EP1326605A1 - Procedes servant a inhiber la proliferation et a induire l'apoptose dans des cellules cancereuses - Google Patents

Procedes servant a inhiber la proliferation et a induire l'apoptose dans des cellules cancereuses

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Publication number
EP1326605A1
EP1326605A1 EP01935770A EP01935770A EP1326605A1 EP 1326605 A1 EP1326605 A1 EP 1326605A1 EP 01935770 A EP01935770 A EP 01935770A EP 01935770 A EP01935770 A EP 01935770A EP 1326605 A1 EP1326605 A1 EP 1326605A1
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European Patent Office
Prior art keywords
leukotriene
alkyl
compound
cancer cells
ethyl
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EP01935770A
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German (de)
English (en)
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EP1326605A4 (fr
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Thomas E. Adrian
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Creighton University
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Creighton University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the subject invention is directed generally to methods for decreasing proliferation of cancer cells, or of inducing apoptosis of cancer cells, or of inducing differentiation of cancer cells into non-cancerous cells and to methods for treating adenocarcinoma in a subject.
  • Pancreatic cancer is one of the most enigmatic and aggressive malignant diseases facing oncologists (Parker et al . , “Cancer Statistics. 1996,” CA Cancer J. Clin. , 46:5-27 (1996) (“Parker”)). It is now the fourth leading cause of cancer death in both men and women in the United States, and the incidence of this disease has significantly increased over the past 20 years (Parker; Trede et al . , "Survival After Pancreaticoduodenectom : 118 Consecutive Resections Without an Operative
  • Pancreatic cancer is responsible for 27,000 deaths per year in the United States . Because of lack of early diagnosis and poor therapeutic responsiveness of pancreatic cancer, less than 2% of patients survive beyond five years, and the median expectation of life after diagnosis of pancreatic cancer is less than 6 months (Horward; Poston; and Black) .
  • Colonic cancer is the second most common form of cancer in the United States (Doll et al . , "Mortality in Relation to Smoking: 20 Years' Observations on Male British Doctors," BMJ, 2:1525-1536 (1976); Hruban et al . , “Molecular Diagnosis of Cancer and Micrometastases, " Adv. Anat. Pathol . , 5:175-178 (1998) ("Hruban”); Figueredo et al . , "Adjuvant Therapy for Stage II Colon Cancer After Complete Resection.
  • Provincial Gastrointestinal Disease Site Group " Cancer Prev.
  • Colonic cancer occurs in more than 138,000 patients and is responsible for more than 55,000 deaths in the United States each year (Wingo) . Up to 70 % of patients with colonic cancer develop hepatic metastasises by the time of death, indicating that non- detectable micro-metastases are present at the time of surgery (Hruban; Figueredo; and Ness) . Furthermore, metastatic cancer is often not responsive to standard chemotherapeutic regimens, resulting in treatment failure (Figueredo and Ness) . The overall response of advanced or non-resectable colorectal cancer patients to chemotherapeutic agents varies from 26 to 44 percent.
  • the present invention relates to a method of decreasing proliferation of adenocarcinoma cancer cells, or of inducing apoptosis of adenocarcinoma cancer cells, or of inducing differentiation of adenocarcinoma cancer cells into non-cancerous cells.
  • a sample which includes adenocarcinoma cancer cells is contacted with a compound under conditions effective to inhibit binding of leukotriene B4 to leukotriene B4 receptor.
  • the present invention also relates to a method of treating adenocarcinoma in a subject.
  • the method includes administering to the subject an amount of a compound effective to inhibit binding of leukotriene B4 to leukotriene B4 receptor.
  • the present invention also relates to a method of decreasing proliferation of adenocarcinoma cancer cells, or of inducing apoptosis of adenocarcinoma cancer cells, or of inducing differentiation of adenocarcinoma cancer cells into non-cancerous cells.
  • the method includes contacting a sample which contains adenocarcinoma cancer cells with a compound having the formula (“Formula I"):
  • R 4 is a R 6 or R 4 is a moiety having one of the following formulae:
  • each R s is independently -COOH, 5-tetrazolyl , - CON(R 9 ) 2 , or -CONHSO 2 R 10 ; each R 7 is hydrogen, C 1 -C 4 alkyl, C 2 -C 5 alkenyl , C 2 -C 5 alkynyl, benzyl, methoxy, -W-R s , -T-G-R 6 , (C x -C 4 , alkyl) -T- (C 1 -C 4 alkylidenyl) -0- , or hydroxy; R 8 is hydrogen or halo; each R 9 is independently hydrogen, phenyl, or
  • R 10 is C- L -C 4 alkyl or phenyl
  • R l ⁇ is R 2 , -W-R 6 , or -T-G-R 6 ; each W is a bond or straight or branched chain divalent hydrocarbyl radical of one to eight carbon atoms ; each G is a straight or branched chain divalent hydrocarbyl radical of one to eight carbon atoms; each T is a bond, -CH 2 -, -O- -NH- -NHCO-
  • the present invention also relates to a method of treating adenocarcinoma in a subject.
  • the method includes administering, to the subject, an therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or solvate thereof .
  • Figures 3A-3D are phase contrast micrograph images.
  • Figure 3B shows the morphological changes induced by 250 nM LY293111 in MiaPaCa-2 pancreatic cancer cells after 12 hours relative to control MiaPaCa-2 pancreatic cancer cells ( Figure 3A) .
  • Figure 3D shows the morphological changes induced by 250 nM LY293111 in LoVo colonic cancer cells relative to control LoVo colonic cancer cells ( Figure 3D) .
  • Figure 5A is a bar graph showing the effects of different concentrations of LTB4 on thymidine incorporation in MiaPaCa-2 cells after 24 hours.
  • Figures 5B and 5C are bar graphs showing the effects of 100 nM of LTB4 on thymidine incorporation in MiaPaCa-2 cells
  • Figure 7 is an image of an agarose gel electrophoresis showing DNA fragmentation reflecting LY293111-induced apoptosis in MiaPaCa-2 cells. Cells were treated with 250, 500, and 1000 nM LY293111 for 48 hours.
  • Figures 8A-8C are dot plots showing TUNEL assay results of MiaPaCa-2 pancreatic cancer cells treated with 250 nM ( Figure 8B) and 500 nM (Figure 8C) LY293111 for 48 hours, as compared to control ( Figure 8A) .
