Classifications

• • 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
  • A61K31/66—Phosphorus compounds
  • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
  • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
• • 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
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/18—Sulfonamides
• • 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
• • 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
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
• • 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
  • A61K31/4164—1,3-Diazoles
• • 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
  • A61K31/4164—1,3-Diazoles
  • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
• • 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
  • A61K31/66—Phosphorus compounds
  • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• the present invention is to the discovery of a new method to treat cell proliferation and cancer in an animal.
• ET- 18-O-CH3 has shown some efficacy in the treatment of patients suffering from leukemias and carcinomas (Berdel et al, Cancer 50: 2011-2015 (1982); Andreesen et al, J. Natl. Can. Inst. 70: 623-627 (1983); Andreesen, Prog. Biochem. Pharmacol. 22: 118-131 (1988)).
• Coenzyme A-independent transacylase is an enzyme responsible for the movement of arachidonate between phospholipid molecular species of inflammatory cells. CoA-IT removes arachidonate from the sn-2 position of 1-acyl-containing phosphoiipids, such as l-acyl-2-arachidonoyl-.m-glycero-3-phosphocholine (l-acyl-2- arachidonoyl-GPC).
• lyso-phospholipid acceptor such as l-alkyl-2-lyso-GPC and l-alkenyl-2-lyso-5 «-glycero-3-phospho- ethanolamine
• a suitable lyso-phospholipid acceptor such as l-alkyl-2-lyso-GPC and l-alkenyl-2-lyso-5 «-glycero-3-phospho- ethanolamine
• This activity is selective for 20 carbon fatty acyl groups and is the mechanism by which inflammatory cells move arachidonate into specific phospholipid pools prior to its release (Winkler and Chilton, Drug News Perspec. 6: 133-138 (1993); Snyder et al, J. Lipid Mediat. 10: 25-31 (1994)).
• the present invention is a method to decreasing, inhibiting or reducing cell proliferation in a mammal, and inducing apoptosis in a mammal, preferably in a human, by inhibition of the enzyme CoA-IT.
• This effect, induced by inhibition of CoA-IT can be accomplished by structurally distinct classes of compounds. Therefore the present invention is to the use of CoA-IT inhibitors for the treatment of cell proliferation, in mammals, preferably humans, in need of such treatment, by administering to said human an effective amount of a CoA-IT inhibitory compound, or pharmaceutically acceptable salt thereof.
• a preferred disease state, for treatment herein, associated with cell proliferation is psoriasis, rheumatoid arthritis or atherosclerosis.
• Another aspect of the present invention is to a method of treating a cancerous cell growth in a mammal, preferably a human, in need of such treatment, which method comprises administering to said mammal an effective amount of a CoA-independent transacylase (CoA-IT) inhibitor, or pharmaceutically acceptable salt thereof; provided that the inhibitor is not l-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18- O-CH3) or an alkyl lysophospholipid analog.
• the CoA-IT inhibitor may be co-administered with an effective amount of ET-I8-O-CH3 or an alkyl lysophospholipid analog.
• a preferred disease state, for treatment herein, associated with cancerous cell growth is leukemia.
• Another aspect of the present invention is to a method of inducing apoptosis in a mammal, in need of such treatment which method comprises administering to said human or mammal an effective amount of a CoA-independent transacylase (CoA-IT) inhibitor, or pharmaceutically acceptable salt thereof; provided that the inhibitor is not l-O- octadecyl-2-O-methyl-5rt-glycero-3-phosphocholine (ET-I8-O-CH3) or an alkyl lysophospholipid analog.
• the CoA-IT inhibitor may be co-administered with an effective amount of ET-I8-O-CH3 or an alkyl lysophospholipid analog.
• Figure 2 demonstrates the kinetics of ET-I8-O-CH3 inhibition of CoA-IT activity.
• U937 microsomes were incubate with the indicated concentrations of ET-I8-O-CH3 and l-alkyl-2-lyso-GPC as described in the Methods section herein. The quantities of product produced in the reactions were determined. A double-reciprocal plot of 1/velocity versus l/[product] is shown.
