WO1995018606A9 - Curcumin, analogues of curcumin and novel uses thereof - Google Patents

Curcumin, analogues of curcumin and novel uses thereof

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Publication number
WO1995018606A9
WO1995018606A9 PCT/US1995/000104 US9500104W WO9518606A9 WO 1995018606 A9 WO1995018606 A9 WO 1995018606A9 US 9500104 W US9500104 W US 9500104W WO 9518606 A9 WO9518606 A9 WO 9518606A9
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acid
curcumin
cells
animal
analogue
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PCT/US1995/000104
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French (fr)
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WO1995018606A1 (en
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Priority to AU15585/95A priority Critical patent/AU687509B2/en
Priority to JP7518587A priority patent/JPH10500657A/en
Priority to EP95907307A priority patent/EP0738143A1/en
Publication of WO1995018606A1 publication Critical patent/WO1995018606A1/en
Publication of WO1995018606A9 publication Critical patent/WO1995018606A9/en

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  • the present invention relates generally to the field of cell proliferative diseases. More specifically, the present invention relates to novel antiproliferative effects of curcumin and analogues thereof. Description of the Related Art
  • Curcumin (diferuloylmethane) is a major active component of the food flavor turmeric (Curcuma longa) .
  • Previously known properties of curcumin in animals include inhibition of both tumor initiation induced by benzo-alpha- pyrene and 7, 12 dimethylbenz-alpha-anthracene and tumor promotion induced by phorbol ester.
  • curcumin exhibits anti-inflammatory properties in vivo . The pharmacological safety of curcumin is demonstrated by the consumption up to 100 g/day.
  • curcumin inhibits neutrophil activation, suppresses mitogen-induced proliferation of blood mononuclear cells, inhibits the mixed lymphocyte reaction, and inhibits proliferation of smooth muscle cells.
  • Curcumin is also a potent scavenger of reactive oxygen species, protects hemoglobin from nitrite-induced oxidation to methe oglobin and inhibits lipid peroxidation. Some of these activities may be responsible for curcumin's ability to protect DNA from free radical-induced damage and hepatocytes against various toxins. In addition, the phorbol ester-induced transcriptional factors c-jun/AP-1 are suppressed by curcumin. Recently curcumin has been shown to be highly effective in inhibiting the type 1 human immunodeficiency virus long terminal repeat-directed gene expression and virus replication.
  • a method for the treatment of pathological cell proliferative diseases comprising administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof.
  • a method of inhibiting the activity of phosphorylase kinase comprising administration to an animal of a pharmacologically effective do ⁇ e of curcumin or an analogue thereof.
  • a method of inhibiting the activity of tyrosine kinase comprising administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof.
  • a method for the treatment of pathological cell proliferative diseases comprising administration to an animal of a pharmacologically effective dose of a flavonoid or an analogue thereof.
  • Figure 1 shows the structure of curcumin and related analogues.
  • Figure 2 shows the dose response curve of curcumin on the growth of hormone-dependent human breast adenocarcinoma tumor cells (Figure 2A; MCF-7 cells) and (Figure 2B; T-47D cells) .
  • 5 x IO 3 cells were plated in 96-well plates overnight at 37° C. The cells were then incubated with either variable concentrations of curcumin (left panel) for 72 hours or for variable times (right panel) with curcumin (2.7 uM or 1 ug/ml) in a total final volume of 0.2 ml. During the last 6 hours, cells were pulsed with tritiated thymidine prior to harvesting. All determinations were made in triplicate. Relative cell viability was calculated as follows: thymidine incorporation in treated cells over thymidine incorporation in untreated cells multiplied by 100.
  • Figure 3 shows the time course of the effect of curcumin on the growth of human breast adenocarcinoma cells, MCF-7. 5 x 10 3 cells were plated in 96-well plates overnight at 37° C , washed and then incubated with different concentrations of curcumin for different times. Viability of cells was examined either by thymidine incorporation (Figure 3A) or by counting viable cell number ( Figure 3B) .
  • Figure 4 shows the dose response (Figure 4A) and time course (Figure 4B) of effect of curcumin on the growth of hormone-independent human breast tumor cells.
  • Figure 5 shows the dose response (Figure 5A) and the time course (Figure 5B) of curcumin on the growth of human promyelo onocytic tumor cells, HL-60. 5 x 10 3 cells were plated in 96-well plates overnight at 37° C. The cells were then incubated with either variable concentrations of curcumin (left panel) for 72 hours or for variable times (right panel) with curcumin (2.7 uM or 1 ug/ml) in a total final volume of 0.2 ml. During the last 6 hours, cells were pulsed with tritiated thymidine prior to harvesting. All determinations were made in triplicate.
  • Relative cell viability was calculated as follows: thymidine incorporation in treated cells over thymidine incorporation in untreated cells multiplied by 100.
  • Figure 6 shows the effect of curcumin on the growth of human glioblastoma U-251 cells (Figure 6A) and on human vascular endothelial cells ( Figure 6B) .
  • Figure 7 shows the additive effects of curcumin and TNF on the growth of human histiocytic ly phoma cell line U- 937.
  • Cells were incubated with either TNF (100 units/ml) or curcumin (1 ug/ml) or both for 72 hours.
  • Figure 8 shows that a continuous presence of curcumin is needed for the growth of human breast adenocarcinoma tumor cells (MCF-7) .
  • Figure 9 shows the effect of curcumin on the activities of various protein kinases.
  • FIG 10 shows the dose response of phosphorylase kinase with curcumin.
  • phosphorylase kinase (134 units/ml) was assayed with phosphorylase b in presence of the indicated concentrations of curcumin.
  • An identical set of incubations were terminated with laemmli sample buffer instead of trichloroacetic acid and subjected to SDS-PAGE. The protein bands were then stained with coomassie brilliant blue and the gels dried.
  • radiolabeled phosphorylase b was detected by autoradiography of the dried gel.
  • Figure IIA shows the Lineweaver-Burke plot analysis of the Inhibition of phosphorylase kinase by curcumin.
  • the incubations contained various concentrations of curcumin as indicated. With each set of curcumin concentrations, the concentration of phosphorylase b was varied. The other two substrates, i.e., Mg 2+ and ATP were present at saturating levels (2 mM and 0.2 mM respectively). The rates of each reaction were calculated as pmol of 32 P incorporated into phosphorylase b per minute.
  • the reciprocal plot was graphed against the relevant concentrations of phosphorylase b as a Lineweaver-Burke plot.
  • Figure 11B shows the slopes of the lines derived from the double reciprocal plot plotted against the relevant concentrations of curcumin in order to derive the K j value for curcumin.
  • Figure 12 shows the structure of two flavonoid compounds useful in the methods of the present invention.
