AU698313B2

AU698313B2 - Use of lipoxygenase inhibitors as anti-cancer therapeutic and intervention agents

Info

Publication number: AU698313B2
Application number: AU22722/95A
Authority: AU
Inventors: Marti Jett; James L. Mulshine
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 1994-03-14
Filing date: 1995-03-14
Publication date: 1998-10-29
Anticipated expiration: 2015-03-14
Also published as: AU2272295A; CA2185448A1; EP0750496A1; WO1995024894A3; WO1995024894A2

Description

4,_~4 WO 95/24894 PCT/US95/03337 1 USE OF LIPOXYGENASE INHIBITORS AS ANTI-CANCER THERAPEUTIC AND INTERVENTION AGENTS Field of the Invention This invention is in the field of the prevention and treatment of cancer. More specifically, this invention relates to the use of 5-lipoxygenase inhibitors or derivatives thereof in preventing and treating cancer.

Background of the Invention Arachidonic acid (AA) is a polyunsaturated fatty acid derived from dietary sources.

Oxygenated AA metabolites participate in a variety of biologic and pathological processes including inflammation, bronchial asthma, and shock. Activation of AA metabolism is initiated by the release of AA from the phospholipid pool by the action of enzymes or other mediators. The released AA can be metabolized by either the lipoxygenase pathway where AA is converted by fatty acid lipoxygenases into hydroperoxy derivatives, giving rise to leukotrienes, or the cycloxygenase pathway where a fatty acid transforms AA rapidly to the prostaglandin

PGG

2 which in turn is further converted enzymatically.

A protein designated 5-lipoxygenase activating protein (FLAP) appears to be necessary for cellular leuketriene synthesis Patent No. 5,182,367; Miller et al. (1990) Nature 343:278-281). FLAP appears to be involved in the translocation of 5-lipoxygenase from the cytosol to the membrane. Other enzymes involved with the sequential metabolism of the lipoxygenase product need to be in close relationship to the 5-lipoxygenase FLAP complex. FLAP and molecules of related structure may be essential to the activity of enzymes in the 7 r ic~ g pathway as well as the other downstream enzymes required for leuketriene biosynthesis.

Inhibitors of the AA 5-lipoxygenase pathway have shown promise in the treatment of inflammation, asthma, and shock, with minimal side effects in preclinical and clinical trials (Batt, Prog. Med. Chem. 29:1-63 (1992); Larson, Ann.Pharmacother. 27:898-903 (1993)). However, their use for treating or preventing cancer has never been described.

The present invention is directed to the use of inhibitors of functional activity for the treatment and prevention of epithelial cell-derived cancers. The present invention is also directed to the use of inhibitors of other enzymes in the lipoxygenase pathway involved in the metabolism of AA for use in the treatment and prevention of epithelial cell-derived cancers.

Summary of the Invention In general the present invention relates to the use of lipoxygenase inhibitors in therapeutic applications, in particular to the prevention and treatment of epithelial cell-derived cancers.

In one aspect of the present invention there is provided a method for treating an epithelial cell-derived cancer in a subject in need of such treatment which comprises administering to the subject an amount of a 20 inhibitor selected from the group consisting of: 4 -chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethyl propanoic acid or a derivative thereof; and zileuton; effective to treat the epithelial cell-derived cancer.

25 A further aspect of the present invention provides a method for preventing an epitheltsi cell-derived cancer in a subject in need of such prevention which comprises administering to the subject an amount of a 5-lipoxygenase inhibitor selected from the group consisting of: 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethyl propanoic acid or a derivative thereof; and zileuton; effective to treat the epithelial cell-derived cancer.

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4r i rj i3t-~ C:\WINWORDULENNYMSPECNKI22722.95.DOC ~i Another aspect of the present invention provides a method for preventing an epithelial cell-derived cancer in a subject in need of such prevention which comprises administering to the subject an amount of an inhibitor of an enzyme that metabolises arachidonic acid effective to prevent an epithelial cell derived cancer.