  • the increase of fluorescence events in the upper right quadrant is due to UTP labeling of fragmented DNA, and the results are representative of three separate experiments.
  • Figures 9A-9F are bar graphs showing the effects of LY293111 on proliferation of pancreatic and colonic cancer cell lines.
  • Figures 9A, 9C, and 9D show the dosage effects of LY293111 on pancreatic cancer cell lines MiaPaCa2, HPAF, and Capan2 , respectively.
  • Figure 9B shows the time dependent effects of LY293111 on pancreatic cancer cell line MiaPaCa2.
  • Figures 9E and 9F show the effects of 0.5 ⁇ M LY293111 on cell number as a function of time for pancreatic cancer cell line MiaPaCa2 ( Figure 9E) and for colonic cancer cell line LoVo ( Figure 9F) .
  • Figures 10A and 10B are images of western blots showing the effects of LTB4 and LY293111, respectively, on Bcl-2 expression in MCF-7 breast cancer cells.
  • Figure 11 is a graph of tumor volume in athymic mice xenografted with human pancreatic cell line AsPc-1 as a function of treatment time with oral LY293111 (A) vs control (•) .
  • the present invention relates to a method of decreasing proliferation of adenocarcinoma cancer cells, or of inducing apoptosis of adenocarcinoma cancer cells, or of inducing differentiation of adenocarcinoma cancer cells into non-cancerous cells.
  • the method includes contacting a sample which includes adenocarcinoma cancer cells with a compound which inhibits binding of leukotriene B4 to leukotriene B4 receptor.
  • inhibit and its other forms (e.g., inhibiting") are meant include any degree of inhibition (e.g., more than about 5%, more than about 10%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 95%, more than about 98%, more than about 99%) up to and including complete prevention (100% inhibition) .
  • degree of inhibition e.g., more than about 5%, more than about 10%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 95%, more than about 98%, more than about 99%
  • the mechanism by which binding of leukotriene B4 to leukotriene B4 receptor is inhibited is not particularly critical to the practice of the present invention.
  • the inhibition can be direct, as in the case where the compound interferes directly with the binding of leukotriene B4 to leukotriene B4 receptor (e.g., by binding directly to leukotriene B4 receptor, as explained in more detail below) , or it can be indirect, as in the case where the compound interferes with either the production of leukotriene B4 and/or leukotriene B4 receptor, as further explained below.
  • the compound can inhibit binding of leukotriene B4 to leukotriene B4 receptor by binding to leukotriene B4 receptor.
  • the binding of the compound to leukotriene B4 receptor is specific, i.e., the compound does not bind substantially to other biological materials that are present in the sample.
  • the IC 50 of the compound to leukotriene B4 receptor be greater than about 1.1 times, more preferably greater than about 1.5 times, still more preferably greater than about 2 times, still more preferably greater than about 5 times, still more preferably greater than about 10 times the IC 50 of the compound to all other biological materials in the sample.
  • the degree of binding of the compound to leukotriene B4 receptor is preferably greater (i.e., has a higher IC 50 ) than the binding of leukotriene B4 to leukotriene B4 receptor, although the desired effect can be achieved in some circumstances even when the degree of binding of the compound to leukotriene B4 receptor is less (i.e., has a lower IC 50 ) than the binding of leukotriene B4 to leukotriene B4 receptor.
  • Binding is meant to include irreversible binding and reversible binding.
  • the binding can be the result of thermodynamic forces (e.g., favorable equilibrium constants) or kinetic considerations (e.g., favorable on/off rate constants), and it can be the result of chemical interactions (e.g., covalent bonding, ionic bonding, hydrogen bonding, van der Waals bonding, chelation, pi-sigma bonding, and/or pi-pi bonding) or physical interactions (e.g., surface phenomena, entrapment, etc.) .
  • binding partner e.g., binding of the compound to a leukotriene B4 receptor
  • binding is also meant to include any interaction of the compound with its binding partner which results in a decrease in the ability of the binding partner to fulfill its physiological role.
  • binding partner is leukotriene B4 receptor
  • binding of the compound to leukotriene B4 receptor is meant to include any interaction or combination of interactions between the compound and leucotriene B4 receptor which result in physical, chemical, and/or other changes in leucotriene B4 receptor which, in turn, decreases the ability of leucotriene B4 receptor to bind to leucotriene B4.
  • a variety of compounds can be used to inhibit binding of leukotriene B4 to leukotriene B4 receptor by binding to leukotriene B4 receptor.
  • Examples of such compounds include those which have the formula (“Formula I”) :
  • R x is C x -C 5 alkyl, C 2 -C 5 alkenyl, C 2 -C 5 alkynyl, C x -C 4 alkoxy, (C ⁇ alkyl) thio, halo, or R 2 -substitutedphenyl .
  • Each of R 2 and R 3 is independently hydrogen, halo, hydroxy, C 1 -C 4 alkyl, C x -C 4 alkoxy, (C x -C 4 alkyl) -S (O) q - , trifluoromethyl , or di- ( ⁇ 3 alkyl)amino.
  • Z is a straight or branched chain alkylidenyl.
  • R 4 is a R 6 (as defined below) , or R 4 is a moiety having one of the following formulae:
  • Each R 6 is independently -COOH, 5-tetrazolyl , -CON(R 9 ) 2 , or -CONHSO 2 R 10 .
  • Each R 7 is hydrogen, C ⁇ alkyl, C 2 -C 5 alkenyl, C 2 -C 5 alkynyl, benzyl, methoxy, -W-R s , -T-G-R 6 , (C, . - ⁇ alkyl) -T- (C 1 -C 4 alkylidenyl) -O- , or hydroxy.
  • R 8 is hydrogen or halo.
  • Each R 9 is independently hydrogen, phenyl, or C x -C 4 alkyl; or R 9 , when taken together with the nitrogen atom to which they are attached, form a morpholino, piperidino, piperazino, or pyrrolidino group.
  • R 10 is C- L -C 4 alkyl or phenyl.
  • R 1X is R 2 , -W-R s , or -T-G-R 6 .
  • Each W is a bond or straight or branched chain divalent hydrocarbyl radical of one to eight carbon atoms .
  • Each G is a straight or branched chain divalent hydrocarbyl radical of one to eight carbon atoms.
  • Each q is independently 0, 1, or 2; p is 0 or 1; and t is 0 or 1.
  • X is -0- or -S-
  • Y is not -0-; when A is -0- or -S-, R 4 is not R 6 ; when A is -0- or -S- and when Z is a bond, Y is not -0- ; and, when p is 0, W is not a bond.