  • the present invention is directed to a method for the treatment of pathological cell proliferative diseases comprising administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof or a flavonoid.
  • the method of present invention may be used to treat either neoplastic diseases and non-neoplastic diseases.
  • neoplastic diseases are ovarian cancer, bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, colon cancer, carcinoma of the kidney and pancreatic cancer.
  • non-neoplastic diseases are selected from the group consisting of psoriasis, benign proliferative skin diseases, ichthyosis, papilloma, basal cell carcinoma, squamous cell carcinoma, restinosis, scleroderma and hemangioma.
  • the methods of the present invention may be used to treat any animal. Most preferably, the methods of the present invention are useful in human.
  • the curcumin and curcumin analogues may be administered in any pharmacologically effective dose.
  • the curcumin and curcumin analogues are administered in a dose of from about 1 microgram to about 100 milligram.
  • curcumin analogues are effective in the methods of the present invention.
  • Representative examples of curcumin analogues are compounds such as: (a) ferulic acid, i.e., 4-hydroxy-3-methoxycinnamic acid (compound #1) and related compounds such as 3 ,4-methylenedioxy cinnamic acid (compound #2) and 3, 4-dimethoxycinnamic acid (compound #3); (b) aromatic ketones, such as 4-(4-hydroxy-3- methoxyphenyl) -3-buten-2-one (compound #4) , zingerone (compound #5) , 4-(3 ,4-methylenedioxyphenyl)-2-butanone (compound #6) , 4-(p-hydroxyphenyl) -3-buten-2-one (compound #7) , 4 -hydroxyva 1 erophenone (compound #8) , 4-hydroxybenzylactone (compound #9) , 4-hydroxybenzophenone (compound #10)
  • Figure 1 shows the structure of these curcumin analogues Representative examples of flavanoids are shown by structures 21 and 22 in Figure 12.
  • the present invention also provide ⁇ a novel method of inhibiting the activity of phosphorylase kinase activity in an animal. This novel method comprises administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof.
  • the present invention also provides a novel method of inhibiting the activity of tyrosine kinase activity in an animal.
  • This novel method comprises administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof.
  • the term "individual” is meant to include animals and humans.
  • biologically inhibiting or “inhibition” of the growth of proliferating cells is meant to include partial or total growth inhibition and also is meant to include decreases in the rate of proliferation or growth of the cells.
  • the biologically inhibitory dose of the composition of the present invention may be determined by assessing the effects of the test element on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell culture or any other method known to those of ordinary skill in the art.
  • compositions of the present invention may be by topical, intraocular, parenteral, oral, intranasal, intravenous, intramuscular, subcutaneous, or any other suitable means.
  • the dosage administered is dependent upon the age, clinical stage and extent of the disease or genetic predisposition of the individual, location, weight, kind of concurrent treatment, if any, and nature of the pathological or malignant condition.
  • the effective delivery system useful in the method of the present invention may be employed in such forms as capsules, tablets, liquid solutions, suspensions, or elixirs, for oral administration, or sterile liquid forms such as solutions, suspensions or emulsions.
  • Any inert carrier is preferably used, such as saline, or phosphate-buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties.
  • delivery systems useful in the method of the present invention may be employed in such sterile liquid forms such as solutions, suspensions or emulsion ⁇ .
  • sterile liquid forms such as solutions, suspensions or emulsion ⁇ .
  • topical use it may be employed in such forms as ointments, creams or sprays.
  • Any inert carrier is preferably used, such as saline, or phosphate-buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties.
  • saline or phosphate-buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties.
  • pathological cancerous and noncancerous cell proliferative conditions for which the compositions and methods of the present invention will provide therapeutic benefits. These pathological conditions may occur in almost all cell types capable of abnormal cell proliferation.
  • the method of the present invention is useful in treating local or disseminated pathological conditions in all or almost all organ and tissue systems of the individual.
  • Purified curcumin inhibited the growth of a wide variety of human tumor cells including myeloid and lymphocytic leukemia, breast carcinoma and lung carcinoma (Table I) . While all breast tumor cell lines examined were highly sensitive to curcumin, other cell types such as kidney, hepatic, and certain epithelial cell types, were resistant.
  • Hydroxycinnamic acids are synthesized via a phase transfer catalyzed Wittig-Horner reaction of acetylated hydroxy aromatic aldehydes with triethylphosphonoacetate. The corresponding saturated analogs are obtained by hydrogenation of the cinnamic acids.
  • Conjugated carbonyl compounds are synthesized by aldol condensation involving reactions on a solid support. This method is adaptable to the synthesis of compounds with sensitive functionality such as carbomethoxy group. Morphologically, most cells are killed by two distinct mechanism, viz; apoptosis and necrosis. Apoptosis is generally characterized as a programmed cell death resulting in membrane blebbing, nuclear condensation, and fragmentation of DNA into 200-bp fragments wherea ⁇ necrotic cell death involves swelling, dissolution of cellular components, and random DNA fragmentation.
  • curcumin had a strong antiproliferative effect on myeloid cells, particularly the promyelocytic Hl-60 and ML-1 cell lines and myelogenous cell lines. Curcumin inhibited B Cell and T Cell Lymphoma cell lines and strongly inhibited brea ⁇ t cell line ⁇ and the lung cell line, A 549. In contrast, the Burkitt lymphoma (Raji) cell line, embryonal kidney (A293 LT) cell line, the epithelioid (HeLa) cell line and the hepatocellular (Hep G2) cell line was not affected by curcumin.
  • Raj Burkitt lymphoma
  • A293 LT embryonal kidney
  • HeLa epithelioid
  • Hep G2 hepatocellular
  • Histiocytic Lymphoma (U-937) 70 ⁇ 4 54
  • B Cell Lymphoma Burkitt Lymphoma (Daudi) 45 ⁇ 5 ND
  • Figures 2A and 2B show the dose response inhibition by curcumin of the growth of hormone-dependent human breast adenocarcinoma tumor cells (MCF-7 cells) and T-47D cells, respectively. For both types of cells, a dose of 1 ug/ml showed almost total inhibition.
  • Figures 3A and 3B show the time course of the antiproliferative effect of curcumin on the growth of MCF-7 cells.
  • Figure 3A shows that a dose of 1 ug/ml of curcumin inhibited the growth by about 80%.
  • Figure 3B shows that the time course of curcumin's effect by thymidine incorporation.
  • Figures 4A and 4B show the dose response and time course of the antiproliferative effect of curcumin on hormone independent human breast tumor cell, SK-BR3 and BT-20, respectively.
  • Figure 5A and 5B show the dose response and time course of the antiproliferative effect of curcumin on promyelomonocytic HL-60 cells.