A further aspect of the present invention provides a method for treating an epithelial cell derived cancer in a subject in need of such treatment which comprises administering to the subject an amount of an inhibitor of an enzyme that metabolises arachidonic acid effective to treat the epithelial cell-derived cancer.

In a yet further aspect of the present invention a method is provided for treating an epithelial cell-derived cancer in a subject in need of such treatment which comprises administering to the subject an amount of a hydroxyurea derivative effective to treat the epithelial cell-derived cancer.

A further aspect of the present invention provides a method for preventing an epithelial cell-derived cancer in a subject in need of such prevention which comprises administering to the subject an amount of a hydroxyurea derivative effective to treat the epithelial cell-derived cancer.

Finally, in another aspect of the present invention there is provided a cj 20 pharmaceutical composition which can be used in the methods described herein.

Brief Description of the Figures Figures and Effect of 5-lipoxygenase inhibitor AA861 on I the in vitro growth of specific lung.

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4/1 cancer cell lines. Fia. classic SCLC cell lines NCI-H209, NCI-H345, and NCI-H510. Fia. 1(b) variant SCLC cell lines NCI-N417 and NCI-H82. Fiq. NSCLC cell lines NCI-H23, A549, and NCI-H1155.

Fiaures and Effect of inhibitor NDGA on the in vitro growth of lung cancer cell lines. Fig. classic SCLC cell lines NCI-H209 and NCI-H345. Fis. variant SCLC cell lines NCI-N417 and NCI-H82. Fig. NSCLC cell lines A549 and NCI- H1155.

Figures 3 and Effect of inhibitor MK-886 on the in vitro growth of lung cancer cell lines. Fia. classic SCLC cell lines NCI-H209 and NCI-H345. Fi variant SCLC cell lines NCI- N417 and NCI-H82. Fiq. NSCLC cell lines A549 and NCI-H1155.

Figure 4. Structures of MK-886 and related structures: L-669,572, L-663,511, L-665,210, L-654,639, L-668,017 and MK-591.

Figure 5. Effect on tumor volume (NCI-H209 xenografts) over time in animals treated with NDGA and control animals receiving phosphate buffered saline (PBS;O) Detailed Description of the Invention The present invention provides a method for treating or preventing an epithelial cell-derived cancer in a subject in need of such treatment or prevention which comprises administering to the subject an amount of a lipoxygenase inhibitor or derivative thereof, effective to treat or prevent the epithelial cell-derived cancer. The present invention also provides a method for treating or preventing an epithelial cell derived cancer in a subject in need of such treatment by administering an effective amount of an inhibitor of other enzymes involved in the metabolism of arachidonic acid in the C 1so' AMENDED SHEET

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pathway which comprises administering to the subject an amount of the inhibitor effective to treat or prevent a epithelial cell derived cancer.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and 'comprises", is not intended to exclude other additives, components, integers or steps.

The term "treatment" includes partial or total inhibition of the cancer growth, as well as partial or total destruction of the cancer cells.

The term "prevention" includes either preventing the onset of clinically evident cancer altogether or preventing the onset of a preclinically evident stage of cancer in individuals at risk, also intended to encompassed by this definition is the prevention of initiation for malignant cells or to arrest or reverse the progression of premalignant cells to malignant cells.

i In both methods above, the epithelial cell-derived cancer (epithelial carcinoma) includes basal cell carcinoma, adenocarcinoma, colon cancer, 15 prostate cancer, renal cell carcinoma, and other known cancers that effect epithelial cells throughout the body. Preferably, the epithelial cell-derived cancer is lung cancer or breast cancer.

The term "subject" for purposes of treatment includes any human or animal subject who has any one of the known epithelial cell-derived cancerns, and 20 preferably is a human subject. For methods of prevention, the subject is any human or an'ral subject, and preferably is a human subject who is at risk for obtaining an epithelium cell-derived cancer. The subject may be at risk due to exposure to carcinogenic agents, being genetically predisposed to have the cancer, and the like.