• Figure 3 demonstrates the effect of ET-I8-O-CH3 (A), diethyl 7-(3,4,5-triphenyl-2-oxo- 2,3-dihydro-imidazol-l-yl)heptane-phosphonate (B, Compound III) and 2-[2-[3-(4- Chloro-3-trifluoromethylphenyl) ureido] -4-trifluoromethyl phenoxy]-4,5- dichlorobenzene sulfonic acid (C, Compound I) on HL-60 cell proliferation.
• HL-60 cells were treated at time 0 with the indicated concentrations of compound and the number of viable cells determined by trypan blue exclusion after 24 and 48 hours.
• Figure 4 demonstrates the effect of ET-I8-O-CH3 on DNA synthesis in HL-60 cells.
• HL-60 cells were treated with various concentrations of compound or vehicle. Twenty- four hours prior to harvesting the cells, they were given a pulse of [ ⁇ Hlthymidine. At the indicated time points, the cells were harvested, washed, lysed and the amount of [ ⁇ H] incorporated into DNA was measured as described in the Methods. The results are the means + SD of triplicate determinations and are representative of 3 experiments.
• Figure 5 demonstrates the effects of ET-I8-O-CH3, 2-[2-[3-(4-Chloro-3- trifluoromethylphenyl)ureido]-4-trifluoromethyl phenoxy]-4,5-dichlorobenzene sulfonic acid (Compound I) and diethyl 7-(3,4,5-tripheny l-2-oxo-2,3-dihydro- imidazol- 1- yl)heptane-phosphonate (Compound III) on DNA fragmentation in HL-60 cells.
• HL-60 cells were treated with the indicated concentration of compound and DNA fragmentation determinate after 24 hours as described in the Methods. Control is vehicle treatment and the arrow indicates the internal standard fragment.
• Figure 6 demonstrates the effects of ET-I8-O-CH3, 2-[2-[3-(4-Chloro-3- trifluoromethylphenyl)ureido]-4-trifluoromethyl phenoxy]-4,5-dichlorobenzene sulfonic acid (Compound I) and diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydro-imidazol-l- yl)heptane-phosphonate (Compound III) on the content of arachidonate in phosphatidylethanolamine subclasses of HL-60 cells.
• HL-60 cells were treated with ET- I8-O-CH3 (25 ⁇ M), Compound I (50 ⁇ M), Compound III (25 ⁇ M) or vehicle for 48 hours and A A content determined as described in the Methods.
• Figure 7 demonstrates the inco ⁇ oration of [3H]thymidine by chronic myelogenous leukemic cells following incubation with inhibitors for 20 hours. - o -
• CoA-IT represents a crucial event in the regulation of 'cancer' cell growth, and in particular apoptosis, Therefore, inhibition of CoA-IT will have a profound therapeutic effect on proliferative diseases and treatment of them thereby.
• ET-I8-O-CH3 was examined for its effect on CoA-IT activity in U937 microsomes by measuring the acylation of l-[-1H]alkyl-2-lyso-GPC to form product, [ 3 H]l-alkyl-2-arachidonoyl-GPC.
• the results ( Figure 1) showed that ET-I8-O-CH3 was a potent inhibitor of CoA-IT activity with an IC5 of 0.5 uM and complete inhibition observed at 3 uM.
• kinetic analysis experiments were performed. These data ( Figure 2) revealed that ET-18- O-CH3 inhibited CoA-IT activity in a manner that was best fit by a model of competitive competition.
• ET-I8-O-CH3 is a CoA-IT inhibitor, interacting with CoA-IT in a specific and predictable manner and that the inhibition of CoA-IT was not caused by a non-specific effect, such as a detergent effect on the microsomal membranes.
• ET-I8-O-CH3 has been shown to block the proliferation of cancer cells. Therefore, another aspect of the present invention is the discovery that ET- I8-O-CH3 is a potent inhibitor of the enzyme CoA-IT and hence is useful for treating a
• CoA-IT mediated disease or disorder in a mammal in need thereof comprises administering to said mammal an effective amount of l-O-octadecyl-2-O- methyl--?n-glycero-3-phosphocholine (ET-I8-O-CH3) as a CoA-IT inhibitor; provided that the disease or disorder is other than inhibiting cell proliferation or inducing apoptosis.
