 0.4-<5mm in diameter. In our opinion, PGHS-2 protein is expressed at a later stage in the polyp-cancer sequence, when polyps are larger in diameter, perhaps greater than or equal to 5mm in size. A recent study by Ladenheim et. al. reports that sulindac did not result in a regression of sporadic colonic polyps; however, they emphasised that their study addressed early sporadic polyps (67% of the polyps were and suggested that response to NSAIDS may be more favourable in polyps at a "particular stage along the adenoma-carcinoma sequence (Ladenheim, Garcia, Titzer, Herzenberg, H., 20 Lavori, Edson, and Omary, B. Effect of sulindac on sporadic colonic polyps. Gastroenterology, 108; 1083-1087, 1995)." The prolonged use of NSAIDS is associated with side effects including renal toxicity, gastrointestinal ulceration and increased bleeding. Current NSAIDS such as aspirin, sulindac and indomethacin, have little selectivity for inhibition of either PGHS-1 or PGHS-2 (Battistini, Botting, 25 and Bakhle, Y. S. COX-1 and COX-2: Toward the development of more selective NSAIDS. Drug News Perspectives, 7; 501-512, 1994, O'Neill, G. Mancini, J. Kargman, Yergey, Kwan, M. Falgueyret, Abramovitz, Kennedy, B. Ouellet, Cromlish, Gulp, Evans, J.

Ford-Hutchinson, A. W. and Vickers, P. J. Overexpression of human prostaglandin G/H synthase-1 and -2 by recombinant vaccinia virus: inhibition by nonsteroidal anti-inflammatory drugs and 30 biosynthesis of 15-hydroxyeicosatetraenoic acid. Mol. Pharmacol., 45; 245-254, 1994, DeWitt, D. L., Meade, E. and Smith, W. L. PGH synthase isoenzyme selectivity: the potential for safer nonsteroidal antiinflammatory drugs. Am. J. Med. (Suppl.), 95; 40S-44S, 1993). It has been suggested that selective inhibitors of PGHS-2 would have useful therapeutic effect with a decreased capacity to induce mechanism-based side effects. Recently, a selective PGHS-2 inhibitor, NS-398, has been shown in the rat to have antiinflammatory, antipyretic and analgesic effects without being ulcerogenic (Futaki, Yoshikawa, Hamasaka, Arai, Higuchi, lizuka, and Otomo, S. NS-398, a [R:\LIBAA]07725.doc:tab novel non-steroidal anti-inflammatory drug with potent analgesic and antipyretic effects, which causes minimal stomach lesions. Gen. Pharmacol., 24; 105-110, 1993; Futaki, Takahashi, Yokoyama, Arai, Higuchi, S. and Otomo, S. NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro. Prostaglandins, 47; 55-59, 1994). The present study examining the expression of PGHS-1 and PGHS-2 in the colon demonstrates that both PGHS isoforms are present in colon tumours.

Mouse polyps ApcA716 knockout mice (developed by Dr. Taketo at Banyu Merck, ref: Oshima, Oshima, Kitagawa, Kobayashi, Itakura, C. and 30 Taketo, M. Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene truncation mutant mice. Proc. Natl. Acad. Sci. USA, 92, 4482-4486, 1995) develop multiple polyps throughout their intestinal tracts. Small intestinal and colonic polyp samples ranging in size from 0.3-5.6mm from ApcA716 knockout mice were collected and immediately frozen from six mice (both males and females) from backcross N1, N2 and N4 generation mice.

Results Expression of PGHS-1 and PGHS-2 in ApcA716 Mouse Polyps PGHS-1 and PGHS-2 protein expression was examined in polyps of increasing size from ApcA716 mice. Fig. 3 is an immunoblot analyses demonstrating PGHS-1 and PGHS-2 expression in S polyps ranging in size from 0.3-5mm in diameter. (Lanes 1-5 correspond to samples derived from 20 polyps of approximately 1-5mm in diameter, respectively.) PGHS-1 protein was demonstrated in all intestinal and colonic control and polyps samples. PGHS-2 immunoreactivity was not detected in control intestine or colon samples. Although low levels of PGHS-2 protein were detected in larger intestinal polyps (3-5mm in diameter) (lanes 3, 4 and higher levels of PGHS-2 immunoreactive species were detected in all colonic polyp samples ranging in size from 1-5mm in diameter (lanes with very high levels of PGHS-2 protein being demonstrated in polyps of 2-3mm in diameter (lanes 2 and Purified sheep PGHS-1 and PGHS-2 protein (5ng, 10ng, and 20ng) were electrophoresed as standards and are labelled accordingly. We believe that the dramatic induction of COX-2 during polyp growth reflects a critical roll for COX-2 in the initiation of the transformation from adenoma to adenocarcinoma.