Inhibitors of the 5-lipoxygenase pathway in the metabolism of arachidonic acid used in the prevention and treatment of epithelial cell derived cancers may inhibit enzyme activity through a variety of mechanisms. By the way of example, the inhibitor may block or reverse the association of the enzyme with the membrane or inhibit the Li <C c~

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WO 95/24894 PCTUS95/03337 6 S translocation of specific enzymes such as via a protein such as FLAP. Alternatively, the inhibitors used in the methods described herein may block the enzyme activity directly by acting as a substrate for the enzyme or by depriving the enzyme of necessary cofactors.

The methods provided herein relate to the use of inhibitors or derivatives thereof in the prevention and treatment of epithelial cell derived cancers. In the preferred embodiments, the inhibitor is 2-(12-Hydroxydodeca-5,10-diynyl)-3,5,6trimethyl-1,4-benzoquinone (AA861) (Yoshimoto, et al.

(1982) Biochemical Biophvsica ACTA 713:470-473; Ashida, Y, et al. (1993) Prostoclandins 26(6):955) or derivatives thereof; Nordihydroguaiaretic acid (NDGA) (Wang, et al.

(1991) Mutation Research 261:153-162; Salari, et al.

(1984) Prostoqlandins Leukotrienes And Medicine 13:53-60) or derivatives thereof; or 3-[l-(4-chlorobenzyl)-3-tbutyl-thio-5-isopropylindol-2-yl]-2, 2-dimethylpropanoic acid (MK886) (Gillard et al. (1989) Can J. Physiol.

Pharmacol 67:456-464; Rouzer, et al (1990) Journal of Biolocical Chemistry 265:1436-1442) or derivatives thereof.

Derivatives are intended to encompass any compounds which are structurally related to AA861, NDGA or MK886 or which possess the substantially equivalent biologic activity of AA861, NDGA or MK886. By way of example, such inhibitors may include, but are not limited to, derivatives which act as co-factor antagonist, better enzymatic substrates or inhibitors of activating peptide function.

1n MK886 as used herein has the formula: r{ t ,t

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a; jn i I~I~LILi-.: WO 95/24894 PCT/US95/03337 7 Derivatives of MK886 are also intended to be encompassed by this invention. In a preferred embodiment a derivative of MK886 may have the general formula: .w N 02 Wherein RI is C 1

-C

6 alkyl or OH or R 2 CO- in which

R

2 is H or CI-C 6 alkyl or quinoline or a long chain hydrocarbon possessing 1-32 carbon atoms and 0-6 double bonds. Examples of long chain hydrocarbons include, but are not limited to, linoleic acid (18:2) or oleic acid Wherein R 3 is CI-C 6 alkyl or R 4 S- or R 5 SO- or R 6

SO

2 and which R 4 or Rs or R 6 may be Ci-C 6 alkyl or quinoline or a long chain hydrocarbon possessing 1-32 carbon atoms and 0-6 double bonds. Examples of long chain hydrocarbon include, but are not limited to, linoleic acid or oleic acid (18:1).

Examples of R 3 include, but are not limited to,

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I WO 95/24894 PCTUS95/03337 S-8t-butyl thio, cyclic propyl methylthio, phenyl sulfonyl, phenyl, methyl, phenyithiol.

Derivatives of MK886 intended to be encompassed by this invention include, but are not limited to, L- 669,572, 3-[1-(p-cholorobenzyl)-5-isopropyl-3-cyclopropylmethylthioindole-2-yl]-2,2-dimethylpropanoic acid; L-663,511 3-[1-(p-cholorobenzyl)-5-isopropyl-3phenysulfonylindol-2-yl)-2,2-dimethylpropanoic acid, L- 665,210, 3-[1-(p-chlorobenzyl)-5-isopropyl-3phenysulfonylindol-2-yl)-2,2-dimethylpropanoic aid; L-654- 639, 3[1-(p-chlorobenzyl)-5-methoxy-3-methylindol-2-yl]- 2,2-dimethylpropanoic acid; and L-668,017 described in Rouzer et al. (1990) Journal of Biological Chemistry 265:1436-1442 which is herein incorporated by reference (Figure In a preferred embodiment, the MK886 derivative is 3-(1-(4-chlorobenzyl)-3-(1-butyl-thio)-5- (quinolin-2-yl-methoxy)-indol-2-yl)-2,2-dimethyl propanoic acid) (MK-591) (Tagari, et al. (1993) Agents Action 40:62- 71).