• E-I8-O-CH3 l-O-octadecyl-2-O- methyl--?n-glycero-3-phosphocholine
• ET-I8-O-CH3 is a potent inhibitor of CoA-IT raised the question of whether other structurally different CoA-IT inhibitors would influence proliferation in a manner comparable to ET-I8-O-CH3.
• Figure 3 illustrates the effects of ET-I8-O-CH3 and two distinct classes of compounds which are CoA-IT inhibitors on the proliferation of HL-60 cells.
• ET-I8-O-CH3 reduced the number of HL-60 cells in culture in a dose and time-dependent manner (Figure 3).
• two other structurally different CoA-IT inhibitors blocked the increases in cell numbers in a comparable fashion to ET-I8-O-CH3 ( Figure 3). All three inhibitors caused a loss of cell viability that was observed between 24 and 48 h.
• One aspect of the present invention is the demonstration that inhibition of CoA-IT has a role in apoptosis, and that inhibitors of CoA-IT, such as ET-I8-O-CH3 , 2-[2-[3-(4- chloro-3 -trifluoromethylphenyl) ureido] -4-trifluoromethyl phenoxy]-4,5- dichlorobenzene sulfonic acid and diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydro-imidazol- l-yl)heptane-phosphonate all caused apoptosis of HL-60 cells.
• inhibitors of CoA-IT such as ET-I8-O-CH3 , 2-[2-[3-(4- chloro-3 -trifluoromethylphenyl) ureido] -4-trifluoromethyl phenoxy]-4,5- dichlorobenzene sulfonic acid and diethyl 7-
• the enzyme CoA-IT orchestrates the movement of arachidonate into these phosphoiipids (Chilton et al, Biochemistry 34: 5403-5410 (1995)).
• CoA-IT inhibitors In addition to the loss from phosphatidyl- ethanolamine, CoA-IT inhibitors also blocked the incorporation of arachidonate into 1- ether-linked phosphatidylcholine . It has long been recognized that many 'cancer' cells contain high levels of 1 -ether- linked phosphoiipids and in particular 1 -alkenyl phosphatidylethanolamine (Snyder et al, Can. Res. 28: 972-978 (1968); and Snyder et al, Can. Res. 29: 251-257 (1969)). Moreover, Chabot et al. (Cancer Res.
• the normal path for inco ⁇ oration of arachidonate into 1 -ether-linked phosphoiipids is for arachidonate acid to be converted to arachidonate-CoA, to be then inco ⁇ orated into 1-acyl-linked phosphoiipids, such as l-acyl-2-arachidonate-phosphocholine.
• the enzyme CoA-IT then moves the arachidonate portion from the 1-acyl-linked phospholipid into 1 -ether-linked phosphoiipids.
• These 1 -ether-linked phosphoiipids are high in cancer cells.
• CoA-IT inhibitors block the movement of arachidonate into these 1 -ether-linked phosphoiipids, depriving cancers cells of a key component for their survival.
• inhibition of CoA-IT induces apoptosis of cancer cells, thus dramatically influencing their growth and proliferation.
• the data support the discovery, detailed herein, that inhibition of CoA-IT activity results in a decrease in cell growth and proliferation, in induction of apoptosis, with therapeutic utility in proliferative and cancerous disorders.
• This invention therefore provides a method, CoA-IT inhibition, for treating proliferative and cancerous disorders.
• alkyl lysophospholipid As used herein, the terms “alkyl lysophospholipid” (ALP), "alkyl lysophospholipid analog” and “ether-linked phospholipid and analogs thereof are well known terms to those of skill in the art and need no additional definitions. Generally these terms will encompass compounds that are ether linked phosphoiipids with alkyl chains in the sn- 1 position of the glycerol backbone.
• These compounds may be derivatives of l-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine, derivatives of l-O- hexadecyl-2-acetal-sn-glycero-3-phosphocholine (PAF), derivatives of ester- lysophospholipid (2-PLC), or derivatives of lysophophatidylcholine (2-LPC).
