REMEDIES FOR CANCER

Abstract: It is provided that type-X sPLA 2 inhibitors are useful in preventing or treating cancer.

Description: Technical Field The present invention relates to a composition for the prevention or treatment of cancer which contains an inhibitor against type-X sPLA 2 (secretary PLA 2 ) as an active ingredient. Background Art It is described in Cell. (1996) 87 803-809 that inhibitors of COX-2 at a downstream position of PLA 2 in arachidonate cascade are useful for the treatment of rectosigmoid cancer. As the method of diagnosing tumor including prostate cancer, a quantitative and qualitative assay to detect sPLA 2 proteins or sPLA 2 mRNA levels is described in WO98/05349 (JP Laid-Open (Tokukai) No. 2001/500847 ). However, neither of the documents describes that compounds having inhibitory activities against type-X sPLA 2 are effective for the treatment of cancer. WO 00/28997A discloses a method for facilitating apoptosis of cancer cells by inhibiting phospholipase activity. WO99/24026 A discloses compositions for the treatment of disorders associated with apoptosis using N-hexerocyclic glyoxylamide compounds. Disclosure of Invention The inventors of the present invention examined the expression of type-X sPLA 2 in various kinds of human pathological tissues with anti-type-X sPLA 2 antibody. They found the elevated expression of type-X sPLA 2 in several tumor tissues. The immunohistochemical analysis of tumor tissues was performed as follows. At first, anti-human type-X sPLA 2 antibody was added to the slides prepared from normal adult tissues or tumor tissues from cancer patients and incubated for several hours. Next, in order to examine the expression of type-X sPLA 2 in the tissues, the expression of type-X sPLA 2 was visualized by using the methods such as the immunohistochemical labeling to detect the type-X sPLA 2 signals. Consequently, the type-X sPLA 2 signals were detected in the slides prepared from tumor tissues, suggesting that the expression of type-X sPLA 2 is elevated in tumor tissues. In addition, the inventors of the present invention performed the experiments for neutralization of type-X sPLA 2 signals: Precisely, before the addition of anti-human type-X sPLA 2 antibody to the slides, the slides were incubated with the purified type-X sPLA 2 protein for several hours. Hereafter, the slides were processed with the same procedures as described above to examine the type-X sPLA 2 signals. Consequently, the type-X sPLA 2 signals were disappeared in the slides prepared from tumor tissues. Thus, the elevated expression of type-X sPLA 2 was confirmed in human tumor tissues. Furthermore, the inventors of the present invention examined the inhibitory effects of sPLA 2 inhibitor against the type-X sPLA 2 -induced release of oleic acid from tumor cell. Consequently, they confirmed that sPLA 2 inhibitors significantly blocked the type-X sPLA 2 -induced release of oleic acid from tumor cells. On the other hand, potential involvement of PGE 2 in the development of tumors has been described in Cancer Research 59, 5093-5096, 1999 . Then, the inventors of the present invention examined the inhibitory effects of sPLA 2 inhibitors against the type-X sPLA 2 -induced PGE 2 production in tumor cells. Consequently, they confirmed that sPLA 2 inhibitors significantly blocked the type-X sPLA 2 -induced PGE 2 production in tumor cells. The inventors of the present invention achieved the following present invention based on the above experimental results. That is to say, the present invention relates to the following I) use of a type -X sPLA 2 inhibitor for the preparation of a medicament for the prevention or treatment of cancer wherein the type-X sPLA 2 inhibitor is a compound represented by the formula: or , its pharmaceutically acceptable salt, or its solvate. II) use according to I) wherein the cancer is colon cancer, lung cancer, liver cancer, gastric cancer, renal cancer, gellbladder cancer, prostatic cancer, pancreatic cancer, testis cancer, ovary cancer or cutaneous cancer. III) use according to I) wherein the cancer is colon cancer. The present invention is illustrated in detail as follows Type-X sPLA 2 inhibitors are compounds which have an inhibitory activity against type-X sPLA 2 and other optional activities such as inhibitory activities against other enzymes or affinities for any receptors. Such inhibitors include any compound having stronger activities against type-X sPLA 2 than that having no such activities in the evaluation test therefore. Especially, type-X sPLA 2 selective inhibitors are preferred as type-X sPLA 2 inhibitors of the present invention. For example, compounds whose IC 50 values against type X sPLA 2 are 1 µM or less in the experiment of Example 2 are preferred. Compounds having IC 50 values 100 nM or less are more preferred. A type-X sPLA 2 inhibitor, a compound having type-X sPLA 2 inhibitory activities, having one or more of chiral center(s), may exist as an optically active member. Likewise, a compound containing alkenyl or alkenylene, may be a cis- or trans-isomer. Mixtures of R- and S-isomers as well as of cis- and trans-isomers, and mixtures of R- and S-isomers containing a racemic mixture are included in the scope of the present invention. An asymmetric carbon atom may exist also in a substituent such as alkyl group. All such isomers and mixtures are included in the present invention. A specified stereoisomer can be manufactured by subjecting to stereospecific reaction well known to those skilled in the art applying a previously separated starting material having an asymmetrical center or by preparing a mixture of stereoisomers and separating the mixture in accordance with a well-known manner. C 1 -C 6 alkylesters (e.g. methyl ester, ethyl ester) as prodrugs may also be used. When a compound having type-X sPLA 2 inhibitory activities has an acidic or basic functional group, a variety of salts having a higher water solubility and more physiologically suitable properties than those of the original compound can be formed. An example of typical pharmaceutically acceptable salts includes salts with alkali metal and alkaline earth metal such as lithium, sodium, potassium, magnesium, aluminum. A salt is easily manufactured from a free acid by either treating an acid in a solution with a base, or