In yet another embodiment of this invention hydroxyurea derivatives are also used as inhibitors of in the prevention and treatment of epithelial cell derived cancers. Examples of hydroxyurea derivatives include, but are not limited to, benzo(b)thien-2-ylethyl)-N-hydroxyurea) (zileuton) (Tagari, et al. (1993) Acgents Action 40:62-71 herein incorporated by reference.

In an alternative embodiment of this invention inhibitors of other enzymes that metabolize arachidonic acid downstream of 5-lipoxygenase, may also be used to prevent or treat epithelial cell derived cancers 3' a subject in need of such treatment in a method which comprises administering to the subject an amount effective to prevent or treat the epithelial cell derived cancer.

Such inhibitors may cffect the activity of the enzyme amtiiil-- WO 95/24394 PCT/US95/03337 -9either directly by acting as a substrate inhibitor or by depriving the enzyme of a cofactor. The inhibitor may also act by targeting proteins such as FLAP which are responsible for the translocation of the enzymes to the membrane where the enzymes are activated.

The compounds utilized in the methods of the present invention may be present in the form of free bases or pharmaceutically acceptable acid addition salts thereof. Examples of suitable acids for salt formation are: methanesulfonic, sulfuric, hydrochloric, phosphoric, acetic, citric, lactic, ascorbic, maleic, and the like.

The administration for the above methods may be affected by means known to those skilled in the a..t such as oral, rectal, topical (including aerosol), intranasal, intravenous, subcutaneous, intramuscular, intrabronchial, intracavitary, or intraperitoneal routes of administration. If the cancer is localized, local administration rather than system administration is preferred. Formulation in a lipid vehicle may be used to enhance bioavailability.

0 For intravenous, intramuscular, subcutaneous, or intraperitoneal administration, the compound may be combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient. Such formulations may be prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. The formulations may be present in 1 unit or multi-dose containers such as sealed ampoules or vials.

1 For oral administration, the formulation may be presented as capsules, tablets, powders, granules or a suspension, with conventional additives such as lactose, 2 1" 1 H r It -'9 a~ e WO 95/24894 PCTUS95/03337 10 0 mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators sujh as corn starch, potato starch or sodium carboxymethyl-cellulose; and with lubricants such as talc or magnesium stearate.

If the cancer is localized in the G.I. tract, the compound may be formulated with acid-stable, baselabile coatings known in the art which begin to dissolve in the high pH small intestine. Formulation to enhance local pharmacologic effects and reduce systemic uptake are preferred.

Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably made isotonic. Preparations for injections may also be formulated by suspending or emulsifying the compounds in non-aqueous solvent, such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol.

Formulations for topical use include known gels, creams, oils, and the like. For aerosol delivery, the compounds may be formulated with known aerosol exipients, such as saline, and administered using commercially available nebulizers. Formulation in a fatty acid source may be used to enhance biocompatibility. Aerosol delivery is the preferred method of delivery for epithelial cancers of the lung for prevention application.

For rectal administration, the active ingredient may be formulated into suppositories using bases which are solid at room temperature and melt or dissolve at body temperature. Commonly used bases include coca butter, glycerinated gelatin, hydrogenated vegetable oil, polyethylene glycols of various molecular weights, and fatty esters of polyethylene stearate.