  • alkyl refers to the straight and branched aliphatic radicals of 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, 2,2- dimethylpropyl , hexyl , and the like. Included within this definition are the terms “0, ⁇ 3 alkyl", “C 1 -C 4 alkyl” and "Ci-Cs alkyl”.
  • C 2 -C 5 alkynyl refers to straight and branched aliphatic residues of 2 to 5 carbon atoms containing one triple bond, such as -C ⁇ CH, -CH 2 -C ⁇ CH, -CH 2 -CH 2 -C ⁇ CH, -CH 2 -CH (CH 3 ) -C ⁇ CH,
  • C ⁇ alkoxy refers to, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy, and tert-butoxy.
  • halo refers to, for example, fluoro, chloro, bromo, and iodo .
  • C ⁇ C ⁇ alkylidenyl refers to a divalent radical derived from a C ⁇ C- ⁇ alkane such as -CH 2 -, -CH(CH 3 )-, -C(CH 3 ) 2 -,-CH(C 2 H 5 ) -, -CH 2 -CH 2 -, -CH 2 -CH (CH 3 ) - , -CH(CH 3 ) -CH 2 -, -CH(CH 3 ) -CH(CH 3 ) -, -CH 2 -CH (C 2 H 5 ) - ,
  • C 4 -C 8 cycioalkyl refers to a cycioalkyl ring of four to eight carbon atoms, such as cyclobutyl, cyclopentyl, cyclohexyl, 4 , 4-dimethylcyclohexyl, cycloheptyl, cyclooctyl, and the like.
  • straight or branched chain divalent hydrocarbyl residue of one to eight carbon atoms refers to a divalent radical derived from a straight or branched alkane, alkene, or alkyne of one to eight carbon atoms.
  • such a moiety can contain one, two, or three double or triple bonds, or combinations of both.
  • this term can be considered an alkylidene group as defined above containing from 1 to 8 carbon atoms optionally containing one to three double or triple bonds, or combinations of the two, limited as noted in the preceding sentence.
  • salts of the compounds of Formula I can also be used.
  • Such salts include those derived from inorganic bases, such as ammonium and alkali and alkaline earth metal hydroxides, carbonates, bicarbonates, and the like, as well as salts derived from basic organic amines, such as aliphatic and aromatic amines, aliphatic diamines, hydroxy alkylamines, and the like.
  • bases useful in preparing the salts useful in the practice of the present invention thus include ammonium hydroxide, potassium carbonate, sodium bicarbonate, calcium hydroxide, methyl amine, diethyl amine, ethylene diamine, cyclohexylamine, ethanolamine, and the like.
  • the potassium and sodium salt forms are particularly preferred.
  • This invention includes using both mono-salt forms, i.e., a 1:1 ratio of a compound of Formula I with a base as previously described, as well as di-salt forms in those instances where a compound of Formula I has two acidic groups .
  • the method of the present invention can be practiced using solvate forms of the compounds of Formula I or salts thereof, such as ethanol solvates, hydrates, and the like.
  • Illustrative compounds which can be used in the practice of the method of the present invention include 2- (2-propyl-3- (3- (2-ethyl-4- (4 -fluorophenyl) -5- hydroxyphenoxy) propoxy) phenoxy) benzoic acid, 3- (2- (3- (2- ethyl-4- (4 -fluorophenyl) -5-hydroxyphenoxy) propoxy) -6- (4- carboxy-phenoxy) phenyl)propionic acid, 1- (4- (carboxy- methoxy) phenyl ) -1- (lH-tetrazol-5-yl) -6- (2-ethyl-4- (4- fluorophenyl ) -5-hydroxyphenoxy) hexane, 3- (4- (7-carboxy-9- oxo-3- (3- (2-ethyl-4- (4-fluorophenyl) -5-hydroxyphenoxy) - propoxy) -9H-xanthene) ) propanoic acid
  • the present invention is practiced with 2- (2- propyl-3- (3- (2-ethyl-4- (4-fluorophenyl) -5- hydroxyphenoxy) propoxy) phenoxy) benzoic acid or a pharmaceutically acceptable salt or solvate thereof.
  • Compounds of Formula I can be synthesized by established methods, such as those set forth in U.S. Patent No. 5,462,954 to Baker et al . and in U.S. Patent No. 5,910,505 to Fleisch et al . , which are hereby incorporated by reference.
  • leukotriene B4 receptor antagonists such as those described in U.S. Patent No. 5,998,454 to Fleisch et al . ; U.S. Patent No. 5,914,340 to Fleisch et al . ; U.S. Patent No. 5,817,864 to Fleisch et al . ; U.S. Patent No. to 6,037,377 to Anderskewitz et al . ; Perchonock et al .
  • Still other compounds which can be used to inhibit binding of leukotriene B4 to leukotriene B4 receptor by binding to leukotriene B4 receptor include antibodies (monoclonal or polyclonal) as well as peptide drugs, such as mimotopes which recognize and bind to the leukotriene B4 receptor. Methods for making and using such antibodies and mimotopes are well known to those skilled in the art and are described below in relation to making antibodies and mimotopes which recognize and bind to leukotriene A4 hydrolase.
  • binding of leukotriene B4 to leukotriene B4 receptor can be inhibited by using compounds which bind to leukotriene B4 to form, for example, a complex which is not capable of binding to leukotriene B4 receptor.
  • the binding of the compound to leukotriene B4 is specific; i.e., the compound does not bind substantially to other biological materials that are present in the sample.
  • the IC 50 of the compound to leukotriene B4 be greater than about 1.1 times, more preferably greater than about 1.5 times, still more preferably greater than about 2 times, still more preferably greater than about 5 times, still more preferably greater than about 10 times the IC 50 of the compound to all other biological materials in the sample.
  • the degree of binding of the compound to leukotriene B4 is preferably greater (i.e., has a higher IC 50 ) than the binding of leukotriene B4 to leukotriene B4 receptor, although the desired effect can be achieved in some circumstances even when the degree of binding of the compound to leukotriene B4 is less (i.e., has a lower IC 50 ) than the binding of leukotriene B4 to leukotriene B4 receptor.
  • binding of leukotriene B4 to leukotriene B4 receptor can be inhibited by inhibiting the production of leukotriene B4.