  • Figure 5A shows that a dose of 1 ug/ml inhibited the growth of HL-cells by about 90%.
  • Figures 6A and 6B show the antiproliferative effects of curcumin on glioblastoma (U251) cells and human vascular endothelial cells (HUVEC) , respectively.
  • Figure 6A shows that a dose of about 2 ug/ml inhibited the growth of U-251 cells by about 60% while Figure 6B show ⁇ that a do ⁇ e of curcumin of about 2 ug/ml inhibited the do ⁇ e of HUVEC cell ⁇ by about 40%.
  • Figure 7 ⁇ how ⁇ the effect of 1 ug/ml of curcumin or
  • ferulic acid and related compounds had a strong antiproliferative effect on promyelocytic cells (HL-60 and ML-1) .
  • ferulic acid inhibited the breast tumor cell lines (BT-20 and T-47D) , the hepatocellular (Hep G2) cell line and the embryonal kidney (A293 LT) cell line.
  • THP-1 Promonocytic (THP-1) 138 95 114 B Cell Lymphoma :
  • Burkitt Lymphoma (Daudi) 184 136 128
  • the aromatic ketones inhibited the promyelocytic (HL-60 and ML-1) cell lines and the myelogenous (KG-1 and KG-la) cell lines. Moreover, antiproliferative effects of the aromatic ketones were seen on the Burkitt Lymphoma (Raji) cell line and the Breast tumor (BT-20, T47D and SK-BR3) cell lines.
  • Aromatic Diketone fCompound 12 Antiproliferative Effect of Aromatic Diketone fCompound 12) on Tumor Cell Lines Cell Line ⁇ Relative Cell Viability (%)
  • HeLa Epithelioid
  • Hepatocellular Hep G2 99 Lung (A549)
  • caffeic acid had a weaker antiproliferative effect on most tumor cell lines than other curcumin analogs.
  • the tumor cell line most sensitive to caffeic acid was the T Cell Lymphoma (Jurkat) cell line.
  • Burkitt Lymphoma (Raji) 96 T Cell Lymphoma : (Jurkat) 48 Epithelial Cells:
  • HeLa Epithelioid
  • Hep G2 Hepatocellular
  • Table VI show ⁇ that cinnamic acid had an antiproliferative effect on T cell lymphoma (Jurkat) cell ⁇ , breast tumor (BT-20 and T-47D) cells, the hepatocellular
  • Hep G2 tumor cell line
  • D407 retinal pigment
  • Myeloid Cells Promonocytic(ML-1) 84
  • T Cell Lymphoma (Jurkat) 36 Epithelial Cells:
  • Table VII shows that aromatic carboxylic acids had antiproliferative effects on promyelocytic (ML-1 and HL-60) cells, myelogenous (KG-1) cells and T cell lymphoma cell ⁇ . In addition, aromatic carboxylic acid ⁇ inhibited embryonal kidney (A293 LT) cell, retinal pigment (D407) cell ⁇ and Breast (BT-20) tumor cell lines.
  • Promonocytic (ML-1) 68 74 80 80 80 Myelogenous (KG-1) 71 120 115 140 Promyelocytic (HL-60) 83 88 82 74 Myelogenous (KG-la) 114 128 105 126 Promonocytic (THP-1) 117 104 110 98
  • HeLa Epithelioid
  • Hep G2 Hepatocellular
  • Hep G2 Hepatocellular
  • A549 194 251 229 329
  • Table VIII shows that aromatic ketocarboxylic acids inhibited promyelocytic (HL-60 and THP-1) tumor cell lines. In addition, aromatic ketocarboxylic acids inhibited T cell lymphoma tumor cells and retinal pigment (D407) cells.
  • Table IX indicate ⁇ the antiproliferative effect ⁇ of aromatic alcohol ⁇ on promyelocytic (HL-60) tumor cell line.
  • aromatic ketocarboxylic acid ⁇ inhibited T cell lymphoma tumor cells and retinal pigment (D407) cells.
  • Table X show ⁇ that flavanoid ⁇ exhibited antiproliferative effect ⁇ on promyelocytic (HL-60 and ML-1) cell ⁇ , myelogenou ⁇ (KG-1 and KG-la) cell ⁇ , hepatocellular (Hep G2) cells, breast tumor cell lines (BT-20, T-47D and MCF-7) and embryonal kidney (A293 LT) cells.
  • Cytosolic protamine kinase and autophosphorylation- activated protein kina ⁇ e were obtained from Dr. Z. Damuni, Department of Biological Science ⁇ , Columbia, South Carolina. These protein kina ⁇ e preparations were judged to be homogeneous based on SDS-PAGE and gel permeation chromatography.
  • Phosphorylase kinase (170 units/mg)
  • phosphorylase b catalytic subunit of protein kinase A (41 units/mg) , Hi ⁇ tone H-l and H-2B
  • protamine sulfate ( ⁇ almine) protamine sulfate
  • myelin basic protein curcumin
  • phosphatidyl-L-serine were obtained from Sigma Chemical Co.
  • Protein kinase C (1200 units/mg) was obtained from Calbiochem Corp.
  • gamma [32 P] ATP was obtained from ICN Biomedical ⁇ , Inc.
  • Protein kina ⁇ e C, protein kinase A, and cytosolic protamine kina ⁇ e were a ⁇ sayed as described by Damuni et al., 1989, Purification and properties of a distinct protamine kinase from the cytosol of bovine kidney cortex. J. Biol . Chem . 264, 6412-6416 with modifications.
  • the assay ⁇ were performed in 0.05 ml mixture ⁇ containing 25 mM Tri ⁇ -HCl, pH 7.3, 10% glycerol, 1 mM Benzamidine, 14 mM B- mercaptoethanol, 0.2 mM phenylmethyl sulfonyl fluoride, 100 ug/ml leupeptin, 4 uM microcystin LR, 2 ug/ml aprotinin, protein kinase, 50 ug histone H-l (PKC) or histone H-2B or 100 ug protamine sulfate, 10 mM MgCl 2 , and 0.2 mM [gamma- 32 P] ATP (200-500 cpm/pmol) .
  • the reaction was initiated by adding MgCl 2 and ATP. After 10 minutes of incubation at 37°C, the reaction was terminated by the addition of 1 ml of 10% trichloroacetic acid (TCA) . Protein in the TCA terminated mixtures was pelleted by centrifugation for 2 minutes in a Beckman centrifuge at 15,000 x g. The pellet wa ⁇ washed five times with TCA, added one ml scintillant and counted for radioactivity in a Packcard liquid scintillation counter. Control tubes were treated in an identical manner except that protein kinase was excluded from the mixture.