allowing an acid to be in contact with an ion exchange resin. Addition salts of the compounds having type-X sPLA 2 inhibitory activities with relatively non-toxic inorganic bases and organic bases, for example, amine cation, ammonium, and quaternary ammonium derived from nitrogenous bases having a basicity sufficient for forming a salt of the compounds of the present invention are included in the definition of "pharmaceutically acceptable salts". (e.g., S. M. Berge et al., "Pharmaceutical Salts, "J. Phar. Sci., 66, 1-19 (1977) ). Furthermore, basic groups of a compound having type-X sPLA 2 inhibitory activities are reacted with a suitable organic or inorganic acid to form salts such as acetates, benzenesulfonates, benzoates, bicarbonates, bisulfates, bitartrate, borates, bromides, camsylates, carbonates, chlorides, clavulanates, citrates, edetates, edisylates, estolates, esylates, fluorides, fumarates, gluceptates, gluconates, glutamates, glycolylarsanilates, hexylresorcinates, hydroxynaphthoates, iodides, isothionates, lactates, lactobionates, laurates, malates, maleates, mandelates, mesylates, methylbromides, methylnitrates, methylsulfates, mucates, napsylates, nitrates, oleates, oxalates, palmitates, pantothenates, phosphates, polygalacturonates, salicylates, stearates, subacetates, succinates, tannates, tartrates, tosylates, trifluoroacetates, trifluoromethanesulfonates, valerates and the like. The solvate includes solvates with organic solvents and/or hydrates. In case of forming a hydrate, a questioned compound may be coordinated with a suitable number of water molecules. The term "pharmaceutically acceptable" means that carriers, diluents, or additives are compatible with other ingredients in a formulation and are not harmful for recipients. The "cancer" means various malignant tumors originated from epithelial cells in various tissues, cells such as colon cancer, lung cancer, liver cancer, gastric cancer, renal cancer, gallbladder cancer, prostate cancer, pancreatic cancer, testis cancer, ovarian cancer, cutaneous cancer, esophagus cancer, laryngeal cancer, breast cancer or uterine cancer and exemplified. Based on the experiments, the inventor of the present invention confirmed the elevated expression of type-X sPLA 2 in various tumor tissues, including colon cancer, lung cancer, liver cancer, gastric cancer, renal cancer, gallbladder cancer, prostate cancer, pancreatic cancer, testis cancer, ovarian cancer or cutaneous cancer. Especially, the present invention is useful for the prevention or treatment of colon cancer, lung cancer, liver cancer, gastric cancer, renal cancer, gallbladder cancer, prostate cancer, pancreatic cancer, testis cancer, ovarian cancer or cutaneous cancer. The term "acidic group" in the present specification means an organic group functioning as a proton donor capable of hydrogen bonding when attached to a basic skeleton through a suitable linking atom (hereinafter defined as "acid linker"). Best Mode for Carrying Out the Invention The present invention relates to the prevention or treatment of cancer by a type-X sPLA 2 inhibitor. The type-X sPLA 2 inhibitors may be known ones and are selected from sPLA2 inhibitors defined in claim 1. Further, the compounds may be identified as type-X sPLA 2 inhibitors by the following procedure. At first, a cell expressing human type-X sPLA 2 is prepared. That is, cDNA sequence encoding human type-X sPLA 2 ( Cupillard et al., J. Biol. Chem, 1997, 272, 15745-15752 ) is inserted into an expression vector for mammalian cells. The obtained expression vector is transfected into the host cell and the cell stably expressing human type-X sPLA 2 is obtained. Next, the above-mentioned transfected cell is cultured in medium and its culture supernatant is used for the measurement of each enzyme activity. In order to identify and evaluate an inhibitor of type-X sPLA 2 , the following chromogenic assay is utilized. A general explanation for this assay is described in " Analysis of Human Synovial Fluid Phospholipase A2 on Short Chain Phosphatidylcholine-Mixed Micelles: Development of a Spectrophotometric Assay Suitable for a Micortiterplate Reader" (Analytical Biochemistry, 204, pp 190-197, 1992 by Laure. J. Reynolds. Lori L. Hughes and Edward A. Dennis . Several compounds of the present invention can be synthesized in accordance with the methods described in PCT/JP00/07024 , EP-620214 (JP Laid-Open (Tokukai) No. 95/010838 , US-5578634 ), EP-620215 (JP Laid-Open (Tokukai) No. 95/025850 , US-5684034 ), EP-675110 (JP Laid-Open (Tokukai) No. 95/285933 , US-5654326 ), WO 96/03120 ( JP Laid-Open No. 98/505336 ), WO 96/03383 ( JP Laid-Open No. 98/505584 ), WO 98/18464 ( EP839806 ), WO99/51605 , WO99/59999 . The composition used for treatment or prevention of cancer in accordance with the present invention may be administered to a patient through a variety of routes including oral, aerosol, rectal, percutaneous, subcutaneous, intravenous, intramuscular, and nasal routes. A formulation according to the present invention may be manufactured by combining (for example, admixing) a curatively effective amount of a compound of the present invention with a pharmaceutically acceptable carrier or diluent. The formulation of the present invention may be manufactured with the use of well-known and easily available ingredients in accordance with a known method. In case of manufacturing a composition of the present invention, active ingredients are admixed or diluted with a carrier, or they are contained in a carrier in the form of capsule, sacheier, paper, or another container. In case of functioning a carrier as a diluent, the carrier is a solid, semi-solid, or liquid material which functions as a medium. Accordingly, a formulation according to the present invention may be produced in the form of tablet, pill, powder medicine, intraoral medicine, elixir agent, suspending agent, emulsifier, dissolving agent, syrup agent, aerosol agent (solid in liquid medium), and ointment. Such a formulation may contain up to 10% of an active compound. It is preferred to formulate a compound having activities for the treatment or prevention of cancer prior to administration. Any suitable carrier well known to those skilled in the art may be used for the formulation. In such formulation, a carrier is in the form of solid, liquid, or