The dosage form and amount can be readily pl n '7 Our Ref: 464542 t i r 1925x r 1- I i DPTnTROrLln???7 WO 95/24Y94 J ,JLI V 'I J 'JVUY I 11 0 established by reference to known cancer treatment or prophylactic regiments. The dosage for the inhibitors or derivatives thereof may be from about 0.1 ng/kg to about 450 mg/kg, most preferred is about 0.5 ng/kg to about 100 mg/kg, and most preferably is about 1 ng/kg to about mg/kg. The serum concentration of the inhibitor may be from about 1 Ag/ml to about 20 Ag/ml. The actual dose will depend upon whether the administration is for treatment or prophylactic purposes, the route of administration, the location of the cancer, as well as the pharmacokinetic properties of the individual treated. The dosad will generally be lower if the compounds are administered locally rather than systemically, and for prevention rather than for treatment. Such treatments may be .administered as often as necessary and for the period of time judged necessary by the treating physician. One of skill in the art will appreciate that the dosage regime or therapeutically effective amount of the inhibitor to be administrated may need to be optimized for each individual.

The administration of the present invention may be for either prevention or treatment purposes. The methods and compositions used herein may be used alone or in conjunction with additional therapies known to those skilled in the a-t in the prevention or treatment of cancer. Alternatively, the methods and compositions described herein may be used as adjunct therapy. By way of example, the 5-lipoxygenase inhibitor may be administered alone or in conjunction with other antineoplastic agents or other growth inhibiting agents or otJaer drugs or nutrients. Alternatively the lipoxygenase inhibitor or derivatives thereof and the inhibitor of other downstream enzymes involved in the metabolism of arachidonic acid may be administered in combination with each other.

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i T /2 WO 95/24894 PCT/US95/03337 12 0 In an alternative embodiment molecular methods may be used to inhibit the 5-lipoxygenase. By way of example antisense constructs generated by methods known to those skilled in the art can be used to target the messenger RNA of the 5-lipoxygenase enzyme.

All books, articles or patents reference herein are incorporated by reference. The present invention is described in the following Experimental Details section, which sets forth specific examples to aid in an understanding of the invention, and should not be construed to liit in any way the invention as defined in the claims which follow thereafter.

Example 1 Materials and Methods Cell lines: The following cell lines were used: S classic small cell lung cancer (SCLC) cell lines NCI-H209, NCI-H345, and NCI-H510; variant SCLC cell lines NCI-N417 and NCI-H82; large cell carcinoma cell line NCI-H1155; adeno carcinoma cell.line NCI-H23; and bronchioalveolear carcinoma cell line A549, breast cancer cell line MCF-7 (American Type Tissue Culture Rockville MD; ATCC) and a color cancer cell line NCI-H630 (ATCC). All cells were grown in RPMI-1640, supplemented with 5% fetal bovine serum (FBS), penicillin and streptomycin (Gibco, Grand Island, NY), and were maintained in a 5% CO 2 atmosphere at 37 0 C. All cell lines were free of mycoplasma contamination.

Biochemical compounds: General lipoxygenase inhibitor Nordihydroguaiaretic acid (NDGA) and lipoxygenase inhibitor AA861, are readily commercially 3 available and were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). inhibitor MK886 and MK-591 were obtained from Merck Frost.

MK886 may be synthesized as described in Gillard, et al. Can. J. Physiol. Pharmacol. 67:456-464 (1989)).

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WO 95/24894 PCTUS95/03337 13 0 Growth studies: A modification (Promega CellTiter 960, Promega Madison, WI) of the semiautomated colorimetric assay, MTT (Nakanishi, et al. Exper. Cell Biol. 56:74-85 (1988)), which quantitates cell numbers based on reduction of a tetrazolium compound by tumor cells as determined by a spectrophotometer (540 nm) was used. All assays were performed in RPMI-1640 mec2ia supplemented with transferrin 10 Ag/ml, insulin gg/ml and selenium 5x10 8 M (TIS) (Sigma Chemicals, St.

Louis, MO). Seeding densities were 1-2x10 4 cells/well, and cells were grown for 5 days. Each experiment was reported as mean optical density corrected for background standard deviation for a minimum of six data points per experiment, with each experiment repeated at least three times.