  • TCA trichloroacetic acid
  • Protein kinase C was assayed as described above except that the incubation mixture also included 0.5 mM CaCl 2 and u40 ug/ml phosphatidyl- L-serine. Phosphorylase kinase was similarly assayed with the following modifications.
  • the assay mixture contained 25 mM Tris-HCl, pH 7.3, 10% glycerol, 1 mM Benzamidine, 14 mM B- ercaptoethanol, 0.2 mM phenylmethyl sulfonyl fluoride, 100 ug/ml leupeptin, 4 uM microcystin LR, 2 ug/ml aprotinin and a protein kinase containing 0.5 mM CaCl 2 . Following incubation for 10 minutes at 37°C, the reaction was terminated with 1 ml of 10% TCA and treated as described above.
  • One unit of protein kinase activity was defined as the amount of enzyme that incorporated 1 nmol of phosphoryl groups into substrate/min. To ensure linearity the extent of incorporation of phosphoryl group ⁇ wa ⁇ limited to ⁇ 1 nmol.
  • Sodium-Dodecyl Gel Electrophoresis Polyacrylamide slab gels (12%) were run in a Biorad protein ® II apparatus at 200 volts con ⁇ tant voltage. Protein band ⁇ were detected by staining with coomassie brilliant blue, dried and autoradiographed.
  • the incubation mixture contained 25 M tris-HCl, pH 7.3, 10% glycerol, 1 mM benzamidine, 14 mM ⁇ - mercaptoethanol, 0.2 mM phenylmethyl ⁇ ulphonyl fluoride, 100 ⁇ g/ml leupeptin, 2 ⁇ g/ml aprotinin, protein kina ⁇ e, 50 ⁇ g polyglutamic acid-tyrosine (4:1) , 10 mM MgCl 2 and 0.2 mM[gamma- 32 P]-ATP (200-500 cpm/pmol) .
  • FIG. 9 illustrates that curcumin inhibited all the kinases examined but to different degrees.
  • Curcumin inhibited by 98%, 40%, 15%, 10%, 1% and 0.5%, respectively.
  • the inhibitory effect was dose-dependent.

Abstract

The present invention provides novel methods for the treatement of pathological cell proliferative diseases. The novel methods of the present invention comprise administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof. The present invention also provides a novel method of inhibiting the activity of phosphorylase kinase and tyrosine kinase and a novel method of treating cell proliferative diseases by administering a pharmacologically effective dose of a flavonoid.

Description

CURCUMIN, ANALOGUES OF CURCUMIN
AND NOVEL USES THEREOF
Background of the Invention
Field of the Invention
The present invention relates generally to the field of cell proliferative diseases. More specifically, the present invention relates to novel antiproliferative effects of curcumin and analogues thereof. Description of the Related Art
Curcumin (diferuloylmethane) is a major active component of the food flavor turmeric (Curcuma longa) . Previously known properties of curcumin in animals include inhibition of both tumor initiation induced by benzo-alpha- pyrene and 7, 12 dimethylbenz-alpha-anthracene and tumor promotion induced by phorbol ester. In addition, curcumin exhibits anti-inflammatory properties in vivo . The pharmacological safety of curcumin is demonstrated by the consumption up to 100 g/day. In vitro , curcumin inhibits neutrophil activation, suppresses mitogen-induced proliferation of blood mononuclear cells, inhibits the mixed lymphocyte reaction, and inhibits proliferation of smooth muscle cells. Curcumin is also a potent scavenger of reactive oxygen species, protects hemoglobin from nitrite-induced oxidation to methe oglobin and inhibits lipid peroxidation. Some of these activities may be responsible for curcumin's ability to protect DNA from free radical-induced damage and hepatocytes against various toxins. In addition, the phorbol ester-induced transcriptional factors c-jun/AP-1 are suppressed by curcumin. Recently curcumin has been shown to be highly effective in inhibiting the type 1 human immunodeficiency virus long terminal repeat-directed gene expression and virus replication.
In the field of chemotherapy for cancer and other cell proliferative diseases, there remains the need and desire in the art for safe, non-toxic and orally effective pharmacological agents. The present invention fulfills this longstanding deficiency in the prior art.
Summary of the Invention In one embodiment of the present invention, there is provided a method for the treatment of pathological cell proliferative diseases comprising administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof. In another embodiment of the present invention, there is provided a method of inhibiting the activity of phosphorylase kinase comprising administration to an animal of a pharmacologically effective doεe of curcumin or an analogue thereof. In another embodiment of the present invention, there is provided a method of inhibiting the activity of tyrosine kinase comprising administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof. In another embodiment of the present invention, there is provided a method for the treatment of pathological cell proliferative diseases comprising administration to an animal of a pharmacologically effective dose of a flavonoid or an analogue thereof. Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure. Brief Description of the Drawings
Figure 1 shows the structure of curcumin and related analogues.
Figure 2 shows the dose response curve of curcumin on the growth of hormone-dependent human breast adenocarcinoma tumor cells (Figure 2A; MCF-7 cells) and (Figure 2B; T-47D cells) . 5 x IO3 cells were plated in 96-well plates overnight at 37° C. The cells were then incubated with either variable concentrations of curcumin (left panel) for 72 hours or for variable times (right panel) with curcumin (2.7 uM or 1 ug/ml) in a total final volume of 0.2 ml. During the last 6 hours, cells were pulsed with tritiated thymidine prior to harvesting. All determinations were made in triplicate. Relative cell viability was calculated as follows: thymidine incorporation in treated cells over thymidine incorporation in untreated cells multiplied by 100.
Figure 3 shows the time course of the effect of curcumin on the growth of human breast adenocarcinoma cells, MCF-7. 5 x 103 cells were plated in 96-well plates overnight at 37° C , washed and then incubated with different concentrations of curcumin for different times. Viability of cells was examined either by thymidine incorporation (Figure 3A) or by counting viable cell number (Figure 3B) .
Figure 4 shows the dose response (Figure 4A) and time course (Figure 4B) of effect of curcumin on the growth of hormone-independent human breast tumor cells.
Figure 5 shows the dose response (Figure 5A) and the time course (Figure 5B) of curcumin on the growth of human promyelo onocytic tumor cells, HL-60. 5 x 103 cells were plated in 96-well plates overnight at 37° C. The cells were then incubated with either variable concentrations of curcumin (left panel) for 72 hours or for variable times (right panel) with curcumin (2.7 uM or 1 ug/ml) in a total final volume of 0.2 ml. During the last 6 hours, cells were pulsed with tritiated thymidine prior to harvesting. All determinations were made in triplicate. Relative cell viability was calculated as follows: thymidine incorporation in treated cells over thymidine incorporation in untreated cells multiplied by 100. Figure 6 shows the effect of curcumin on the growth of human glioblastoma U-251 cells (Figure 6A) and on human vascular endothelial cells (Figure 6B) .