a mixture thereof. For instance, a compound having type-X sPLA 2 inhibitory activities is dissolved into 4% dextrose/0.5% sodium citrate aqueous solution so as to be 2 mg/mL concentration for intravenous injection. Solid formulation includes powder, tablet, and capsule. Solid carrier consists of one or more of material(s) for serving also as fragrant, lubricant, dissolving agent, suspension, binder, tablet disintegrator, capsule. A tablet for oral administration contains a suitable excipient such as calcium carbonate, sodium carbonate, lactose, calcium phosphate together with a disintegrator such as corn starch, alginic acid and/or a binder such as gelatin, acacia, and a lubricant such as magnesium stearate, stearic acid, talc. In a powder medicine, a carrier is a finely pulverized solid which is blended with finely pulverized active ingredients. In a tablet, active ingredients are admixed with a carrier having required binding power in a suitable ratio, and it is solidified in a desired shape and size. Powder medicine and tablet contain about 1 to about 99% by weight of the active ingredients being novel compounds according to the present invention. An example of suitable solid carriers includes magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth gum, methyl cellulose, sodium carboxymethylcellulose, low-melting wax, and cocoa butter. An axenic liquid formulation contains suspending agent, emulsifier, syrup agent, and elixir agent. Active ingredients may be dissolved or suspended into a pharmaceutically acceptable carrier such as sterile water, a sterile organic solvent, a mixture thereof and the like. Active ingredients may be dissolved frequently into a suitable organic solvent such as propylene glycol aqueous solution. When finely pulverized active ingredients are dispersed into aqueous starch, sodium carboxymethylcellulose solution, or suitable oil, the other compositions can be prepared. The dosage varies with the conditions of the disease, administration route, age and body weight of patient. In the case of intravenous administration, the dosage can generally be between 0.01 to 10 mg/kg/h for adult, preferably 0.1 to 1 mg/kg/h. Example Example 1 Preparation of cells expressing human type-X sPLA 2 and their culture supernatants cDNA sequence encoding human type-X sPLA 2 ( Cupillard et al., J. Biol. Chem, 1997, 272, 15745-15752 ) was inserted into the downstream region of the promoter of pSVL SV40 Late Promoter Expression Vector (Amersham Pharmacia Biotech Inc.) that is an expression vector for mammalian cells. The obtained expression vector was transfected into the host CHO cells with a LipofectAMINE reagent (Gibco BRL Inc.) according to the manufacture's instruction to obtain the CHO cells stably expressing human type-X sPLA 2 . The transfected cell was cultured in α-MEM medium containing 10% fetal calf serum for 3 days and its culture supernatant was used for the measurement of each enzyme activity. Example 2 Inhibition test In order to identify and evaluate an inhibitor of type·X sPLA 2 . the following chromogenic assay is utilized. This assay has been applied for high volume screening using a 96-well microtiterplate. A general explanation for this assay is described in " Analysis of Human Synovial Fluid Phospholipase A2 on Short Chain Phosphatidylcholine-Mixed Micelles: Development of a Spectrophotometric Assay Suitable for a Micortiterplate Reader" (Analytical Biochemistry, 204, pp 190-197, 1992 by Laure. J. Reynolds. Lori L. Hughes and Edward A. Dennis . Test compounds (or solvent blank) were added according to the alignment of plates that had been previously set. Human type-X sPLA 2 was incubated (30 min at 40 °C (15 µl/well)) with diheptanoylthio PC (1 mM) in the presence of Triton X-100 (0.3 mM) and 5,5'-dithiobis(2-nitrobenzoic acid) (125 µM) in Tris-HCl buffer (25 mM, pH 7.5) containing CaCl 2 (10 mM), KCI (100 mM), and bovine serum albumin (1.0 mg/mL). The changes in the absorbance at 405 nm were measured and the inhibition activities were calculated. The IC 50 value was determined by plotting the log concentration of the above-mentioned compounds (1)-(19) with respect to their inhibition values within 10% to 90% inhibitory range. Results of the type-X sPLA 2 inhibition test is shown in the following Table 1. Table 1 Compound No. IC 50 (nM) Compound No. IC 50 (nM) 1 10 11 10 2 10 12 16 3 5 13 19 4 27 14 9 5 12 15 17 6 17 16 7 7 5 17 12 8 3 18 16 9 13 19 26 10 12 Example 3 Immunohistochemical analysis in human colon cancer tissues with anti-type-X sPLA 2 antibody In this experiment, anti-type-X sPLA 2 antibody that was described in " The Journal of Biological Chemistry Vol. 274, No. 48, pp.34203-34211 1999 " was used. Paraffin embedded preparations of human colon cancer tissues and corresponding normal tissues were purchased from Biochain Inc. (San Leandro, CA). The tissue sections in the slides were dewaxed, treated in methanol containing 0.3% H 2 O 2 for 30 min to remove the endogenous peroxidase activity and incubated with 5% normal goat serum for 20 min. The slides were then incubated with anti-type-X sPLA 2 antibody (6 µg/mL) in PBS containing 0.1% bovine serum albumin for 14 hr at 4°C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG antibody for 30 min followed by treatment with peroxidase labelled avidin-biotin complex reagent (Vector Laboratories). After washing, the samples were processed with 200 µg/mL diaminobenzidine hydrochloride substrate dissolved in 50 mmol/L Tris-HCl (pH 7.6) containing 0.006% H 2 O 2 for 10 min resulting in the appearance of color dependent on the peroxidase activity to visualize the type-X sPLA 2 expression in the tissue preparations. In addition, the nuclei were counterstained with 0.4% hematoxylin solution. Positive signals representative for type-X sPLA 2 expression was visualized as a dark-brownish color of diaminobenzidine deposit. The neutralization of type-X sPLA 2 specific signals was conducted by incubating anti-type-X sPLA 2 antibody with purified type-X sPLA 2 protein (60 µg/mL) for 2 hr before the addition to the slides. Consequently, positive signals representative for type-X sPLA 2 expression were not obviously detected in normal colon, but strongly detected in the tumor cells in human colon adenocarcinoma tissues. Since the addition of type-X sPLA 2 protein resulted