• PAF l-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine
• PLC ester- lysophospholipid
• 2-LPC lysophophatidylcholine
• Ether- lysophospholipids are also referred to as alkyllysoPC's, or ALPs
• alkylphosphocholines are also referred to as APCs
• HPC hexadecyl phosphocholine
• Suitable compounds encompassed by this term include but are not limited to, those described in Schick et al., Lipids, 22 (11) 904-910 (1987); such as those described in Morris- Natschke et al., J. Med. Chem., 29 (10) 2114-2117 (1986); Goto et al., Anticancer Research, 14:357362 (1994); Langen et al., Anticancer Research, 12:2109-2112 (1992); Volger et al., Lipids, 28(6) 511-516 (1993); Workman et al., Biochem.
• l-[3H]alkyl-2-lyso-GPC (30-60 Ci mmol) was purchased from New England Nuclear (Boston, MA). Common laboratory chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). l-alkyl-2-lyso-GPC was purchased from Biomol (Plymouth Meeting, PA). Silica gel G plates were from Analtech Inc. (Newark, DE). Essentially fatty acid-free bovine serum albumin was obtained from Calbiochem (San Diego, CA). Compounds ET-I8-O-CH3 was obtained from Biomol and was made as a 100 mM stock in
• Indomethacin was obtained from Sigma Chemicals.
• Arachidonyl- trifluoromethyl ketone was purchased from Cayman Chemicals, (Ann Arbor, MI). These compounds were made as stocks (5-100 mM) in dimethylsulfoxide (DMSO) and diluted in DMSO to provide final concentrations, with DMSO concentrations ranging from 0.1-1%.
• TNF (Sigma) was made as stock in distiller water and diluted to provide the desired concentrations.
• HL-60 cells were obtained from American Type Culture Collection and grown in RPMI- 1640 media supplemented with 10% fetal bovine serum at 37°, 5% CO2. Cells were seeded in T-flasks at 0.03 to 0.08 x 10 ⁇ cells / ml and used for experiments at 0.5 to 0.6 x l ⁇ 6 cells / ml. The cell concentration at the time of drug exposure was found to be important parameter (Fujiwara et al, 1994). Proliferation and apoptosis
• the functional status of cells was measured in three ways. First cell viability was assessed by the ability of the cells to exclude trypan blue stain as shown in Figure 3.
• a second, more sensitive and quantitative method was to measure DNA synthesis, using the inco ⁇ oration of [ ⁇ HJthymidine into newly-formed DNA as the index of synthesis.
• HL-60 cells were treated with various concentrations of compounds or vehicle and kept in cell culture for different lengths of time (1 to 3 days). Cells were then given a pulse of [- ⁇ HJthymidine (1 ⁇ Ci) then removed from culture 24 hours later, washed 2X with PBS and the treated with 0.2 N NaOH for 30 min followed by addition of 15% trichloroacetic acid for 2 hrs. The total sample from each well was filtered through GF/C Watman glass microfilters under vaccum and washed 3X with 5% trichloroacetic acid. The inco ⁇ oration of [*-*H]thymidine into DNA bound to the filter was quantatied using liquid scintillation spectroscopy; see Figure 4 herein.
• the final method determines not only the amount of cell death, but also provides insight into mechanism.
• cellular DNA is broken down into oligo-nucleosomal fragments which can be discerned on gels (Kerr et al, Cancer 73: 2013-2026, (1994)).
• Cells were treated for various times (3 - 24 hours) with different concentrations of compounds or vehicle.
• the washed cells were lysed by resuspending them in 200 ⁇ L of cold, sterile detergent buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2% Triton X-l 00) and incubated on ice 30 min.
• RNA and RNA were enzymatically degraded by incubation with 75 ⁇ g / mL RNAse A for 1 hour at 37° , then 200 ⁇ g/mL Proteinase K and 0.5% SDS (final) for 1 hour at 37°.
• An internal standard of DNA was added during the cell lysis and its recovery used as a confirmation of efficient extraction of cellular DNA fragments.
• the samples were extracted twice with an equal volume of cold, buffer saturated phenol, once with phenol / chloroform / isoamyl alcohol (25:24:1, v/v), and once with chloroform. NaCl (300 mM final) and cold ethanol were added, the solution was vortexed and let stand overnight.
• CoA-IT activity was measured as described previously (Winkler et al, Biophys. Acta Lipids Lipid Metab. 1081: 339-346, (1991)) in a total volume of 100 ⁇ l.