Results AA861 was tested in a dose range of 0.5-10 AM on three classic SCLC cell lines, two variant SCLC cell lines, and three NSCLC cell lines. The effect of AA861 on the in vitro growth of the specific lung cancer cell lines is presented in Figures and AA861 significantly inhibited growth on all of the SCLC cell lines, and two out of three of the NSCLC ccll lines. A bronchioalveolear carcinoma, A549, was repeatedly resistant to AA861. The reason for this resistance is unclear. NDGA also was tested on four SCLC cell lines and two NSCLC cell lines, and the results are presented in Figures and NDGA inhibited the growth of all of the lung cancer cell lines tested. MK886 also was tested on four SCLC cell lines and two NSCLC cell lines, and the results are presented in Figures 3(a), and MK886 showed similar inhibition r~sults as NDGA. AA861 inhibited growth of MCF-7 cells relative to controls (absence of AA861) to 70% and H630 to NDGA inhibited growth of MCF-7 cells relative to controls

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(Referred to in PCT Gazette No. 46/1995, Section II) c i ~1V-IYa~ll*UT~.~~ WO 95/24894 PCT/US95/03337 14 0 80% and H630 cells 50% relative to controls. MK886 inhibited growth of MCF7 cells 100% relative to controls.

Example 2 In Vivo Models of Cancer and Carcinogeneses The therapeutic potential of the inhibitor, NDGA, was assessed in a heterotransplant animal model. Using conventional methodology small cell lung cancer cells (cell line NCI-209) were transplanted into nude mice. Experimental animals received a 0.1% solution of NDGA in water ad libitum and drank about 5cc of water/day. Control animals received phosphate buffered saline. The xenografts of the small cell lung cancer line, NCI-209, showed a significant reduction in heterotransplant size (compared to controls) at 2.5 to weeks after cell line engraftment in mice receiving orally administered NGDA. (see Figure 5 and Table I) Table I. Weight of nude mice (grams) Treatment Mouse Tumor None 26.5 1.3 1.29 0.07 NDGA 25.9 1.9 0.60 0.19* The mean weight S.D. of 4 determinations is indicated; p 0.05, using students t-test.

AA861 and derivatives thereof and MK886 and derivatives thereof may be also be tested in the heterotransplant model. Suggested serum concentration of the inhibitor in experimental animals may be from about 1 Ag/ml to about 20 g/ml. In addition to treatment models, in vivo models of cancer prevention may also be assessed for the therapeutic potential of the inhibitors or derivatives thereof.

All publications mentioned hereinabove are hereby incorporated by reference in their entirety. While the foregoing invention has been described in some detail 136a51 p- r t- nF-- A 'Il essential to the activity of enzymes in the r

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WO 95/24894 PCTUS95/03337 15 for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of the disclosure that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.

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Claims

1. A method for treating an epithelial cell-derived cancer in a subject in need of such treatment which comprises administering to the subject an amount of a lipoxygenase inhibitor selected from the group consisting of:

3-[1-(4-chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethyl propanoic acid or a derivative thereof; and zileuton; effective to treat the epiethelial cell-derived cancer. 2. A method according to claim 1 wherein the derivative of chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethyl propanoic acid is selected from the group including L-669,572, L-663,511, L-665,210, L-654,639, L- 668,017 and MK-591. 3. A method according to claim 2 wherein the derivative of chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethyl propanoic acid is (3-(1-(4-chlorobenzyl)-3-(t-butyl-thio)-5-(quinoline-2-yl-methoxy)-indol-2-yl)-2,2- dimethyl propionic acid (MK-591).

4. A method according to any one of claims 1 to 3, wherein the epithelial cell- derived cancer is lung cancer, colon cancer, or breast cancer. 20 5. A method according to any one of claims 1 to 4, wherein the administration is affected by oral, rectal, topically by aerosol, intravenous, subcutaneous, intramuscular, intrabronchial, intracavitary, or intraperitoneal administration.