in abolishment of the signals, they were verified as the specific signals for type-X sPLA 2 . In addition, there was no positive signal when IgG prepared from non-immunized rabbit was used. Taken together, these findings suggest that the expression of type-X sPLA 2 protein was greatly elevated in human colorectal cancer tissues. Example 4 Immunohistochemical analysis in human lung cancer tissues with anti-type-X sPLA 2 antibody In this experiment, anti-type-X sPLA 2 antibody which was described in " The Journal of Biological Chemistry Vol. 274, No. 48, pp.34203-34211 1999 " was used. Paraffin embedded preparations of human lung cancer tissues and corresponding normal tissues were purchased from Biochain Inc. (San Leandro, CA). The tissue sections in the slides were dewaxed, treated in methanol containing 0.3% H 2 O 2 for 30 min to remove the endogenous peroxidase activity and incubated with 5% normal goat serum for 20 min. The slides were then incubated with anti-type-X sPLA 2 antibody (6 µg/mL) in PBS containing 0.1% bovine serum albumin for 14 hr at 4°C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG antibody for 30 min followed by treatment with peroxidase labelled avidin-biotin complex reagent (Vector Laboratories). After washing, the samples were processed with 200 µg/mL diaminobenzidine hydrochloride substrate dissolved in 50 mmol/L Tris-HCl (pH 7.6) containing 0.006% H 2 O 2 for 10 min resulting in the appearance of color dependent on the peroxidase activity to visualize the type-X sPLA 2 expression in the tissue preparations. In addition, the nuclei were counterstained with 0.4% hematoxylin solution. Positive signals representative for type-X sPLA 2 expression was visualized as a dark-brownish color of diaminobenzidine deposit. The neutralization of type-X sPLA 2 specific signals was conducted by incubating anti-type-X sPLA 2 antibody with purified type-X sPLA 2 protein (60 µg/mL) for 2 hr before the addition to the slides. Consequently, positive signals representative for type-X sPLA 2 expression were weakly observed in type II pneumocytes in normal human lung. In contrast, the positive signals were strongly detected in tumor cells in the lung tissues prepared from patients of lung cancer. Since the addition of type-X sPLA 2 protein resulted in abolishment of the signals, they were verified as the specific signals for type-X sPLA 2 . In addition, there was no positive signal when IgG prepared from non-immunized rabbit was used. Taken together, these findings suggest that the expression of type-X sPLA 2 protein was greatly elevated in human lung cancer tissues. Example 5 Immunohistochemical analysis in human liver cancer tissues with anti-type-X sPLA 2 antibody In this experiment, anti-type-X sPLA 2 antibody which was described in " The Journal of Biological Chemistry Vol. 274, No. 48, pp.34203-34211 1999 " was used. Paraffin embedded preparations of human liver cancer tissues and corresponding normal tissues were purchased from Biochain Inc. (San Leandro, CA). The tissue sections in the slides were dewaxed, treated in methanol containing 0.3% H 2 O 2 for 30 min to remove the endogenous peroxidase activity and incubated with 5% normal goat serum for 20 min. The slides were then incubated with anti-type-X sPLA 2 antibody (6 µg/mL) in PBS containing 0.1% bovine serum albumin for 14 hr at 4°C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG antibody for 30 min followed by treatment with peroxidase labelled avidin-biotin complex reagent (Vector Laboratories). After washing, the samples were processed with 200 µg/mL diaminobenzidine hydrochloride substrate dissolved in 50 mmol/L Tris-HCl (pH 7.6) containing 0.006% H 2 O 2 for 10 min resulting in the appearance of color dependent on the peroxidase activity to visualize the type-X sPLA 2 expression in the tissue preparations. In addition, the nuclei were counterstained with 0.4% hematoxylin solution. Positive signals representative for type-X sPLA 2 expression was visualized as a dark-brownish color of diaminobenzidine deposit. The neutralization of type-X sPLA 2 specific signals was conducted by incubating anti-type-X sPLA 2 antibody with purified type-X sPLA 2 protein (60 µg/mL) for 2 hr before the addition to the slides. Consequently, positive signals representative for type-X sPLA 2 expression were weakly observed in hepatic lobule and Kupffer's satellate cells in normal human liver. In contrast, the positive signals were strongly detected in tumor cells in the liver tissues prepared from patients of liver cancer. Since the addition of type-X sPLA 2 protein resulted in abolishment of the signals, they were verified as the specific signals for type-X sPLA 2 . In addition, there was no positive signal when IgG prepared from non-immunized rabbit was used. Taken together, these findings suggest that the expression of type-X sPLA 2 protein was greatly elevated in human liver cancer tissues. Example 6 Immunohistochemical analysis in human gastric cancer tissues with anti-type-X sPLA 2 antibody In this experiment, anti-type-X sPLA 2 antibody which was described in " The Journal of Biological Chemistry Vol. 274, No. 48, pp.34203-34211 1999 " was used. Paraffin embedded preparations of human gastric cancer tissues and corresponding normal tissues were purchased from Biochain Inc. (San Leandro, CA). The tissue sections in the slides were dewaxed, treated in methanol containing 0.3% H 2 O 2 for 30 min to remove the endogenous peroxidase activity and incubated with 5% normal goat serum for 20 min. The slides were then incubated with anti-type-X sPLA 2 antibody (6 µg/mL) in PBS containing 0.1% bovine serum albumin for 14 hr at 4°C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG antibody for 30 min followed by treatment with peroxidase labelled avidin-biotin complex reagent (Vector Laboratories). After washing, the samples were processed with 200 µg/mL diaminobenzidine hydrochloride substrate dissolved in 50 mmol/L Tris-HCl (pH 7.6) containing 0.006% H 2 O 2 for 10 min resulting in the appearance of color dependent on the peroxidase activity to visualize the type-X sPLA 2 expression in the tissue preparations. In addition, the nuclei were counterstained with 0.4% hematoxylin solution. Positive signals representative for type-X sPLA 2 expression was visualized as a dark-brownish color