  • Production of leukotriene B4 can be inhibited, for example, by inhibiting one or more of the steps in the leukotriene B4 synthetic pathway.
  • the compound can be one which inhibits the conversion of leukotriene A4 to leukotriene B4.
  • the conversion of leukotriene A4 to leukotriene B4 by can be inhibited by contacting the sample with a compound that binds to leukotriene A4 to form, for example, a complex which is not susceptible or is less susceptible (than leukotriene A4) to hydrolysis by leukotriene A4 hydrolase .
  • the conversion of leukotriene A4 to leukotriene B4 by can be inhibited by contacting the sample with a compound that inhibits the activity of leukotriene A4 hydrolase.
  • leukotriene A4 hydrolase can be inhibited by contacting the sample with a compound that binds leukotriene A4 hydrolase.
  • leukotriene A4 hydrolase inhibitors can be traditional small molecules or pharmaceutically acceptable salts thereof.
  • leukotriene A4 hydrolase inhibitors examples include 3-oxiranyl benzoic acids and derivatives thereof; Rhone-Poulenc Rorer RP-64996; and 2-amino-3-phenylpropane derivatives, such as (2S) -2-amino ⁇ 3-phenylpropyl mercaptan, 2 -amino-3- (4 ' -benzyloxy) phenylpropyl mercaptan, and (2S) -3- (4- (2-naphthylmethyloxy) phenyl) - 1, 2-diamino-propane HCl, and its corresponding 4 ' -benzyloxy derivative.
  • (2S) -2-amino ⁇ 3-phenylpropyl mercaptan 2 -amino-3- (4 ' -benzyloxy) phenylpropyl mercaptan
  • (2S) -3- (4- (2-naphthylmethyloxy) pheny
  • leukotriene A4 hydrolase inhibitors are described in, for example, U.S. Patent No. 5,990,326 to Djuric et al . ; U.S. Patent No. 5,990,148 to Isakson et al . ; U.S. Patent No. 5,455,271 to Yuan et al . ; International Patent Publictaion Nos . W096/11192 and WO96/1099; Evans et al . , Prostaglandins , Leukotrienes and Medicine, 23:167-171 (1986); Yuan et al , "Isolation and Identification of Metabolites of Leukotriene A4 Hydrolase Inhibitor SC-57461 in Rats," Drug Metab.
  • Leukotriene A4 hydrolase inhibitors can also be in the form of peptide drug products. See, generally, Bevan et al . , Trends in Biotechnology, 13 (3) : 115-121 (1995); Sepetov et al . , Proc . Nat ' 1 Acad. Sci . USA, 92:5426-5430 (1995); O'Connor et al . , Cancer Chemother. Pharmacol . , 34:225-229 (1994); and Webber et al . , J. Med. Chem. , 36:733-746 (1993), which are hereby incorporated by reference.
  • Drugs such as peptide drugs
  • Drugs can be made using various methods known in the art .
  • One such method utilizes the development of epitope libraries and biopanning of bacteriophage libraries. Briefly, attempts to define the binding sites for various monoclonal antibodies have led to the development of epitope libraries. Parmley et al . , Gene , 73:305-318- (1988) ("Parmley"), which is hereby incorporated by reference, describe a bacteriophage expression vector that could display foreign epitopes on its surface. This vector could be used to construct large collections of bacteriophage which could include virtually all possible sequences of a short (e.g. six-amino-acid) peptide.
  • a short e.g. six-amino-acid
  • Parmley which is hereby incorporated by reference, also describes biopanning, which is a method for affinity- purifying phage displaying foreign epitopes using a specific antibody. See, also, e.g., Parmley; Cwirla et al., Proc. Nat ' 1 Acad. Sci . USA, 87:6378-6382 (1990); Scott et al., Science, 249:386-390 (1990); Christian et al., J. Mol. Biol., 227:711-718 (1992); and Smith et al . , Methods in Enzvmology, 217:228-257 (1993) ("Smith”), which are hereby incorporated by reference.
  • sequences of these and other mimotopes do not identify a continuous linear native sequence or necessarily occur in any way in a naturally- occurring molecule, e.g., a naturally occurring protein.
  • the sequences of the mimotopes merely form a peptide which functionally mimics a binding site on a naturally- occurring protein.
  • the mimotope described in Balass mimics the binding site of the acetylcholine receptor.
  • mimotopes are short peptides.
  • the availability of short peptides which can be readily synthesized in large amounts and which can mimic naturally-occurring sequences (i.e. binding sites) can be exploited in the method of the present invention.
  • mimotopes to a monoclonal antibody that recognizes leukotriene A4 hydrolase can be identified.
  • the sequences of these mimotopes represent short peptides which can then be used in various ways, for example as peptide drugs that bind to leukotriene A4 hydrolase and decrease the activity of leukotriene A4 hydrolase.
  • the peptide drugs can be chemically synthesized.
  • Antibodies to leukotriene A4 hydrolase represent another class of compounds that are useful for inhibiting the conversion of leukotriene A4 to leukotriene B4 by inhibiting the activity of leukotriene A4 hydrolase.
  • Antibodies as used herein are meant to include antibody fragments, such as Fab, Fab2 , and Fd fragments, as well as humanized forms. Humanized forms of the antibodies may be generated using one of the procedures known in the art, such as chimerization. ' The antibody binds to leukotriene A4 hydrolase, decreasing activity of the leukotriene A4 hydrolase. Suitable antibodies include polyclonal antibodies and monoclonal antibodies .
  • Monoclonal antibodies that bind to leukotriene A4 hydrolase can be produced by hybridomas .
  • a hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
  • Such methods include subcutaneous or intraperitoneal injection of the enzyme.
  • the amount of the enzyme used for immunization will vary based on the animal which is immunized, the antigenicity pf the enzyme, and the site of injection.
  • the enzyme which is used as an immunogen may be modified or administered in an adjuvant in order to increase the enzyme's antigenicity.
  • Methods of increasing the antigenicity of an enzyme are well known in the art and include, but are not limited to, coupling the antigen with a heterologous protein (such as a globulin or beta-galactosidase) or including an adjuvant during immunization.
  • spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Ag 15 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
  • myeloma cells such as SP2/0-Ag 15 myeloma cells
  • any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas using, for example, an ELISA assay, a western blot analysis, or a radioimmunoassay . See, e.g., Lutz et al . , Ex . Cell Res. , 175:109-124 (1988), which is hereby incorporated by reference . Hybridomas secreting the desired antibodies are cloned, and the class and subclass are determined using procedures known in the art, such as those set described in Campbell, which is hereby incorporated by reference. For polyclonal antibodies, antibody containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.