• Microsomes (5-20 ⁇ g protein) from a 100,000 x g, 60 min centrifugation (Winkler et al, supra) were diluted in PBS with 1 mM EGTA to the desired protein concentration. The reaction was initiated by addition of l-[ ⁇ H]alkyl-2-lyso-GPC (0.1 ⁇ Ci/tube) and 1 ⁇ M final cold l-alkyl-2-lyso-GPC in assay buffer with 0.25 mg/ml fatty acid-poor bovine serum albumin. The reaction was run for 10 min at 37°. The reaction was stopped, the lipids extracted (Bligh and Dyer, Can. J. Biochem. Physiol.
• ET-I8-O-CH3 The effects of ET-I8-O-CH3 on the kinetics of the CoA-IT reaction were examined.
• Various concentrations of ET-I8-O-CH3 (0-15 ⁇ M) were mixed with variable concentrations of substrate l-alkyl-2-lyso-GPC (0.01-3 ⁇ M).
• U937 microsomes containing CoA-IT activity were added and the reaction allowed to proceed for 3 minutes at 37°.
• the amount of product formed was determined as described above and was not more than 5% of added substrate at any concentration. Analysis of the results was performed using the method of Cleland,W., Methods Enzymol. 63: 103-138, (1979); see Figure 2 herein.
• HL-60 cells were exposed to compounds or vehicle and incubated for 48 hrs. At that time, incubation was terminated by extraction (Bligh and Dyer, Can. J. Biochem. Physiol. 37: 911-917 (1959)), the phospholipid classes in the organic phase were separated and collected by normal phase HPLC (Chilton, Methods Enzymol. 187: 157- 166 (1990)).
• the phosphoiipids were converted into diradylglycerols by addition of 20- 80 units of Bacillus cereus phospholipase C (Sigma Type XIII) in 100 mM Tris HCl buffer (pH 7.4) for 2.5 -6 hr, then converted into l,2-diradyl-3-acetylglycerols by incubation with acetic anhydride and pyridine (Chilton, Methods Enzymol. 187: 157- 166 (1990)).
• the phospholipid subclasses were separated by TLC in benzene / hexane / ethyl ether (50:45:4, v/v).
• the content of arachidonate in the phospholipid subclasses was determined after base hydrosysis, performed by using 2 M potassium hydroxide in 75% ethanol (30 min, 60°C). The reaction was stopped by addition of an equal amount of water and the reaction mixture was adjusted to pH 3 with HCl. Free fatty acids were extracted with ethyl ether, solvents removed under nitrogen and the free fatty acids converted to pentafluorobenzylesters and analyzed by GC/MS (Hubbard et al., Prostaglandins 22: 349-355 (1986)). Octadeuterioarachidonic acid was used as an internal standard and mole quantities determined using a standard curved obtained using known quantities of arachidonic acid; see Figure 6 herein. Protein analysis
• Protein concentrations were determined using the method of Bradford. M., Anal. Biochem. 72: 248-254, (1976), with reagents purchased from Bio-Rad (Hercules, CA).
• the present invention demonstrates that inhibition of CoA-IT by diverse, structurally unrelated compounds can produce an apoptotic response in cancer cells, taken directly from human patients.
• Leukemic cells from these patients were treated with inhibitors of CoA-IT and proliferation measured by monitoring the inco ⁇ oration of [3H]thymidine into newly synthesized DNA.
• two structurally different inhibitors of CoA-IT activity inhibited proliferation of human cancer cells.
• inco ⁇ oration of [3H]thymidine by chronic myelogenous leukemic cells is shown following incubation with the noted inhibitors for 20 hours.
• Leukemic cells from a human patient were incubated with vehicle (DMSO), diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydro- imidazol-l-yl)heptane-phosphonate (Compound III) or 2-[2-[3-(4-Chloro-3- trifluoromethylphenyl) ureido] -4-trifluoromethyl phenoxy]-4,5-dichlorobenzene sulfonic acid (Compound I) at the indicated concentrations. After 20 hours of treatment, the inco ⁇ oration of [3H]thymidine into DNA was determined as described in the Methods Section herein.