of diaminobenzidine deposit. The neutralization of type-X sPLA 2 specific signals was conducted by incubating anti-type-X sPLA 2 antibody with purified type-X sPLA 2 protein (60µg/mL) for 2 hr before the addition to the slides. Consequently, positive signals representative for type-X sPLA 2 expression were not obviously detected in normal gaster, but strongly detected in tumor cells in the gastric tissues prepared from patients of gastric cancer. Since the addition of type-X sPLA 2 protein resulted in abolishment of the signals, they were verified as the specific signals for type-X sPLA 2 . In addition, there was no positive signal when IgG prepared from non-immunized rabbit was used. Taken together, these findings suggest that the expression of type-X sPLA 2 protein was greatly elevated in human gastric cancer tissues. Example 7 Immunohistochemical analysis in human renal cancer tissues with anti-type-X sPLA 2 antibody. In this experiment, anti-type-X sPLA 2 antibody which was described in " The Journal of Biological Chemistry Vol. 274, No. 48, pp.34203-34211 1999 " was used. Paraffin embedded preparations of human renal cancer tissues and corresponding normal tissues were purchased from Biochain Inc. (San Leandro, CA). The tissue sections in the slides were dewaxed, treated in methanol containing 0.3% H 2 O 2 for 30 min to remove the endogenous peroxidase activity and incubated with 5% normal goat serum for 20 min. The slides were then incubated with anti-type-X sPLA 2 antibody (6 µg/mL) in PBS containing 0.1% bovine serum albumin for 14 hr at 4°C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG antibody for 30 min followed by treatment with peroxidase labelled avidin-biotin complex reagent (Vector Laboratories). After washing, the samples were processed with 200µg/mL diaminobenzidine hydrochloride substrate dissolved in 50 mmol/L Tris-HCl (pH 7.6) containing 0.006% H 2 O 2 for 10 min resulting in the appearance of color dependent on the peroxidase activity to visualize the type-X sPLA 2 expression in the tissue preparations. In addition, the nuclei were counterstained with 0.4% hematoxylin solution. Positive signals representative for type-X sPLA 2 expression was visualized as a dark-brownish color of diaminobenzidine deposit. The neutralization of type-X sPLA 2 specific signals was conducted by incubating anti-type-X sPLA 2 antibody with purified type-X sPLA 2 protein (60 µg/mL) for 2 hr before the addition to the slides. Consequently, positive signals representative for type-X sPLA 2 expression were weakly observed in glomerular mesangial cells in normal kidney. In contrast, the positive signals were strongly detected in tumor cells in the kidney prepared from patients of renal cancer. Since the addition of type-X sPLA 2 protein resulted in abolishment of the signals, they were verified as the specific signals for type-X sPLA 2 . In addition, there was no positive signal when IgG prepared from non-immunized rabbit was used. Taken together, these findings suggest that the expression of type-X sPLA 2 protein was greatly elevated in human renal cancer tissues. Example 8 Immunohistochemical analysis in human gallbladder cancer tissues with anti-type-X sPLA 2 antibody In this experiment, anti-type-X sPLA 2 antibody which was described in " The Journal of Biological Chemistry Vol. 274, No. 48, pp.34203-34211 1999 " was used. Paraffin embedded preparations of human gallbladder cancer tissues and corresponding normal tissues were purchased from Biochain Inc. (San Leandro, CA). The tissue sections in the slides were dewaxed, treated in methanol containing 0.3% H 2 O 2 for 30 min to remove the endogenous peroxidase activity and incubated with 5% normal goat serum for 20 min. The slides were then incubated with anti-type-X sPLA 2 antibody (6 µg/mL) in PBS containing 0.1% bovine serum albumin for 14 hr at 4°C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG antibody for 30 min followed by treatment with peroxidase labelled avidin-biotin complex reagent (Vector Laboratories). After washing, the samples were processed with 200 µg/mL diaminobenzidine hydrochloride substrate dissolved in 50 mmol/L Tris-HCl (pH 7.6) containing 0.006% H 2 O 2 for 10 min resulting in the appearance of color dependent on the peroxidase activity to visualize the type-X sPLA 2 expression in the tissue preparations. In addition, the nuclei were counterstained with 0.4% hematoxylin solution. Positive signals representative for type-X sPLA 2 expression was visualized as a dark-brownish color of diaminobenzidine deposit. The neutralization of type-X sPLA 2 specific signals was conducted by incubating anti-type-X sPLA 2 antibody with purified type-X sPLA 2 protein (60 µg/mL) for 2 hr before the addition to the slides. Consequently, positive signals representative for type-X sPLA 2 expression were not obviously detected in normal gallbladder tissues, but strongly detected in tumor cells in the gallbladder tissues prepared from patients of gallbladder cancer. Since the addition of type-X sPLA 2 protein resulted in abolishment of the signals, they were verified as the specific signals for type-X sPLA 2 . In addition, there was no positive signal when IgG prepared from non-immunized rabbit was used. Taken together, these findings suggest that the expression of type-X sPLA 2 protein was greatly elevated in human gallbladder cancer tissues. Example 9 Immunohistochemical analysis in human prostate cancer tissues with anti-type-X sPLA 2 antibody In this experiment, anti-type-X sPLA 2 antibody which was described in " The Journal of Biological Chemistry Vol. 274, No. 48, pp.34203-34211 1999 " was used. Paraffin embedded preparations of human prostate cancer tissues and corresponding normal tissues were purchased from Biochain Inc. (San Leandro, CA). The tissue sections in the slides were dewaxed, treated in methanol containing 0.3% H 2 O 2 for 30 min to remove the endogenous peroxidase activity and incubated with 5% normal goat serum for 20 min. The slides were then incubated with anti-type-X sPLA 2 antibody (6 µg/mL) in PBS containing 0.1% bovine serum albumin for 14 hr at 4°C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG antibody for 30 min followed by treatment with peroxidase labelled avidin-biotin complex reagent (Vector Laboratories). After washing, the samples were