6. A method according to any one of claims 1 to 5, wherein the amount is between about 1 ng/kg and about 10 mg/kg.

7. A method for preventing an epithelial cell-derived cancer in a subject in need of such prevention which comprises administering to the subject an amount of a 5-lipoxygenase inhibitor selected from the group consisting of: 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethyl propanoic acid or a derivative thereof; and zileuton; effective to prevent the epithelial cell-derived cancer. C S' C. C C Cr C C Cto C i I: F i. i; i: i: i rt~ P i_ W O095/24 894 PCTIUS95/03337 Qe NWORDIENNYMSPECNKIl22722-95.DOC 17

8. A method according to claim 7, wherein the derivati, of chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethy propanoic acid is selected from the group including L-669,572, L-663,511, L-665,210, L-654,639, L- 668,017 and MK-591.

9. A method according to claim 8, wherein the derivative is chlorobenzyl)-3-(t-butyl-thio)-5-(quinoline-2-yl-methoxy)-indol-2-yl)-2,2-dimethy propionic acid (MK-591). A method according to any one of claims 7 to 9, wherein the epithelial cell- derived cancer is selected from the group including lung cancer, colon cancer or breast cancer.

11. A method according to any one of claims 7 to Iu wherein the administration is affected by oral, rectal, topically by aerw< intravenous, subcutaneous, intramuscular, intrabronchial, intracavitary, or intraperitoneal administration.

12. A method according to any one of claims 7 to 11, wherein the amount is between about 1 ng/kg and about 10 mg/kg.

13. A pharmaceutical composition for the treatment of epithelial cell cancer C comprising a 5-lipoxygenase inhibitor selected from the group consisting of: 3-[1 -(4-chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethyl propanoic acid or a derivative thereof; and zileuton; and a pharmaceutically acceptable carrier, diluent or excipient.

14. A pharmaceutical composition according to claim 13 containing chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethylpropanoic acid or a derivative thereof. A pharmaceutical composition according to clairrm 13 wherein the derivative of 3-[1 -(4-chlorobenzyl)-3-t-butyl-thio-W isopropyl-indol-2-yl]-2,2-dimethylpropanoic acid is selected from the group including L-669,572, L-663,511, L-665,210, L- 654,639, L-668,017, -(4-chlorobenzyl)-3-(t-butyl-thio)-5-(quinoline-2-yl- methoxy)-indol-2-yl)-2,2-dimethyl propionic acid (MK-591). CWNORDIENNYMIPECNKT2272295.DOC C',IMNWORDJENNWSPECNKM722-95.DMis TE I Ii amount of an inhibitor of other enzymes involved in the metabolism of arachidonic acid in the 5-lipoxygenase U iT 160 AMENDED SHEET IPEA/EP >18

16. A method of treating epithelial cell cancer in a subject in need thereof said method including administering an effective amount of a pharmaceutical composition according to any one of claims 13 to

17. A method according to claim 16 wherein the epithelial cancer is selected from the group including lung cancer, colon cancer or breast cancer.

18. A method according to claim 16 or 17, wherein the composition is administered orally, rectally, topically by aerosol, intravenously, subcutaneously, intramuscularly, intrabronchially, intracavitary, or intraperitoneally.

19. A method according to any one of claims 16 to 18, wherein the amount of active compound in the pharmaceutical composition is in the range of 1 ng/kg to mg/kg. A method according to any one of claims 16 to 19, wherein the amount of inhibitor is in the range of 1 ng/kg to 10 mg/kg.

21. A method for treating an epithelial cell derived carrier in a subject which comprises administering to the subject an amount of an inhibitor of an enzyme that metabolises arachidonic acid effective to treat the epithelial cell-derived cancer.

22. A method for preventing an epithelial cell-derived cancer in a subject which comprises administering to the subject an amount of an inhibitor of an enzyme that metabolises arachidonic acid effective to treat the epithelial cell-derived cancer.