• vehicle DMSO
• Compound I 2-[2-[3-(4-Chloro-3- trifluoromethylpheny
• CoA-IT inhibitors cells were examined for the presence of DNA fragments, a hallmark of apoptosis. This was accomplished by labeling strand breads in the DNA with fluorescein dTUP with terminal transferase. Fluorecent analysis of DNA fragmentation, was performed for human leukemic cells treated with vehicle (DMSO) or diethyl 7- (3,4,5-triphenyl-2-oxo-2,3-dihydro-imidazol-l-yl)heptane-phosphonate (Compound III) (25 ⁇ M) for 24 hours. The cells were stained for DNA strand breaks using a kit from Boehringer Mannheim (Cat. No 1684795) according to the manufacture's instructions.
• DMSO vehicle
• Compound III diethyl 7- (3,4,5-triphenyl-2-oxo-2,3-dihydro-imidazol-l-yl)heptane-phosphonate
• the compounds of the present invention will generally be administered in a standard pharmaceutical composition obtained by admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• they may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsule, ovules or lozenges either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavouring or colouring agents.
• They may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously.
• parenteral administration they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
• a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
• the choice of form for administration as well as effective dosages will vary depending, inter alia, on the condition being treated. The choice of mode of administration and dosage is within the skill of the art.
• the compounds of the present invention can be formulated as liquids, for example syrups, suspensions or emulsions, tablets, capsules and lozenges.
• a liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent.
• a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent.
• a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations.
• suitable pharmaceutical carrier(s) include magnesium stearate, starch, lactose, sucrose and cellulose.
• a composition in the form of a capsule can be prepared using routine encapsulation procedures.
• pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
• a suitable pharmaceutical carrier(s) for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
• the composition is in unit dose form such as a tablet or capsule.
• Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.
• a sterile aqueous carrier or parenterally acceptable oil for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.
• the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.
• a typical suppository formulation comprises a compound or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
• a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
• each dosage unit for oral administration contains from about 1 to 250 mg (and for parenteral administration contains preferably from 0.1 to 25 mg) of the compound, or a pharmaceutically acceptable salt thereof calculated as the free base.
• the pharmaceutically acceptable compounds of the invention will normally be administered to a subject in a daily dosage regimen.
• a daily dosage regimen for an adult patient this may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compound, or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered 1 to
• Disease states which could benefit from the inhibition of cell proliferation include, but are not limited to, rheumatoid arthritis, psoriasis, atherosclerosis, and cancers such as leukemia or solid tumors.
• the sodium salt of 2-[2-[3,5-Bis (trifluoromethyl)phenyl- sulfonamido]-4-trifluoromethylphenoxy]benzoic acid may also be prepared.
• Ethyl 6-( 1.4.5-triphenylimidazol-2-oxy)hexanoate A mixture of ethyl-6- bromohexanoate (13.38g), 1,4,5-triphenylimidazole (6.24g), potassium carbonate (13.82g) and butan-2-one (250ml) was stirred under nitrogen at reflux for 24 hours. The mixrture was filtered and the filtrate was evaporated. The residue was chromatographed on silica gel eluted with 10% ethyl acetate to give ethyl 6-(l,4,5-triphenylimidazol-2-one-
• the compound may also be synthesized in accordance with the procedures indicated in DE 3,228, 271 Al whose disclosure is inco ⁇ orated herein by reference in its entirety.
• EXAMPLE 6 5-( 1 AS-Triphenylimidazol- ⁇ -one-S-yllvalerate a) Ethyl 5-(l,4,5-triphenylimidazol-2-yloxy)valerate l,4,5-Triphenylimidazol-2-one was treated with ethyl 5-bromovalerate and potassium carbonate in butanone to give after chromatographic work-up ethyl 5-( 1,4,5- triphenylimidazol-2-yloxy)valerate, m.p.
• 3-phosphocholine 5-lipoxygenase
• PLA2 phospholipase A2
• PBS phosphate buffered saline
• TLC thin layer chromatography
• TNF tumor necrosis factor ⁇ .

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• 1996
  • 1996-07-24 WO PCT/US1996/012257 patent/WO1997004765A1/en not_active Application Discontinuation
  • 1996-07-24 JP JP9507752A patent/JPH11511130A/ja active Pending
  • 1996-07-24 EP EP96925501A patent/EP0841910A4/de not_active Withdrawn

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