*i [R:\LBAA07725.doc:tab

Claims (15)

1. A method of treating or preventing colonic adenomas in a mammalian patient, the method comprising administering to a patient in need of such treatment or prevention, a compound that is a potent inhibitor of PGHS-2 in an amount that is effective for treating or preventing colonic adenomas.

2. A compound that is a potent inhibitor of PGHS-2 when used in treating or preventing colonic adenomas in a mammalian patient.

3. Use of a compound that is a potent inhibitor of PGHS-2 in the manufacture of a medicament for treating or preventing colonic adenomas in a mammalian patient.

4. The method according to claim 1, the compound according to claim 2 or the use according to claim 3, wherein said compound is a non-steroidal anti-inflammatory agent ("NSAID"). The method, compound or use according to claim 4, wherein said NSAID is selected from aspirin, ibuprofen, indomethacin, sulindac, dolobid, diclofenac, naproxen, piroxicam, etodolac, ketoprofen, flurbiprofen, meloxicam, flosulide and nabumetone.

6. The method, compound or use according to claim 5 wherein said NSAID possesses a specificity for inhibiting PGHS-2 over PGHS-1 of at least 50 fold as measured by the ratio of ICso for the inhibition of PGHS-2 over ICso for the inhibition of PGHS-1 as measured whole cell assay.

7. A method of treating or preventing colonic adenomas in a mammalian patient, the method comprising administering to a patient in need of such treatment or prevention a compound of 20 formula I o 0 /o H3CS'N" A 0 Oc wherein A, B and C are each independently selected from the group consisting of: hydrogen F, *o CI, Br or I, methyl or ethyl, CF3 vinyl, OCH 3 or OCF 3 SCH 3 or SCF3, CN, N 3 Swith the proviso that at least one of A, B and C must be hydrogen. 25 8. A compound of formula I as defined in the method according to claim 7 when used in treating or preventing colonic adenomas in a mammalian patient. S9. Use of a compound of formula I as defined in the method according to claim 7 in the manufacture of a medicament for treating or preventing colonic adenomas in mammalian patient. The method according to claim 7, the compound according to claim 8 or the use according to claim 9, wherein A and B are each independently selected from the group consisting of hydrogen, F, Cl, Br or I, methyl or ethyl, CF 3 vinyl, SCH3 or SCF 3 CN, N3, c R and C is hydrogen. [R:LIBAA]07725.dactab

11. The method, compound or use according to claim 10 wherein the compound is of the formula H3 C 1 N" A 0 S( H wherein B is hydrogen, F, CI, Br or I, methyl or ethyl, CF 3 vinyl, -OCF 3 or SCH 3

12. The method, compound or use according to claim 11 wherein the compound is of the formula S'O H 0 H 3 C 'N' O wherein A is F, Cl, Br, and B is F, Cl, Br.