processed with 200 µg/mL diaminobenzidine hydrochloride substrate dissolved in 50 mmol/L Tris-HCl (pH 7.6) containing 0.006% H 2 O 2 for 10 min resulting in the appearance of color dependent on the peroxidase activity to visualize the type-X sPLA 2 expression in the tissue preparations. In addition the nuclei were counterstained with 0.4% hematoxylin solution. Positive signals representative for type-X sPLA 2 expression was visualized as a dark-brownish color of diaminobenzidine deposit. The neutralization of type-X sPLA 2 specific signals was conducted by incubating anti-type-X sPLA 2 antibody with purified type-X sPLA 2 protein (60 µg/mL) for 2 hr before the addition to the slides. Consequently, positive signals representative for type-X sPLA 2 expression were not obviously detected in normal prostates, but strongly detected in tumor cells in the prostate tissues prepared from patients of prostate cancer. Since the addition of type-X sPLA 2 protein resulted in abolishment of the signals, they were verified as the specific signals for type-X sPLA 2 . In addition, there was no positive signal when IgG prepared from non-immunized rabbit was used. Taken together, these findings suggest that the expression of type-X sPLA 2 protein was greatly elevated in human prostate cancer tissues. Example 10 Immunohistochemical analysis in human pancreatic cancer tissues with anti-type-X sPLA 2 antibody In this experiment, anti-type-X sPLA 2 antibody which was described in " The Journal of Biological Chemistry Vol. 274, No. 48, pp.34203-34211 1999 " was used. Paraffin embedded preparations of human pancreatic cancer tissues and corresponding normal tissues were purchased from Biochain Inc. (San Leandro, CA). The tissue sections in the slides were dewaxed, treated in methanol containing 0.3% H 2 O 2 for 30 min to remove the endogenous peroxidase activity and incubated with 5% normal goat serum for 20 min. The slides were then incubated with anti-type-X sPLA 2 antibody (6 µg/mL) in PBS containing 0.1% bovine serum albumin for 14 hr at 4°C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG antibody for 30 min followed by treatment with peroxidase labelled avidin-biotin complex reagent (Vector Laboratories). After washing, the samples were processed with 200 µg/mL diaminobenzidine hydrochloride substrate dissolved in 50 mmol/L Tris.HCl (pH 7.6) containing 0.006% H 2 O 2 for 10 min resulting in the appearance of color dependent on the peroxidase activity to visualize the type-X sPLA 2 expression in the tissue preparations. In addition, the nuclei were counterstained with 0.4% hematoxylin solution. Positive signals representative for type-X sPLA 2 expression was visualized as a dark-brownish color of diaminobenzidine deposit. The neutralization of type-X sPLA 2 specific signals was conducted by incubating anti-type-X sPLA 2 antibody with purified type-X sPLA 2 protein (60 µg/mL) for 2 hr before the addition to the slides. Consequently, positive signals representative for type-X sPLA 2 expression were not obviously detected in normal pancreas, but strongly detected in tumor cells in the pancreas prepared from patients of pancreatic cancer. Since the addition of type-X sPLA 2 protein resulted in abolishment of the signals, they were verified as the specific signals for type-X sPLA 2 . In addition, there was no positive signal when IgG prepared from non-immunized rabbit was used. Taken together, these findings suggest that the expression of type-X sPLA 2 protein was greatly elevated in human pancreatic cancer tissues. Example 11 Immunohistochemical analysis in human testis cancer tissues with anti-type-X sPLA 2 antibody In this experiment, anti-type-X sPLA 2 antibody which was described in " The Journal of Biological Chemistry Vol. 274, No. 48, pp.34203-34211 1999 " was used. Paraffin embedded preparations of human testis cancer tissues and corresponding normal tissues were purchased from Biochain Inc. (San Leandro, CA). The tissue sections in the slides were dewaxed, treated in methanol containing 0.3% H 2 O 2 for 30 min to remove the endogenous peroxidase activity and incubated with 5% normal goat serum for 20 min. The slides were then incubated with anti-type-X sPLA 2 antibody (6 µg/mL) in PBS containing 0.1% bovine serum albumin for 14 hr at 4°C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG antibody for 30 min followed by treatment with peroxidase labelled avidin-biotin complex reagent (Vector Laboratories). After washing, the samples were processed with 200 µg/mL diaminobenzidine hydrochloride substrate dissolved in 50 mmol/L Tris-HCl (pH 7.6) containing 0.006% H 2 O 2 for 10 min resulting in the appearance of color dependent on the peroxidase activity to visualize the type-X sPLA 2 expression in the tissue preparations. In addition, the nuclei were counterstained with 1% methyl green dye in 0.1 mol/L sodium acetate (pH 4.0). Positive signals representative for type-X sPLA 2 expression was visualized as a dark-brownish color of diaminobenzidine deposit. The neutralization of type-X sPLA 2 specific signals was conducted by incubating anti-type-X sPLA 2 antibody with purified type-X sPLA 2 protein (60 µg/mL) for 2 hr before the addition to the slides. Consequently, positive signals representative for type-X sPLA 2 expression were weakly observed in some parts of the seminiferous tubules of normal testis. In contrast, the positive signals were strongly detected in malignant cells present in the seminiferous tubules and/or the epithelium of seminal vesicles in the testis prepared from patients of testis cancer. Since the addition of type-X sPLA 2 protein resulted in abolishment of the signals, they were verified as the specific signals for type-X sPLA 2 . In addition, there was no positive signal when IgG prepared from non-immunized rabbit was used. Taken together, these findings suggest that the expression of type-X sPLA 2 protein was greatly elevated in human testis cancer tissues. Example 12 Immunohistochemical analysis in human ovarian cancer tissues with anti-type-X sPLA 2 antibody In this experiment, anti-type-X sPLA 2 antibody which was described in " The Journal of Biological Chemistry Vol. 274, No. 48, pp.34203-34211 1999 " was used. Paraffin embedded preparations of human ovarian cancer tissues and corresponding normal tissues were purchased from