  • the conversion of leukotriene A4 to leukotriene B4 by can be inhibited by contacting the sample with a nucleic acid compound that binds to a nucleic acid molecule encoding the leukotriene A4 hydrolase, thus decreasing expression of the leukotriene A4 hydrolase.
  • Suitable nucleic acid molecules include, for example, antisense nucleic acid molecules and ribozymes .
  • Antisense nucleic acid molecules are complementary to at least a portion of the mRNA encoding leukotriene A4 hydrolase. Nucleic acid and amino acid sequences of leukotriene A4 hydrolases are known. See, for example,
  • Antisense nucleic acid molecules can be RNA or single-stranded DNA and can be complementary to the entire mRNA molecule encoding leukotriene A4 hydrolase (i.e., of the same nucleotide length as the entire molecule) . It may be desirable, however, to work with a shorter molecule.
  • the antisense molecule can be complementary to a portion of the entire mRNA molecule encoding the leukotriene A4 hydrolase.
  • These shorter antisense molecules are capable of hybridizing to the mRNA encoding the entire molecule and, preferably, consist of at least fifteen nucleotides and up to about 100 nucleotides.
  • These antisense molecules can be used to reduce levels of leukotriene A4 hydrolase, by contacting the cells with an RNA or single-stranded DNA molecule that is complementary to at least a portion of the mRNA of the leukotriene A4 hydrolase (e.g., by introducing an antisense molecule into the sample) .
  • the antisense molecule can base-pair with the mRNA of the leukotriene A4 hydrolase, preventing translation of the mRNA into protein.
  • an antisense molecule to the leukotriene A4 hydrolase can prevent translation of mRNA encoding the leukotriene A4 hydrolase into a functional leukotriene A4 hydrolase, thereby decreasing the activity of leukotriene A4 hydrolase in the cell. .
  • Antisense molecules are contacted with the cells by any suitable means.
  • the cells are adenocarcinoma cancer cells, such as prostate cancer cells, lung cancer cells, stomach cancer cells, breast cancer cells, colon cancer cells, and pancreatic cancer cells.
  • the antisense RNA molecule is contacted with the cells by injecting the antisense RNA molecule directly into the cellular cytoplasm, where the RNA interferes with translation.
  • a vector can also be used for introducing the antisense molecule into a cell. Such vectors include, for example, various plasmid and viral vectors.
  • the antisense molecules could also be introduced into a cell using liposomes.
  • antisense molecules For a general discussion of antisense molecules and their use, see Han et al., Proc. Nat'l Acad. Sci. USA, 88:4313-4317 (1991) ("Han”); and Rossi, British Medical Bulletin 51(1) :217- 225 (1995), which are hereby incorporated by reference.
  • a special category of antisense RNA molecules known as ribozymes, having recognition sequences complementary to specific regions of the mRNA encoding the leukotriene A4 hydrolase, can also be used to inhibit the activity of leukotriene A4 hydrolase. Ribozymes not only complex with target sequences via complementary antisense sequences but also catalyze the hydrolysis, or cleavage, of the template mRNA molecule.
  • Expression of a ribozyme in a cell can inhibit gene expression (such as the expression of the leukotriene A4 hydrolase) . More particularly, a ribozyme having a recognition sequence complementary to a region of a mRNA encoding the leukotriene A4 hydrolase can be used to decrease expression of leukotriene A4 hydrolase. A cell with a first level of expression of leukotriene A4 hydrolase is selected, and, then, the ribozyme is introduced into the cell. The ribozyme in the cell decreases expression of leukotriene A4 hydrolase in the cell, typically because mRNA encoding the leukotriene A4 hydrolase is cleaved and cannot be translated.
  • Ribozymes can be contacted with the cancer cells in accordance with the methods of the present invention by any suitable means.
  • the ribozyme can be injected directly into the cellular cytoplasm, where the ribozyme cleaves the mRNA and thereby interferes with translation.
  • a vector can be used to introduce the ribozyme into a cell.
  • Such vectors include various plasmid and viral vectors .
  • the DNA encoding the ribozyme does not need to be "incorporated" into the genome of the host cell; instead, for example, the ribozyme-encoding DNA molecule could be expressed in a host cell infected by a viral vector, with the vector expressing the ribozyme.
  • ribozyme molecules could also be introduced into a cell using liposomes.
  • liposomes For a general discussion of ribozymes and their use, see, for example, Sarver et al . , Science, 247:1222-1225 (1990); Chrisey et al . , Antisense Research and Development, l(l):57-63 (1991); Rossi et al . , AIDS Research and Human Retroviruses, 8(2):183-189 (1992); and Christoffersen et al., Journal of Medicinal Chemistry, 38 (12) : 2023-2037 (1995), which are hereby incorporated by reference.
  • levels of leukotriene A4 hydrolase in an adenocarcinoma cancer cell can be decreased by introducing an antisense or ribozyme construct into the cell.
  • An antisense construct blocks translation of mRNA encoding leukotriene A4 hydrolase into the leukotriene A4 hydrolase.
  • a ribozyme construct cleaves the mRNA encoding the leukotriene A4 hydrolase thus also prevents expression of functional leukotriene A4 hydrolase.
  • various gene therapy techniques can be utilized to introduce the antisense or ribozyme construct into the desired cell.
  • the construct may need to be targeted to the desired cells (e.g., the prostate, lung, stomach, pancreatic, colon, or breast cancer cells) by known methods, since, in other cells of the subject, decreased expression of leukotriene A4 hydrolase may not be desired.
  • binding of leukotriene B4 to leukotriene B4 receptor can be inhibited by inhibiting the production of leukotriene B4.
  • production of leukotriene B4 can be inhibited, for example, by inhibiting one or more of the steps in the leukotriene B4 synthesis pathway.
  • Another method for inhibiting the leukotriene B4 synthesis pathway involves inhibiting the production of leukotriene A4 , for example, by contacting the sample of adenocarcinoma cancer cells with a 5-lipoxygenase inhibitor.
  • 5- lipoxygenase inhibitor means any compound that directly or indirectly inhibits the activity of 5-lipoxygenase.