13. The method, compound or use according to claim 12 wherein the compound is of the formula N5H S H H3C, 3 H F 3C N F H 3 C' N C H SH e. i\ S CI HS-Br O o o o or a pharmaceutically H YH 5 H 0 0 0 or a pharmaceutically S. acceptable salt thereof. is5 14. A method of treating or preventing colonic adenomas in a mammalian patient, the method comprising administering to a patient in need of such treatment or prevention a compound of formula II 2 II [R:\LIBAA]07725.doc:tab or a pharmaceutically acceptable salt thereof wherein: X-Y-Z-is selected from the group consisting of: -CH 2 CH 2 CH 2 -C(O)CH 2 CH2-, -0H 2 CH 2 -CR 5 (R 5 -C(O)-0-0R 5 (R 5 -CH 2 -NR 3 -CH2-, -CR5(R 5 )-NR 3 -CR 4 =CR 4 -S-CR 4 =CR 4 CH=N-S-, -N=CR 4 (in) -O-CR 4 -N=0R 4 -N=CR 4 and -S-CR4=N-, C(0)-NR3-CR 5 (R 5 -NR 3 -CH=CH- provided R 1 is other than -S(0) 2 Me, -CH=CH-NR3- provided R 1 is other than -S(0) 2 Me, when side b is -a double bond, and sides a and c are single bonds; and X- Y-Z is selected from the group consisting of: =CH-0-CH=, and =CH-NR 3 =N-S-CH=, when sides a and c are double bonds and side b is a single bond; R 1 is selected from the group consisting of S(0) 2 CH3, S(0) 2 NH 2 S(0) 2 NHC(O)CF 3 S(0)(NH)CH 3 S(0)(NH)NH2, S(0)(NH)NHC(0)0F3, (g) P(0)(CH 3 )OH and P(O)(CH3)NH 2 R 2 is selected from the group consisting of C1.6alkyl, C3-_7 cycloalkyl, mono-, di- or tni-substituted phenyl wherein the substituent is selected from the group consisting of hydrogen, halo, Ci-6alkoxy, Ci-6alkylthio, CN, CF3, C1_6alkyl, (8) N 3 -CO2H, (10) -C02-Cl~alkyl, (11) -C(R5)(R 6 (12) -C(R 5 )(R 6 )-0-C14alkyl, and (13) -Ci-6alkyl- C0 2 -R 5 mono-, di- or tni-substituted heteroaryl wherein the heteroaryl is a monocyclic aromatic ring of 5 atoms, said ring having one hetero atom which is S, 0, or N, and optionally 1, 2, or 3 additional 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 or 3 additional N atoms; said substituents are selected from the group consisting of hydrogen, halo, including fluoro, chloro, bromo and iodo, C1_6alkyl, C,- 6alkoxy, Cl-6alkylthio, CN, CF3, N 3 -C(R5)(R 6 (10) -C(R 5 )(R 6 )-0-CI~alkyl; R 3 is selected from the group consisting of hydrogen, CF 3 CN, Ci-6alkyl, hydroxyCl-6alkyl, and -C(0)-CI-6alkyl, optionally substituted -CI-5 alkyl-Q, -Cl- 3 alkyl-0-Cl-3alkyl-Q, -Ci_ 3 alkyl-S-Ci.3alkyl-Q, -CI-5 alkyl-0-Q, or -CIIs alkyl-S-Q, wherein the substituent resides on the alkyl and the substituent is Ci-3alkyl; -Q R 4 and R 4 are each independently selected from the group consisting of hydrogen, OF 3 CN, C1_6alkyl, and (h) optionally substituted -01.5 alkyl-Q, -0-C1.5 alkyl-Q, -S-C1_5 alkyl-Q, -Ol.3alkyl-0-O1-3alkyl- Q, -Cl. 3 alkyl-S-Cl-3alkyl-Q, -01-5 alkyl-0-Q, -01.5 alkyl-S-Q, wherein the substituent resides on the alkyl and the substituent is C1_3alkyl, and R 5 R 5 and R 6 R 7 and R 8 are each independently selected from the group consisting of hydrogen, Cl.6alkyl, or R 5 and R 6 or R 7 and R 8 together 30 with the carbon to which they are attached form a monocyclic saturated carbon ring of 3, 4, 5, 6 or 7 atoms; Q is 002H, C0 2 -CI.4alkyl, tetrazolyl-5-yl, C(R7)(R8)(OH), or C(R7)(R 8 )(0-CI~alkyl); provided that when X-Y-Z is -S-CR 4 =CR 4 then R 4 and R 4 are other than CF3. A compound of formula 11 as defined in the method according to claim 14 when used in treating or preventing colonic adenomas in a mammalian patient.