Figure 7 shows the additive effects of curcumin and TNF on the growth of human histiocytic ly phoma cell line U- 937. Cells were incubated with either TNF (100 units/ml) or curcumin (1 ug/ml) or both for 72 hours.
Figure 8 shows that a continuous presence of curcumin is needed for the growth of human breast adenocarcinoma tumor cells (MCF-7) . Figure 9 shows the effect of curcumin on the activities of various protein kinases. Preparations of phosphorylase kinase (Phos K, 134 units/ml) , protein kinase C (PkC, 6.8 units/ml), protein kinase A catalytic subunit (PkA, 5 units/ml) , cytosolic protamine kinase (cPK, 500 units/ml) , autophosphorylation-activated kinase (AK, 500 units/ml) and cellular tyrosine kinase(pp60c"src; 8 units/ml) were assayed with phosphorylase b, histone H-l, histone H-2B, protamine sulfate, myelin basic protein and poly glutamic acid-tyrosine, respectively, as substrates in the presence of the indicated concentrations of curcumin. Controls were treated in an identical manner except that dimethylsulfoxide was substituted for curcumin.
Figure 10 shows the dose response of phosphorylase kinase with curcumin. In Figure 10A, phosphorylase kinase (134 units/ml) was assayed with phosphorylase b in presence of the indicated concentrations of curcumin. An identical set of incubations were terminated with laemmli sample buffer instead of trichloroacetic acid and subjected to SDS-PAGE. The protein bands were then stained with coomassie brilliant blue and the gels dried. In Figure 10B, radiolabeled phosphorylase b was detected by autoradiography of the dried gel.
Figure IIA shows the Lineweaver-Burke plot analysis of the Inhibition of phosphorylase kinase by curcumin. The incubations contained various concentrations of curcumin as indicated. With each set of curcumin concentrations, the concentration of phosphorylase b was varied. The other two substrates, i.e., Mg2+ and ATP were present at saturating levels (2 mM and 0.2 mM respectively). The rates of each reaction were calculated as pmol of 32P incorporated into phosphorylase b per minute. The reciprocal plot was graphed against the relevant concentrations of phosphorylase b as a Lineweaver-Burke plot. Figure 11B shows the slopes of the lines derived from the double reciprocal plot plotted against the relevant concentrations of curcumin in order to derive the Kj value for curcumin.
Figure 12 shows the structure of two flavonoid compounds useful in the methods of the present invention. Detailed Description of the Invention The present invention is directed to a method for the treatment of pathological cell proliferative diseases comprising administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof or a flavonoid. The method of present invention may be used to treat either neoplastic diseases and non-neoplastic diseases. Representative examples of neoplastic diseases are ovarian cancer, bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, gliomas, fibrosarcomas, retinoblastomas, melanomas, soft tissue sarcomas, osteosarcomas, colon cancer, carcinoma of the kidney and pancreatic cancer.
Representative examples of non-neoplastic diseases are selected from the group consisting of psoriasis, benign proliferative skin diseases, ichthyosis, papilloma, basal cell carcinoma, squamous cell carcinoma, restinosis, scleroderma and hemangioma.
The methods of the present invention may be used to treat any animal. Most preferably, the methods of the present invention are useful in human.
Generally, to achieve the antiproliferative or phosphorylase kinase inhibitory effectε, the curcumin and curcumin analogues may be administered in any pharmacologically effective dose. Preferably, the curcumin and curcumin analogues are administered in a dose of from about 1 microgram to about 100 milligram.
A wide variety of curcumin analogues are effective in the methods of the present invention. Representative examples of curcumin analogues are compounds such as: (a) ferulic acid, i.e., 4-hydroxy-3-methoxycinnamic acid (compound #1) and related compounds such as 3 ,4-methylenedioxy cinnamic acid (compound #2) and 3, 4-dimethoxycinnamic acid (compound #3); (b) aromatic ketones, such as 4-(4-hydroxy-3- methoxyphenyl) -3-buten-2-one (compound #4) , zingerone (compound #5) , 4-(3 ,4-methylenedioxyphenyl)-2-butanone (compound #6) , 4-(p-hydroxyphenyl) -3-buten-2-one (compound #7) , 4 -hydroxyva 1 erophenone (compound #8) , 4-hydroxybenzylactone (compound #9) , 4-hydroxybenzophenone (compound #10) , 1, 5-biε(4-dimethyla inophenyl) -1,4-pentadien- 3-one (compound #11) ; (c) aromatic diketones such as 6-hydroxydibenzoylmethane (compound #12) ; (d) caffeic acid compounds such as 3, 4-dihydroxycinnamic acid (compound #13); (e) cinnamic acid (compound #14) ; (f) aromatic carboxylic acids, such as 3 , 4-dihydroxyhydrocinnamic acid (compound #15) , 2-hydroxycinnamic acid (compound #16) , 3-hydroxycinnamic acid (compound #17) and 4-hydroxycinnamic acid (compound #18) ; (g) aromatic ketocarboxylic acids such as 4-hydroxyphenylpyruvic acid (compound #19) ; (h) aromatic alcohols such as 4- hydroxyphenethyl alcohol (compound #20) . Figure 1 shows the structure of these curcumin analogues Representative examples of flavanoids are shown by structures 21 and 22 in Figure 12. The present invention also provideε a novel method of inhibiting the activity of phosphorylase kinase activity in an animal. This novel method comprises administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof.
The present invention also provides a novel method of inhibiting the activity of tyrosine kinase activity in an animal. This novel method comprises administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof.
The term "individual" is meant to include animals and humans.
The term "biologically inhibiting" or "inhibition" of the growth of proliferating cells is meant to include partial or total growth inhibition and also is meant to include decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose of the composition of the present invention may be determined by assessing the effects of the test element on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell culture or any other method known to those of ordinary skill in the art.
Administration of the compositions of the present invention may be by topical, intraocular, parenteral, oral, intranasal, intravenous, intramuscular, subcutaneous, or any other suitable means. The dosage administered is dependent upon the age, clinical stage and extent of the disease or genetic predisposition of the individual, location, weight, kind of concurrent treatment, if any, and nature of the pathological or malignant condition. The effective delivery system useful in the method of the present invention may be employed in such forms as capsules, tablets, liquid solutions, suspensions, or elixirs, for oral administration, or sterile liquid forms such as solutions, suspensions or emulsions. Any inert carrier is preferably used, such as saline, or phosphate-buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties.