Biochain Inc. (San Leandro, CA). The tissue sections in the slides were dewaxed, treated in methanol containing 0.3% H 2 O 2 for 30 min to remove the endogenous peroxidase activity and incubated with 5% normal goat serum for 20 min. The slides were then incubated with anti-type-X sPLA 2 antibody (6 µg/mL) in PBS containing 0.1% bovine serum albumin for 14 hr at 4°C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG antibody for 30 min followed by treatment with peroxidase labelled avidin-biotin complex reagent (Vector Laboratories). After washing, the samples were processed with 200 µg/mL diaminobenzidine hydrochloride substrate dissolved in 50 mmol/L Tris-HCl (pH 7.6) containing 0.006% H 2 O 2 for 10 min resulting in the appearance of color dependent on the peroxidase activity to visualize the type-X sPLA 2 expression in the tissue preparations. In addition, the nuclei were counterstained with 1% methyl green dye in 0.1 mol/L sodium acetate (pH 4.0). Positive signals representative for type-X sPLA 2 expression was visualized as a dark-brownish color of diaminobenzidine deposit. The neutralization of type-X sPLA 2 specific signals was conducted by incubating anti-type-X sPLA 2 antibody with purified type-X sPLA 2 protein (60 µg/mL) for 2 hr before the addition to the slides. Consequently, positive signals representative for type-X sPLA 2 expression were not obviously detected in normal ovary, but strongly detected in the malignant cells present in the epithelium of ovarian follicles and/or oviducts in the ovary prepared from patients of ovarian cancer. Since the addition of type-X sPLA 2 protein resulted in abolishment of the signals, they were verified as the specific signals for type-X sPLA 2 . In addition, there was no positive signal when IgG prepared from non-immunized rabbit was used. Taken together, these findings suggest that the expression of type-X sPLA 2 protein was greatly elevated in human ovarian cancer tissues. Example 13 Immunohistochemical analysis in human cutaneous cancer tissues with anti-type-X sPLA 2 antibody In this experiment, anti-type-X sPLA 2 antibody which was described in " The Journal of Biological Chemistry Vol. 274, No. 48, pp.34203-34211 1999 " was used. Paraffin embedded preparations of human cutaneous cancer tissues (malignant melanoma) and corresponding normal tissues were purchased from Biochain Inc. (San Leandro, CA). The tissue sections in the slides were dewaxed, treated in methanol containing 0.3% H 2 O 2 for 30 min to remove the endogenous peroxidase activity and incubated with 5% normal goat serum for 20 min. The slides were then incubated with anti-type-X sPLA 2 antibody (6 µg/mL) in PBS containing 0.1% bovine serum albumin for 14 hr at 4°C. After washing with PBS, they were incubated with biotin-conjugated goat anti-rabbit IgG antibody for 30 min followed by treatment with peroxidase labelled avidin-biotin complex reagent (Vector Laboratories). After washing, the samples were processed with 200 µg/mL diaminobenzidine hydrochloride substrate dissolved in 50 mmol/L Tris-HCl (pH 7.6) containing 0.006% H 2 O 2 for 10 min resulting in the appearance of color dependent on the peroxidase activity to visualize the type-X sPLA 2 expression in the tissue preparations. In addition, the nuclei were counterstained with 1% methyl green dye in 0.1 mol/L sodium acetate (pH 4.0). Positive signals representative for type-X sPLA 2 expression was visualized as a dark-brownish color of diaminobenzidine deposit. The neutralization of type-X sPLA 2 specific signals was conducted by incubating anti-type-X sPLA 2 antibody with purified type-X sPLA 2 protein (60 µg/mL) for 2 hr before the addition to the slides. Consequently, positive signals representative for type-X sPLA 2 expression were weakly observed in the melanocytes of stratum spinosum and/or stratum basale in skin epidermis in normal human cutis. In contrast, the positive signals were strongly detected in hypertrophied melanocytes in the cutis prepared from patients of cutaneous cancer (malignant melanoma). Since the addition of type-X sPLA 2 protein resulted in abolishment of the signals, they were verified as the specific signals for type-X sPLA 2 . In addition, there was no positive signal when IgG prepared from non-immunized rabbit was used. Taken together, these findings suggest that the expression of type-X sPLA 2 protein was greatly elevated in human cutaneous cancer (malignant melanoma) tissues. Example 14 Effects of sPLA 2 inhibitors on human type-X sPLA 2 -induced oleic acid release in human colon carcinoma cell lines, HT-29 cells Compound (1) and compound (19) was used as test compounds. Human colon carcinoma cell lines, HT-29 cells (obtained from ATCC) were cultured in DMEM supplemented with 10% fetal calf serum. The cells were washed by phosphate-buffered saline (PBS), detached from culture plates by treatment with trypsin/EDTA solution and further washed by PBS. The resulting cells were resuspended in Hanks' buffered saline containing 0.1% bovine serum albumin (BSA) at a density of 12.5 x 10 6 cells/mL. Aliquots of the cell suspension (0.4 mL) were transferred into polypropylene tubes and test compounds dissolved in DMSO solution (final concentration; 10 µM) were added. After preincubation for 10 min at 37°C, 100 nM purified human type-X sPLA 2 enzyme ( Hanasaki et al. J. Biol. Chem. (1999) 274. 34203-34211 ) was added (final volume of 0.5 mL). After incubation for 30 min at 37°C, the reaction was stopped by the addition of 2 mL Dole's reagent (heptane : 2-propanol : 1 M H 2 SO 4 = 10 : 40 : 1, v/v/v). According to the method of Tojo et al. (J. lipid Res. (1993) 34, 837-844 ), the released fatty acids were extracted, labeled with 9-anthryldiazomethane (Funakoshi Co.), and the oleic acid was quantified by reverse-phase high performance chromatography (LiChroCART 125-4 Superspher 100 RP-18 column (Merck). From each data, the value in the absence of type-X sPLA 2 was subtracted. The amount of released oleic acid in the presence of each test compound was expressed as the percentage of the increased content by the addition of type-X sPLA 2 enzyme. As shown in Table 2, each test compound significantly inhibited the type-X sPLA 2 -induced oleic acid release. Example 15 Effects of sPLA 2 inhibitors on type-X sPLA 2 -induced PGE 2 production in human colon