  • 5-lipoxygenase inhibitors include inhibitors of 5-lipoxygenase-activating protein (e.g., 3- (1- (4-chlorobenzyl) -3-t-butyl-thio-5-isopropyl-2-yl) -2,2- dimethylpropanoic acid and/or nordihydroguaiaretic acid) and nucleic acid molecules which decrease expression of 5-lipoxygenase-activating protein (e.g., 3- (1- (4-chlorobenzyl) -3-t-butyl-thio-5-isopropyl-2-yl) -2,2- dimethylpropanoic acid and/or nordihydroguaiaretic acid) and nucleic acid molecules which decrease expression of 5-lipoxygenase-activating protein (e.g., 3- (1- (4-chlorobenzyl) -3-t-butyl-thio-5-isopropyl-2-yl) -2,2- dimethylpropanoic acid and/or nord
  • 5-lipoxygenase 5-lipoxygenase. Further details with regard to these compounds are discussed in applicant's copending U.S. Patent Application Serial No. 09/111,343, which is hereby incorporated by reference.
  • the method of the present invention can be practiced with compounds that inhibit the binding of leukotriene B4 to leukotriene B4 receptor but that do not inhibit the production of leukotriene A4.
  • the method of the present intention can be practiced with compounds that are not 5-lipoxygenase inhibitors, that are not inhibitors of 5-lipoxygenase- activating protein, that are not 3- (1- (4-chlorobenzyl) -3- t-butyl-thio-5-isopropyl-2 -yl) -2 , 2-dimethylpropanoic acid, and/or that are not nordihydroguaiaretic acid.
  • the method of the present invention is carried out with a compound which inhibits binding of leukotriene B4 to leukotriene B4 receptor.
  • This can be carried out, alternatively or additionally to the various methods set forth above, by decreasing expression of leukotriene B4 receptor.
  • the methods discussed above with respect to decreasing expression of leukotriene A4 hydrolase are equally applicable here.
  • the compound can be a nucleic acid molecule which binds to a nucleic acid molecule encoding leukotriene B4 receptor, such as, for example, antisense nucleic acid molecules and ribozymes targeted to the nucleic acid molecule encoding leukotriene B4 receptor.
  • leukotriene B4 receptor which can be used is to design suitable antisense nucleic acid molecules and ribozymes are disclosed in, for example, Martin et al . , "Leukotriene Binding, Signaling, and Analysis of HIV Coreceptor Function in Mouse and Human Leukotriene B4 Receptor-transfected Cells, " J. Biol . Chem. ,
  • the method of the subject invention is based on the finding that these compounds decrease proliferation of adenocarcinoma cancer cells, and/or induce apoptosis of adenocarcinoma cancer cells, and/or induce differentiation of adenocarcinoma cancer cells into non-cancerous cells.
  • proliferation decreases proliferation of adenocarcinoma cancer cells
  • apoptosis induces differentiation of adenocarcinoma cancer cells into non-cancerous cells.
  • differentiation are readily understood in the art.
  • Illustrative methods for assaying for proliferation, apoptosis, or differentiation are provided in the examples which follow and are also described in applicant's copending U.S. Patent
  • the method of the present invention can be used in vitro to screen for compounds which are potentially useful in treating adenocarcinoma cancer (such as prostate, lung, stomach, breast, colon, and/or pancreatic cancer); to evaluate a compound's efficacy in treating adenocarcinoma cancer; or to investigate the mechanism by which a compound combats adenocarcinoma cancer (e.g., whether it does so by inducing apoptosis, by inducing differentiation, by decreasing proliferation, etc) .
  • adenocarcinoma cancer such as prostate, lung, stomach, breast, colon, and/or pancreatic cancer
  • a compound's efficacy in treating adenocarcinoma cancer or to investigate the mechanism by which a compound combats adenocarcinoma cancer (e.g., whether it does so by inducing apoptosis, by inducing differentiation, by decreasing proliferation, etc) .
  • a compound has been identified as a compound that inhibits binding of leukotriene B4 to leukotriene B4 receptor
  • one skilled in the art can apply the method of the present invention in vitro to evaluate the degree to which the compound induces apoptosis, induces differentiation, and/or decreases proliferation of cancer cells; or one skilled in the art can apply the method of the present invention to determine whether the compound operates by inducing apoptosis, by inducing differentiation, by decreasing proliferation, or by a combination of these methods .
  • the method of the present invention can be used in vivo to treat adenocarcinoma cancers, such as prostate cancer, lung cancer, stomach cancer, pancreatic cancer, breast cancer, and colon cancer.
  • adenocarcinoma cancers such as prostate cancer, lung cancer, stomach cancer, pancreatic cancer, breast cancer, and colon cancer.
  • contacting can be carried out by administering a therapeutically effective amount of the compound to the human subject, for example, by directly injecting the compound into a tumor. Details with regard to administering compounds in accordance with the method of the present invention are described below.
  • the present invention in another aspect thereof, relates to a method of treating adenocarcinomas, such as prostate cancer, lung cancer, stomach cancer, breast cancer, pancreatic cancer, colon cancer, or other cancers involving epithelial cells, in a subject.
  • the method includes administering to the subject an amount of a compound effective to inhibit binding of leukotriene B4 to leukotriene B4 receptor.
  • Suitable subjects include, for example mammals, such as rats, mice, cats, dogs, monkeys, and humans.
  • Suitable human subjects include, for example, those which have previously been determined to be at risk of having prostate cancer, lung cancer, stomach cancer, pancreatic cancer, colon cancer, and/or breast cancer and those who have been diagnosed as having prostate cancer, lung cancer, stomach cancer, pancreatic cancer, colon cancer, and/or breast cancer.
  • the subject does not suffer from oral squamous cell carcinoma or would not otherwise be indicated as a candidate for treatment of oral squamous cell carcinoma.
  • the compounds which inhibit binding of leukotriene B4 to leukotriene B4 receptor are administered to the subject preferably under conditions effective to decrease proliferation and/or induce apoptosis and/or induce differentiation of the adenocarcinoma cancer cells in the event that they develop.
  • Such preventive (which is not used in the absolute 100% sense) therapy can be useful in high risk individuals as long as the adverse side effects of the administration of compounds which inhibit binding of leukotriene B4 to leukotriene B4 receptor are outweighed by the potential benefit of prevention.
  • any of the compounds described above can be used in the treatment method of the present invention.
  • compounds which inhibit binding of leukotriene B4 to leukotriene B4 receptor such as traditional chemicals and peptide drugs disclosed herein that bind directly to leukotriene B4 receptor, may be administered alone or in combination with compatible carriers as a composition.