Preferably, delivery systems useful in the method of the present invention may be employed in such sterile liquid forms such as solutions, suspensions or emulsionε. For topical use it may be employed in such forms as ointments, creams or sprays. Any inert carrier is preferably used, such as saline, or phosphate-buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties. There are a wide variety of pathological cancerous and noncancerous cell proliferative conditions for which the compositions and methods of the present invention will provide therapeutic benefits. These pathological conditions may occur in almost all cell types capable of abnormal cell proliferation. Among the cell types which exhibit pathological or abnormal growth are (1) fibroblasts, (2) vascular endothelial cells and (3) epithelial cells. It can be seen from the above that the method of the present invention is useful in treating local or disseminated pathological conditions in all or almost all organ and tissue systems of the individual. Purified curcumin inhibited the growth of a wide variety of human tumor cells including myeloid and lymphocytic leukemia, breast carcinoma and lung carcinoma (Table I) . While all breast tumor cell lines examined were highly sensitive to curcumin, other cell types such as kidney, hepatic, and certain epithelial cell types, were resistant. The comparison of the antiproliferative effects of curcumin with tumor necrosis factor (TNF) , a cytokine produced primarily by the cells of the immune system, showed that curcumin at 1 ug/ml was at least as effective as TNF at 0.2 ug/ml (10,000 units/ml) (Table I). A human promyelomonocytic cell line, HL-60, which is highly resistant to TNF, was found to be also sensitive to curcumin. Structural Analogues of Curcumin Structural analogues of curcumin are derivatized.
Hydroxycinnamic acids are synthesized via a phase transfer catalyzed Wittig-Horner reaction of acetylated hydroxy aromatic aldehydes with triethylphosphonoacetate. The corresponding saturated analogs are obtained by hydrogenation of the cinnamic acids. Conjugated carbonyl compounds are synthesized by aldol condensation involving reactions on a solid support. This method is adaptable to the synthesis of compounds with sensitive functionality such as carbomethoxy group. Morphologically, most cells are killed by two distinct mechanism, viz; apoptosis and necrosis. Apoptosis is generally characterized as a programmed cell death resulting in membrane blebbing, nuclear condensation, and fragmentation of DNA into 200-bp fragments whereaε necrotic cell death involves swelling, dissolution of cellular components, and random DNA fragmentation.
The following examples are provided for the sole purpose of illustrating various embodiments of the present invention and are not meant to limit the present invention in any fashion.
EXAMPLE 1 Antiproliferative Effects of Curcumin
5 x 103 cells were plated overnight at 37°C and then incubated with curcumin (2.7 uM) or TNF (0.2 ug/ml). After 66 hours at 37°C, cells were pulsed with tritiated thymidine for 6 hours prior to harvesting. All determinations were made in triplicate.
As shown in Table I, curcumin had a strong antiproliferative effect on myeloid cells, particularly the promyelocytic Hl-60 and ML-1 cell lines and myelogenous cell lines. Curcumin inhibited B Cell and T Cell Lymphoma cell lines and strongly inhibited breaεt cell lineε and the lung cell line, A 549. In contrast, the Burkitt lymphoma (Raji) cell line, embryonal kidney (A293 LT) cell line, the epithelioid (HeLa) cell line and the hepatocellular (Hep G2) cell line was not affected by curcumin.
TABLE I Antiproliferative Effects of Curcumin and TNF
Relative Cell Viability (% of Control) Cell Line Curcumin TNF
Mγeloid Cells:
Promyelocytic (HL-60) 10 ± 0 100
Promonocytic (ML-1) 33 + 1 59 Myelogenous (KG1) 44 + 1 43
Myelogenous (KG-la) 46 ± 3 60
Histiocytic Lymphoma (U-937) 70 ± 4 54
Promonocytic (THP-1) 86 ± 4 81
B Cell Lymphoma : Burkitt Lymphoma (Daudi) 45 ± 5 ND
Burkitt Lymphoma (Raji) 100 + 3 76
T Cell Lγmphoma (Jurkat) 23 + 1 7
Epithelial Cells:
Breast (BT-20) 1 + 0 24 Breast (BT-20 TNF R) 8 ±_ _0 60
Breast (SK-BR3) 6 ± 1 52
Breast (MCF-7) 9 ± 1 1
Breast (T-47 D) 13 ± 0 70
Breast (ZR-75-1) 26 ± 1 6 Lung adenocarcinoma (A549) 16 ± 1 ND
Small cell lung carcinoma (H596) 63 + 2 ND
Retinal Pigment (D407) 41 + 4 ND
Embryonal Kidney (A293-LT) 100 + 3 ND Epithelioid (HeLa) 101 ± 5 55
Hepatocellular (Hep G2) 138 + 2 83
Melanoma Cells:
Tumorigenic & metastatic (SB-CL-1) 3 + 1 ND Nontumorigenic & nonmetastatic
(SB-CL-2) 2 ± 1 ND
Tumorigenic but nonmetastatic
(SB-CL-3) 1 + 1 ND
Tumorigeni./ . eta. adrenals (SB-CL-1A) 3 + 1 ND
Tumorigen. /meta. brain (SB-CL-1B) 1 + 1 ND
Glioblastoma Cells:
Glial (U-251) 103 + 3 ND
Normal Cells: Human umbilical vein endothelial cells 89 + 3 ND
Bovine arterial endothelial cells 94 + 9 20
Human foreskin fibroblasts 55 + 7 311
Figures 2A and 2B show the dose response inhibition by curcumin of the growth of hormone-dependent human breast adenocarcinoma tumor cells (MCF-7 cells) and T-47D cells, respectively. For both types of cells, a dose of 1 ug/ml showed almost total inhibition.
Figures 3A and 3B show the time course of the antiproliferative effect of curcumin on the growth of MCF-7 cells. Figure 3A shows that a dose of 1 ug/ml of curcumin inhibited the growth by about 80%. Figure 3B shows that the time course of curcumin's effect by thymidine incorporation.
Figures 4A and 4B show the dose response and time course of the antiproliferative effect of curcumin on hormone independent human breast tumor cell, SK-BR3 and BT-20, respectively.
Figure 5A and 5B show the dose response and time course of the antiproliferative effect of curcumin on promyelomonocytic HL-60 cells.
Figure 5A shows that a dose of 1 ug/ml inhibited the growth of HL-cells by about 90%.
Figures 6A and 6B show the antiproliferative effects of curcumin on glioblastoma (U251) cells and human vascular endothelial cells (HUVEC) , respectively.