carcinoma cell lines. HT-29 cells HT-29 cells were seeded into 24-well plates at a density of 2.5×10 5 cells/well. After incubation for 24 h, the cells were washed with PBS and incubated with 30 ng/mL of Tumor necrosis factor-α (R&D Systems, Inc.) in DMEM medium supplemented with 10% fetal bovine serum for 18 h. After washing with PBS, the cells were preincubated with or without test compounds (at a final concentration of 10 µM ; dissolved in DMSO) in Hanks' buffered saline containing 0.1% BSA for 10 min at 37°C, and then stimulated with 100 nM purified human type-X sPLA 2 enzyme in a final volume of 0.5 mL. After incubation for 3 h at 37°C, the culture supernatant was collected following the centrifugation for the removal of floating cells, and its PGE 2 content was quantified with an enzyme-immunoassay kit (Cayman Chemicals Co.). From each data, the value in the absence of type-X sPLA 2 was subtracted. The amount of PGE 2 in the presence of each test compound was expressed as the percentage of the PGE 2 content produced by the addition of type-X sPLA 2 enzyme. As shown in Table 2, each test compound significantly blocked the type-X sPLA 2 -induced PGE 2 production. Potential involvement of PGE 2 in the progression of tumors has been described in Cancer Research 59, 5093-5096, 1999 . Since the compounds in the present invention significantly block the type-X sPLA 2 -induced PGE 2 production, they can be applied as antitumor agents. Table 2 Compound (10 µM) Oleic acid release PGE 2 production No treatment group 100±2.8 100±22.7 (1) -3.0±3.9** -4.1±12.6** (19) 1.8±4.6** 42.1±9.7** Student's t-test (*P < 0.05, **P < 0.01) Formulation Example It is to be noted that the following Formulation Examples 1 to 8 are mere illustration of the invention. The term "active ingredient" means the compounds having an inhibitory activity against type-X PLA 2 , the prodrugs thereof, their pharmaceutical acceptable salts, or their hydrate as defined in claim 1. Formulation Example 1 Hard gelatin capsules are prepared using of the following ingredients: Dose (mg/capsule) Active ingredient 250 Starch, dried 200 Magnesium stearate 10 Total 460 mg Formulation Example 2 A tablet is prepared using of the following ingredients: Dose (mg/tablet) Active ingredient 250 Cellulose, microcrystals 400 Silicon dioxide, fumed 10 Stearic acid 5 Total 665 mg The components are blended and compressed to form tablets each weighing 665 mg. Formulation Example 3 An aerosol solution is prepared containing the following components: Weight Active ingredient 0.25 Ethanol 25.75 Propellant 22 (chlorodifluoromethane) 74.00 Total 100.00 The active compound is mixed with ethanol and the admixture added to a portion of the propellant 22, cooled to -30 °C and transferred to filling device. The required amount is then fed to stainless steel container and diluted with the reminder of the propellant. The valve units are then fitted to the container. Formulation Example 4 Tablets, each containing 60 mg of active ingredient, are made as follows. Active ingredient 60 mg Starch 45 mg Microcrystals cellulose Polyvinylpyrrolidone (as 10% solution in water) 35 mg 4 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1 mg Total 150 mg The active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve, and (0.355 mm) mixed thoroughly. The aqueous solution containing polyvinylpyrrolidone is mixed with the resultant powder, and the admixture then is passed through a No. 14 mesh U.S. sieve (1.4 mm). The granules so produced are dried at 50°C and passed through a No. 18 mesh U.S. sieve (1.0 mm). The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through No. 60 mesh U.S. sieve (0.250 mm), are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg. Formulation Example 5 Capsules, each containing 80 mg of active ingredient, are made as follows: Active ingredient 80 mg Starch 59 mg Microcrystals cellulose 59 mg Magnesium stearate 2mg Total 200 mg The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 45 mesh U.S. sieve (0.355 mm), and filled into hard gelatin capsules in 200 mg quantities. Formulation Example 6 Suppository, each containing 225 mg of active ingredient, are made as follows: Active ingredient 225 mg Saturated fatty acid glycerides 2000 mg Total 2225 mg The active ingredient is passed through a No. 60 mesh U.S. sieve (0.251 mm) and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2g capacity and allowed to cool. Formulation Example 7 Suspensions, each containing 50 mg of active ingredient per 5 mL dose, are made as follows: Active ingredient 50 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25 mL Benzoic acid solution 0.10 mL Flavor q.v. Color q.v. Purified water to total 5 mL The active ingredient is passed through a No. 45 U.S. sieve (0.355 mm), and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution and flavor are diluted with a portion of the water and added, with stirring. Sufficient water is then added to produce the required volume. Formulation Example 8 An intravenous formulation may be prepared as follows: Active ingredient 100 mg Saturated fatty acid glycerides 1000 mL The solution of the above ingredients generally is administered intravenously to a subject at a rate of 1 mL per minute. Industrial Applicability It is provided that the type X sPLA 2 inhibitors of claim 1 are useful in preventing or treating cancer.

Claims: Use of a type-X sPLA 2 inhibitor for the preparation of a medicament for the prevention or treatment of cancer, wherein the type-X sPLA 2 inhibitor is a compound represented by the formula: or a C1-C6 alkyl ester thereof, its pharmaceutically acceptable salt, or its solvate. Use as claimed in claim 1, wherein the cancer is colon cancer, lung cancer, liver cancer, gastric cancer, renal cancer, gallbladder cancer, prostatic cancer, pancreatic cancer, testis cancer, ovary cancer or cutaneous cancer. Use as claimed in claim 1, wherein the cancer is colon cancer.

Inventors: HANASAKI KOHJI, IKEDA MINORU, ONO TAKASHI, HANASAKI, KOHJI, IKEDA, MINORU, ONO, TAKASHI, HANASAKI, Kohji, Shionogi & Co., Ltd., IKEDA, Minoru, Shionogi & Co., Ltd., ONO, Takashi, Shionogi & Co., Ltd.
Applicants: ANTHERA PHARMACEUTICALS INC, ANTHERA PHARMACEUTICALS, INC.
Application year: 2001
Main classification: A61K45
IPC codes: A61P35, A61K31, C07D405, C07D209, C07D487, A61K45, C07D471