  • Compatible carriers include suitable pharmaceutical carriers or diluents. The diluent or carrier ingredients should be selected so that they do not diminish the therapeutic effects of the compounds used in the present invention.
  • the compositions herein may be made up in any suitable form appropriate for the desired use. Examples of suitable dosage forms include oral, parenteral, or topical dosage forms.
  • Suitable dosage forms for oral use include tablets, dispersible powders, granules, capsules, suspensions, syrups, and elixirs.
  • Inert diluents and carriers for tablets include, for example, calcium carbonate, sodium carbonate, lactose, and talc.
  • Tablets may also contain granulating and disintegrating agents, such as starch and alginic acid; binding agents, such as starch, gelatin, and acacia; and lubricating agents, such as magnesium stearate, stearic acid, and talc. Tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption.
  • Inert diluents and carriers which may be used in capsules include, for example, calcium carbonate, calcium phosphate, and kaolin.
  • Suspensions, syrups, and elixirs may contain conventional excipients, for example, methyl cellulose, tragacanth, sodium alginate; wetting agents, such as lecithin and polyoxyethylene stearate; and preservatives, such as ethyl-p-hydroxybenzoate .
  • Dosage forms suitable for parenteral administration include solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions which can be dissolved or suspended in sterile injectable medium immediately before use . They may contain suspending or dispersing agents known in the art. Examples of parenteral administration are intraventricular, intracerebral , intramuscular, intravenous, intraperitoneal, rectal, and subcutaneous administration .
  • these formulations can include other active materials, particularly, actives which have been identified as useful in the treatment of prostate, lung, stomach, breast, colon, pancreatic cancers and/or other adenocarcinomas.
  • actives can be broad-based anti- cancer agents, such that they also are useful in treating other types of cancers (i.e., in addition to adenocarcinomas) or they may be more specific, for example, in the case where the other active is useful for treating adenocarcinomas but not useful for treating oral squamous cell carcinoma.
  • the other actives can also have non-anti-cancer pharmacological properties in addition to their anti-andenocarcinoma properties.
  • the other actives can have anti-inflammatory properties, or, alternatively, they can have no such anti-inflammatory properties.
  • the actual preferred amount of compound to be administered according to the present invention will vary according to the particular compound, the particular composition formulated, and the mode of administration. Many factors that may' modify the action of the compound (e.g., body weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the subject, drug combinations, and reaction sensitivities and severities) can be taken into account by those skilled in the art. Administration can be carried out continuously or periodically within the maximum tolerated dose. Optimal administration rates for a given set of conditions can be ascertained by those skilled in the art using conventional dosage administration tests.
  • DMEM, MEM, and McCoy's 5A media penicillin-streptomycin solution, trypsin-EDTA solution, proteinase K, propidium iodide, RNAse A, and phenol/chloroform were purchased from Sigma Chemicals (St. Louis, Missouri).
  • Fetal bovine serum (“FBS”) was obtained from Atlanta Biologicals (Norcross, Georgia) .
  • APO-BRDU kits for TUNEL assay were from purchased from Pharmingen (San Diego, California) .
  • Leukotriene B4 (“LTB4") was obtained from Cayman Chemicals (Ann Arbor, Michigan).
  • the LTB4 antagonist 2- (2-propyl-3- (3- (2- ethyl-4- (4 -fluorophenyl) -5-hydroxyphenoxy) propoxy) - phenoxy) benzoic acid (hereinafter "LY293111”), was provided by Eli Lilly and Company (Indianapolis, Indiana).
  • pancreatic cancer cell lines were used: MiaPaCa-2 and PANC-1 (poorly- differentiated) ; Capan-1 and Capan-2 (well- differentiated) ; and HPAF and AsPC-1 (pleomorphic) . These were purchased from American Type Culture Collection (Rockville, Maryland) . PANC-1 and MiaPaCa-2 were grown in DMEM media; HPAF and AsPC-1 were grown in MEM media; and Capan-1 and Capan-2 were grown in McCoy's media. Media were supplemented with 10% FBS, and the cells were grown as monolayers in a humidified atmosphere of 95% 0 2 and 5% C0 2 ' at 37°C.
  • the cells were regularly seeded into 75cm 2 flask with media changed every other day. For experiments, cells were grown to 80% confluence, digested with trypsin-EDTA, and plated either in 12 or 24-well plates at a concentration of 50,000/ml, as appropriate. DNA synthesis by [ 3 H] -Thymidine incorporation.
  • Cells were plated in 12 or 24-well 'plates . After reaching 70% confluence, they were incubated in serum- free media for 24 hours, -which was then replaced with fresh serum-free media with or without the appropriate concentration of leukotriene B4 receptor antagonist LY293111 (62.5-1000 nM) , LTB4 (100-400 nM) , or both. After the appropriate period of culture, cellular DNA synthesis was assayed by adding 0.5 ⁇ Ci/well of [ 3 H] - methyl-thymidine and incubating for another 2 hours. Then, the cells were washed twice with PBS, fixed with
  • TCA trichloroacetic acid
  • Pancreatic cancer cells were seeded into 24-well microplates and incubated at 37°C. After 24 hours, cells were cultured in serum- free media with or without 250 nM LY293111 for 24, 48, and 72 hours. At the end of each time period, the cells were trypsinized to produce a single cell suspension, and the cell number in each well was determined using a Zl- Coulter Counter (Luton, Bedfordsire, United Kingdom) .
  • Pancreatic cancer cells grown in 75cm 2 flasks were treated with different concentrations of LY293111 for different periods of time. They were then viewed by phase-contrast microscopy, and photographs were taken.
  • RNAse A 0.3 mg/ml
  • 10 ml proteinase K 10 mg/ml
  • pellets containing DNA were then resuspended in 50 ml of TE buffer, subjected to electrophoresis on a 1.8% agarose gel containing ethidium bromide (1 mg/ml) , visualized by UV illumination, and analyzed using a Molecular Analyst Gel Documentation system (Bio-rad Laboratories, Hercules, California) .
  • TUNEL assay Apoptosis was measured by flow cytometry using a terminal deoxynucleotidyl transferase- mediated deoxyuridine triphosphate nick-end labeling
  • TUNEL terminal deoxynucleotidyl transferase
  • Bonferroni ' s corrections for multiple comparisons as appropriate. This analysis was performed with the Prism software package (GraphPad, San Diego, California) . Data are expressed as mean ⁇ SEM.