Figure 6A shows that a dose of about 2 ug/ml inhibited the growth of U-251 cells by about 60% while Figure 6B showε that a doεe of curcumin of about 2 ug/ml inhibited the doεe of HUVEC cellε by about 40%. Figure 7 εhowε the effect of 1 ug/ml of curcumin or
100 units/ml of TNF both inhibited the growth of the U-9371 cell line. However, as seen by Figure 7, curcumin and TNF together exhibited a synergistic antiproliferative effect by inhibiting about 95% of the growth of these cells. Figure 8 showε that curcumin must be present for several hours before its antiproliferative effects are seen.
EXAMPLE 2 Antiproliferative Effects of Ferulic Acid
For the studies shown in Table II, 5xl03 cells were plated overnight at 37°C and then incubated with ferulic acid (lug/ml) . After 68 h at 37°C, cells were pulsed with thymidine for 6 hourε prior to harveεting. Thymidine incorporation by untreated cellε was expreεεed as 100%. All determinations were made in triplicate. The variation between the triplicate was less then 10%.
As is shown in Table II, ferulic acid and related compounds had a strong antiproliferative effect on promyelocytic cells (HL-60 and ML-1) . In addition, ferulic acid inhibited the breast tumor cell lines (BT-20 and T-47D) , the hepatocellular (Hep G2) cell line and the embryonal kidney (A293 LT) cell line.
TABLE II
Antiproliferative Effect of Ferulic acid on Tumor Cell Lines
Cell Lines Relative Cell Viability ( )
Compound# 2
Myeloid Cells:
Promyelocytic (HL-60) 37 79 Promonocytic (ML-1) 72 84 92
Myelogenous (KG-la) 86 79 90
Histiocytic Lymphoma (U-937) 108 90 111
Myelogenous (KG-1) 113 96 103
Promonocytic (THP-1) 138 95 114 B Cell Lymphoma :
Burkitt Lymphoma ( Raj i ) 126 112 85
Burkitt Lymphoma (Daudi) 184 136 128
T Cell Lymphoma: (Jurkat) 78 37 49
Epithelial Cells: Breast (BT-20) 36 49 32
Breast (T-47 D) 77 68 55
Breast (MCF-7) 102 108 115
Breast (SK-BR3) 129 124 86
Hepatocellular (Hep G2) 71 90 85 Epithelioid (HeLa) 109 95 100
Embryonal Kidney (A293 LT) 77 64 87
Retinal Pigment (D407) 116 72 85
Lung (A549) 131 88 88
EXAMPLE 3 Antiproliferative effectε of aromatic ketoneε
In the experi entε illuεtrated in Table III, 5xl03 cells were plated overnight at 37°C and then incubated with aromatic ketones (1 ug/ml) . After 68 hours at 37°C, cells were pulsed with tritiated thymidine for 6 hours prior to harvesting. Thymidine incorporation by untreated cells waε expreεεed as 100%. All determinations were made in triplicate. The variation between the triplicate was less then 10%.
As is shown in Table III, the aromatic ketones inhibited the promyelocytic (HL-60 and ML-1) cell lines and the myelogenous (KG-1 and KG-la) cell lines. Moreover, antiproliferative effects of the aromatic ketones were seen on the Burkitt Lymphoma (Raji) cell line and the Breast tumor (BT-20, T47D and SK-BR3) cell lines.
TABLE III Antiproliferative Effect of Aromatic Ketones on Tumor Cell Lines
Cell Lines Relative Cell Viability (%)
Compounds 6 7 8 9 10 11 Myeloid Cells: Promyelocytic (HL-60) 22 128 115 117 108 121 76 88 Myelogenous (KG-1) 56 133 128 120 131 136 105 129 Myelogenous (KG-la) 65 104 118 113 119 121 113 121 Promonocytic (ML-1) 72 96 94 71 101 120 83 86 Promonocytic (THP-1) 109 104 114 105 132 130 85 114 Histiocytic Lym. (U-937) 115 104 100 121 91 94 129 143 fl Cell Lymphoma : Burkitt Lymphoma (Raji) 63 126 111 112 117 75 195 193 Burkitt Lymphoma (Daudi) 111 159 176 229 166 151 144 175 T Cell Lymphoma:
(Jurkat) 74 66 55 48 57 48 55 60 Epithelial Cells: Breast (BT 20) 25 11 27 37 18 27 103 119 Breast (T47 D) 36 51 51 50 57 51 - - Breast (SK-BR3) 39 26 49 131 91 80 - - Breast (MCF-7) 117 100 111 89 104 122 110 83 Hepato- (Hep G2) 83 144 109 186 144 99 105 93 Epithelioid (HeLa) 97 97 96 99 98 103 95 93 Embryonal Kidney
(A293 LT) 98 50 100 84 70 94 129 115 Lung (A549) 134 29 49 103 115 116 489 325 Retinal Pigment (D407) 229 195 235 93 63 83 87 76 EXAMPLE 4
Antiproliferative Effects of Aromatic Diketones
In the experiments shown in Table IV, 5xl03 cells were plated overnight at 37°C and then incubated with aromatic diketone (lug/ml) . After 68 hours at 37°C, cells were pulsed with thymidine for 6 hours prior to harvesting. Thymidine incorporation by untreated cells was expressed as 100%. All determinations were made in triplicate. The variation between the triplicate was leεε then 10%. As shown in Table IV, the aromatic diketone showed a strong antiproliferative effect on all myeloid tumor cell lines tested except the Histiocytic Lymphoma (U-937) cell line. In addition, the aromatic ketone inhibited B cell and T Cell lymphoma tumor cell lineε and the Breaεt tumor cell lineε with the exception of SK-BR3) .
TABLE IV
Antiproliferative Effect of Aromatic Diketone fCompound 12) on Tumor Cell Lines Cell Lineε Relative Cell Viability (%)
Myeloid Cells:
Promyelocytic (HL-60) 6
Promonocytic (ML-l) 11
Myelogenous (KG-1) 21
Myelogenous (KG-la) 23 Promonocytic (THP-1) 62
Histiocytic Lymphoma (U-937) 94 B Cell Lymphoma :
Burkitt Lymphoma (Daudi) 25
Burkitt Lymphoma (Raji) 57 T Cell Lymphoma (Jurkat) 16
Epithelial Cells:
Breast (BT-20) 42
Breast (MCF-7) 51
Breast(T-47 D) 73 Breaεt (SK-BR3) 114
Embryonal Kidney (A293 LT) 69
Epithelioid (HeLa) 77
Hepatocellular (Hep G2) 99 Lung (A549) 83
Retinal Pigment (D407) 107
EXAMPLE 5
Antiproliferative Effectε of Caffeic Acid
In the experimentε shown in Table V, 5xlθ3 cells were plated overnight at 37°C and then incubated with caffeic acid (lug/ml) . After 68 hours at 37°C, cells were pulsed with thymidine for 6 hours prior to harvesting. Thymidine incorporation by untreated cells was expresεed aε 100%. All determinations were made in triplicate. The variation between the triplicate was less then 10%.