16. Use of a compound of formula 11 as defined in the method according to claim 14 in the manufacture of a medicament for treating or preventing colonic adenomas in a mammalian patient. [R:\LIBAA]07725.doc~tab

17. The method according to claim 14, the compound according to claim 15 or the use according to claim 16, wherein the compound is of formula III R 5 R R 0o 0 0 III or a pharmaceutically acceptable salt thereof wherein: R 1 is selected from the group consisting of (a) S(0) 2 CH 3 S(0)2NH 2 S(0) 2 NHC(O)CF 3 S(O)(NH)CH 3 S(O)(NH)NH 2 (f) S(O)(NH)NHC(O)CF 3 P(O)(CH 3 )OH and P(O)(CH 3 )NH 2 R 2 is selected from the group consisting of C3-7 cycloalkyl, mono-, di- or tri-substituted phenyl wherein the substituent is selected from the group consisting of hydrogen, halo, Ci-6alkoxy, Cl.ealkylthio, CN, CF3, C-6alkyl, N 3 -CO2H, (10) -CO2-C14alkyl, (11) -C(H)(R 6 (12) -C(H)(R 6 )-O-Ci- to 4alkyl, and (13) -Cl.alkyl-CO2H; mono-, di- or tri-substituted heteroaryl wherein the 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 additional 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 or 3 additional N atoms; said substituents are selected from the group consisting of hydrogen, halo, including fluoro, chloro, bromo and iodo, Cilalkyl, C1.6alkoxy, Ci. 6 alkylthio, CN, CF3, N3, -C(H)(R 6 (10) -C(H)(R 6 O-C-4alkyl; R 5 and R5' are each methyl or ethyl, R 6 is selected from the group consisting of (a) hydrogen, Ciealkyl.

18. The method, compound or use according to claim 17 wherein R 1 is selected from the group consisting of S(0) 2 CH 3 S(0) 2 NH2, S(O) 2 NHC(0)CF3, S(0)NHCH3, (e) 20 S(O)NHNH 2 and S(0)NHNHC(0)CF3; R 2 is selected from the group consisting of C-4alkyl, C 3 7cycloalkyl, mono- or di-substituted phenyl wherein the substituent is selected from the group consisting of hydrogen, fluoro, chloro, and bromo, C.4alkoxy, Ci4alkylthio, CN, (6) CF3, C14alkyl, N 3 -CO 2 H, (10) -CO2-Cl-3alkyl, (11) -C(H)(R 6 (12) -C(H)(R 6 3alkyl. 25 19. The method, compound or use according to claim 18 wherein R 2 is selected from the group consisting of cyclohexyl, and mono- or di-substituted phenyl, and wherein the substituents are selected from the group consisting of hydrogen, halo, C14alkoxy, Ci. 4alkylthio, CN, CF 3 Ci4alkyl, N3, and -C(H)(R 6 Rs and R 5 are each independently selected from the group consisting of methyl or ethyl, R 6 is selected from the group consisting of hydrogen, methyl or ethyl. The method, compound or use according to claim 19 wherein R 1 is selected from the group consisting of S(0) 2 CH3, S(0) 2 NH2, S(0)NHCH 3 and S(0)NHNH2; R 2 is selected from the group consisting of mono- or di-substituted phenyl wherein the substituents are selected [R:\BAA]07725.docab from the group consisting of hydrogen,(2) halo, selected from the group consisting of fluoro, chioro and bromo, Cl-3alkoxy, Cl-3alkythio, CN, and C1-3alkyl.

21. The method, compound or use according to claim 20 wherein R 1 is selected from the group consisting of S(O)2CH3, S(O) 2 NH 2 S(O)NHCH3, and S(O)NHNH2; R 2 is mono- or di-substituted phenyl wherein the substituents are selected from the group consisting of hydrogen, halo, selected from the group consisting of fluoro, chloro and bromo, methoxy, and methyl.

22. The method, compound or use according to claim 21 wherein R 1 is selected from the group consisting of S(O) 2 CH 3 and S(O) 2 NH2, R 2 is mono- or di-substituted phenyl wherein the substituents are selected from the group consisting of hydrogen, halo, selected from the group consisting of fluoro, chloro and bromo.

23. The method, compound or use according to claim 22 wherein the compound is selected from 3-(3-fluorophenyl)-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone, 3-(3,4-difluorophenyl)-4- (4-(methylsulfonyl)phenyl)-2-(5H)-furanone, 3-(3,4-dichlorophenyl)-4-(4-(nmethylsulfonyl)phenyl)-2- 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone and 5,5-dimethyl-3-(3- fluorophenyl)-4-(methylsulfonyl)phenyl)-2-(5H)-furanone or a pharmaceutically acceptable salt thereof. Dated 10 September 1999 MERCK CO., INC. MERCK FROSST CANADA INC. Patent Attorneys for the Applicants/Nominated Persons SPRUSON FERGUSON [R1\1BAA07725.doc~tab