  • Example 2 Effect of LY293111 on Pancreatic Cancer Cells
  • Experiments carried out with 5 other pancreatic cancer cell lines HPAF, Capan-1, Capan-2, AsPC-1, and PANC-1) also showed that LY293111 inhibited proliferation, in a concentration-dependent fashion, after 24 hours.
  • LY293111 inhibited the proliferation by at least 50% at a concentration of 250 nM and by more than 95% at 1000 nM.
  • Phase-contrast microscopy revealed marked morphological changes in treated MiaPaCa-2 pancreatic cancer cells (Figure 3B) compared with non-treated controls ( Figure 3A) , as early as 4 hours after treatment with 250 nM LY293111. Over time, the treated cells became rounded and exhibited membrane blebbing, chromatin condensation, nuclear fragmentation and finally detached from the microplate. These morphological changes have been previously interpreted as reflecting apoptosis. Similar results were seen for colonic cell line LoVo, as shown in Figure 3C (treated with LY293111) and Figure 3D (control) .
  • Figure 5A shows the effects of different concentrations of LTB4 on thymidine incorporation in MiaPaCa-2 cells after 24 hours.
  • Figures 5B and 5C show the effects of 100 nM of LTB4 on thymidine incorporation in MiaPaCa-2 cells ( Figure 5B) and HPAF cells ( Figure 5C) for 24, 48, and 72 hours. At a concentration of 100 nM for 72hours, LTB4 more than doubled thymidine incorporation compared with control .
  • LTB4 abolished the inhibitory effect of the leukotriene B4 receptor antagonist, LY293111, on the proliferation of MiaPaCa-2 ( Figure 6A) and HPAF ( Figure 6B) pancreatic cancer cell lines.
  • LY293111 After 24 hours treatment with 100 nM LTB4, there was a 25% increase of proliferation compared with control, and LY293111 at 250 nM caused 50% decrease of proliferation.
  • LY293111 When treated with the combination of LTB4 and its antagonist, LY293111, proliferation was restored to almost the control level .
  • Example 6 DNA Fragmentation Assay A DNA fragmentation assay was used to study apoptosis of pancreatic cancer cells treated with LY293111. After treatment of MiaPaCa-2 cells with 250, 500 and 1000 nM LY293111 for 48 hours, a distinctive DNA ladder was seen after running cellular DNA on 1.8% agarose gel, while no such ladder was seen in control cells ( Figure 7) . Similar results were obtained in the other pancreatic cancer cell lines tested.
  • Example 7 TUNEL Assay LY293111-induced apoptosis of pancreatic cancer cell lines was also evaluated using the TUNEL assay.
  • Figures 9A-9F The effect of LY293111 on cancer cell proliferation, was investigated by measuring thymidine incorporation and cell number.
  • LY293111 caused a profound concentration-dependent and time-dependent inhibition of proliferation of all pancreatic, colonic, and breast cancer cell lines studied.
  • Figures 9A-9F The results are presented in Figures 9A-9F.
  • Figures 9A and 9B pertain to pancreatic cancer cell line MiaPaCa2;
  • Figure 9C pertains to pancreatic cancer cell line HPAF;
  • Figure 9D pertains to pancreatic cancer cell line Capan2.
  • Each of Figures 9A, 9C, and 9D show the dosage effects of LY293111 on these pancreatic cancer cell lines.
  • Figure 9B shows the time dependent effects of LY293111 on the pancreatic cancer cell line MiaPaCa2.
  • Figures 9E and 9F shows the effect of 0.5 ⁇ M LY293111 on cell number vs control as a function of time for pancreatic cancer cell line MiaPaCa2 ( Figure 9E) and for colonic cancer cell line LoVo ( Figure 9F) .
  • LTB4 100 nM was found to increase expression of the pro-apoptotic protein, Bcl-2, in MCF-7 breast cancer cells, as revealed by western blotting using Bcl-2 specific antisera.
  • LY293111 500 nM inhibited expression of the pro-apoptotic protein, Bcl-2, in MCF-7 breast cancer cells.
  • Example 10 Effect of LY293111 on Growth of AsPc-1 Human Pancreatic Tumor Cell Line Xenografted Into Athymic Mice
  • Athymic mice received a subcutaneous injection of 3 million cancer cells in 50 ⁇ l of serum-free media.
  • the highly malignant and aggressive pancreatic tumor cell line, AsPc-1 was used in this experiment.
  • the tumors were allowed to establish and grow for four days prior to treatment with 0.01 % LY293111 in the drinking water.
  • Figure 11 shows the size of the tumor (in mm 3 ) as a function of treatment time . The results show that mice receiving LY293111 in their drinking water had smaller tumors than those in the control group.

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Abstract

L'invention concerne des procédés servant à diminuer la prolifération de cellules cancéreuses d'adénocarcinome ou à induire l'apoptose de ces cellules ou à induire la différenciation de cellules cancéreuses d'adénocarcinome en cellules non cancéreuses. Un de ces procédés consiste à mettre en contact les cellules cancéreuses d'adénocarcinome avec un composé dans des conditions efficaces pour que ce composé inhibe la fixation de leucotriène B4 au récepteur de leucotriène B4. Un autre de ces procédés consiste à mettre en contact les cellules cancéreuses d'adénocarcinome avec acide 2-(2-propyl-3-(3-(2-éthyl-4-(4-fluorophényl)-5-hydroxyphénoxy)propoxy)phénoxy)benzoïque ou un de ses sels, solvates ou congénères acceptables sur le plan pharmaceutique. Elle concerne également des procédés de traitement des adénocarcinomes chez un patient. Un de ces procédés consiste à administrer au patient une quantité d'un composé servant à inhiber la fixation de leucotriène B4 au récepteur de leucotriène B4. Un autre procédé consiste à administrer acide 2-(2-propyl-3-(3-(2-éthyl-4-(4-fluorophényl)-5-hydroxyphénoxy)propoxy)phénoxy)benzoïque ou un de ses sels, solvates ou congénères acceptables sur le plan pharmaceutique.
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MXPA02010974A (es) 2004-09-06
NO20025343L (no) 2003-01-09
KR20030019372A (ko) 2003-03-06
WO2001085166A1 (fr) 2001-11-15
CA2408622A1 (fr) 2001-11-15
JP2003532675A (ja) 2003-11-05

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