23. A pharmaceutical composition comprising an inhibitor of an enzyme that metabolises arachidonic acid and a pharmaceutica!ly acceptable diluent, excipient or carrier. 25 24. A method of treatment of epithelial cell-derived cancer in a subject in need thereof including administration of a composition according to claim 23. A method of preventing epithelial cell-derived cancer in a subject in need thereof including administration of a composition according to claim 23.

26. A method for treating an epithelial cell-derived cancer in a subject which comprises administering to the subject an amount of an inhibitor of a 5- lipoxygenase downstream enzyme that metabolises arachidonic acid effective to treat the epithelial cell-derived cancer. 1WIVNWORDDUENNYMSPECNKn22722-95,DOC CI *4 *i 4 S. CI C' C 4 I 44 F 44 4cv C i c (C r C 4 c CCr CC 19

27. A method for preventing an epithelial cell-derived cancer in a'subject which comprises administering to the subject an amount of an inhibitor of a lipoxygenase downstream enzyme that metabolises arachidonic acid effective to treat the epithelial cell-derived cancer.

28. A pharmaceutical composition comprising an inhibitor of a downstream enzyme that metabolises arachidonic acid a pharmaceutically acceptable carrier, diluent or excipient.

29. A method of treatment of an epithelial cell-derived cancer in a subject in need thereof including administration of a composition according to claim 28.

30. A method of prevention of epithelial ceil-derived cancer in a subject including administration of a composition according to claim 28.

31. The use of a 5-lipoxygenase inhibitor selected from the group consisting of: 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethyl propanoic acid or a derivative thereof; and zileuton; in the preparation of a medicament for the prevention or treatment of epithelial cell-derived cancer in a subject.

32. A use according to claim 31 wherein the 5-lipoxygenase is chlorobenzyl)-3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethyl propanoic acid or a derivative thereof.

33. A use according to claim 32 wherein the derivative of 3-[1-(4-chlorobenzyl)- 3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethyl propanoic acid is selected from the group including L-669,572, L-663,511, L-665,210, L-654,639, L-668,017 and MK-591. 25 34. A use according to claim 33 wherein the derivative of 3-[1-(4-chlorobenzyl)- 3-t-butyl-thio-t-isopropyl-indol-2-yl]-2,2-dimethyl propanoic acid is chlorobenzyl)-3-(t-butyl-thio)-5-(quinoline-2-yl-methoxy)-indol-2-yl)-2,2-dimethy propionic acid (MK-591). A use according to any one of claims 31 to 34 wherein the epithelial cell derived cancer is lung cancer, colon cancer or breast cancer.

36. A use according to any one of claims 31 to 35 wherein the preparation produces a medicament in a form suitable for oral, rectal, aerosol, intravenous, S:\WINWORDUENNYMSPECNKIJ22722-95.DOC I j i; i 431L~ql No DRT I ccmana plicanon INTERNATIONAL SEARCH REP( li i: B subcutaneous, intramuscular, intrabronchial, intracavitary, or intraperitoneal administration.

37. A use according to any one of claims 31 to 36 wherein the medicament is in a form suitable to provide between about 1 mg/kg and about 10 mg/g to the subject.

38. Use of an inhibitor of an enzyme that metabolises arachidonic acid in the preparation of a medicament for the prevention or treatment of epithelial cell- derived cancer.

39. Use of an inhibitor of a 5-lipoxygenase downstream enzyme that metabolises arachidonic acid for the preparation of a medicament for the prevention or treatment of epitheliai cell derived cancer. A method according to claim 1 substantially as hereinbefore described with reference to example 1. DATED: 26 August, 1998 C 4 4 4 44 44c 4 4* 4 4,f 4s 44 4 4 445 I+ 4 4 h cC PHILLIPS ORMONDE FITZPATRICK Attorneys for: THE GOVERNMENT OF THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES. 7f c r. C:\WINWORDUENNYMPECNKI22722-95.DOC I' I