As is shown in Table V, caffeic acid had a weaker antiproliferative effect on most tumor cell lines than other curcumin analogs. The tumor cell line most sensitive to caffeic acid was the T Cell Lymphoma (Jurkat) cell line.
TABLE V
Antiproliferative Effect of Caffeic Acid (Compound 13
Cell Lines Relative Cell Viability ( % ) Myeloid Cells :
Promyelocytic (HL-60) 80 Myelogenous(KG-la) 89
Histiocytic Lymphoma (U-937) 92
Promonocytic (THP-1) 95
Promonocytic(ML-1) 99
Myelogenous (KG-l) 108 B Cell Lymphoma :
Burkitt Lymphoma (Daudi) 93
Burkitt Lymphoma (Raji) 96 T Cell Lymphoma : (Jurkat) 48 Epithelial Cells:
Breast (T-47 D) 76
Breast (BT-20) 87 Breast (MCF-7) 98
Breast (SK-BR3) 105
Lung (A549) 86
Embryonal Kidney (A293 LT) 89
Epithelioid (HeLa) 100 Hepatocellular (Hep G2) 102
Retinal Pigment (D407) 110
EXAMPLE 6
Antiproliferative Effects of Cinnamic Acid
In the experiments shown in Table VI, 5xl03 cells were plated overnight at 37°C and then incubated with cinnamic acid (lug/ml) . After 68 hours at 37°C, cells were pulsed with thymidine for 6 h prior to harvesting. Thymidine incorporation by untreated cells was expressed as 100%. All determinations were made in triplicate. The variation between the triplicate waε less then 10%.
Table VI showε that cinnamic acid had an antiproliferative effect on T cell lymphoma (Jurkat) cellε, breast tumor (BT-20 and T-47D) cells, the hepatocellular
(Hep G2) tumor cell line and the retinal pigment (D407) cell line.
TABLE VI
Antiproliferative Effect of Cinnamic Acid (Compound 1 Cell Lines Relative Cell Viability f%)
Myeloid Cells: Promonocytic(ML-1) 84
Promonocytic (THP-l) 86
Promyelocytic (HL-60) 91 Myelogenous(KG-la) 98
Histiocytic Lymphoma (U-937) 100
Myelogenous (KG-1) 121 B Cell Lymphoma : Burkitt Lymphoma (Raji) 88
Burkitt Lymphoma (Daudi) 113
T Cell Lymphoma : (Jurkat) 36 Epithelial Cells:
Breast (BT-20) 34 Breast (T-47 D) 54
Breast (SK-BR3) 87
Breast (MCF-7) 108
Hepatocellular (Hep G2) 45
Epithelioid (HeLa) 99 Retinal Pigment (D407) 56
Embryonal Kidney (A293 LT) 52
Lung (A549) 107
EXAMPLE 7
Antiproliferative Effects of Carboxylic Acids For the experiment shown in Table VII, 5xl03 cells were plated overnight at 37°C and then incubated with aromatic carboxylic acid (lug/ml) . After 68 hours at 37°C, cells were pulsed with thymidine for 6 hours prior to harvesting. Thymidine incorporation by untreated cells was expressed as 100%. All determinations were made in triplicate. The variation between the triplicate was less then 10%.
Table VII shows that aromatic carboxylic acids had antiproliferative effects on promyelocytic (ML-1 and HL-60) cells, myelogenous (KG-1) cells and T cell lymphoma cellε. In addition, aromatic carboxylic acidε inhibited embryonal kidney (A293 LT) cell, retinal pigment (D407) cellε and Breast (BT-20) tumor cell lines. TABLE VII
Antiproliferative Effect of Aromatic Carboxylic Acids
Cell Lines Relative Cell Viability (%)
Compounds 15 16 17 18 Myeloid Cells :
Promonocytic (ML-1) 68 74 80 80 Myelogenous (KG-1) 71 120 115 140 Promyelocytic (HL-60) 83 88 82 74 Myelogenous (KG-la) 114 128 105 126 Promonocytic (THP-1) 117 104 110 98
Histiocytic Lymphoma (U-937) 118 125 115 123 B Cell Lymphoma: Burkitt Lymphoma (Raji) 142 154 141 170 Burkitt Lymphoma (Daudi) 109 191 176 178 T Cell Lymphoma: (Jurkat) 43 66 46 46 Epithelial Cells: Embryonal Kidney (A293 LT) 37 140 107 98 Retinal Pigment (D407) 59 63 89 72 Breast (BT 20) 81 90 89 Breast (MCF7) 111 104 107 103
Epithelioid (HeLa) 91 81 100 108 Hepatocellular (Hep G2) 92 123 107 125 Lung (A549) 194 251 229 329
EXAMPLE 8 Antiproliferative Effects of Aromatic Ketocarboxylic Acidε
In the experiment shown in Table VIII, 5xl03 cells were plated overnight at 37°C and then incubated with aromatic ketocarboxylic acid (lug/ml) . After 68 hours at 37°C, cells were pulsed with thymidine for 6 hours prior to harvesting. Thymidine incorporation by untreated cells was expressed as
100%. All determinationε were made in triplicate. The variation between the triplicate waε leεs then 10%.
Table VIII shows that aromatic ketocarboxylic acids inhibited promyelocytic (HL-60 and THP-1) tumor cell lines. In addition, aromatic ketocarboxylic acids inhibited T cell lymphoma tumor cells and retinal pigment (D407) cells.
TABLE VIII Effect of a ketocarboxylic acid (Compound 19)
Cell Lines Relative Cell Viability (%) Myeloid Cells:
Promyelocytic (HL-60) 72
Promonocytic (THP-1) 83 Promonocytic(ML-1) 95
Myelogenous (KG-la) 107
Myelogenous (KG-1) 112
Histiocytic Lymphoma (U-937) 118
B Cell Lymphoma : Burkitt Lymphoma (Raji) 197
Burkitt Lymphoma (Daudi) 135
T Cell Lymphoma : (Jurkat) 50
Epithelial Cells :
Retinal Pigment (D407) 83 Epithelioid (HeLa) 97
Hepatocellular (Hep G2) 98
Breast (MCF-7) 100
Breast (BT-20) 107
Embryonal Kidney (A293 LT) 176 Lung (A549) 286
EXAMPLE 9
Antiproliferative Effects of Aromatic Alcohols
In the experiments shown in Table IX, 5xl03 cells were plated overnight at 37°C and then incubated with aromatic alcohol (lug/ml) . After 68 hours at 37°C, cells were pulsed with thymidine for 6 hours prior to harvesting. Thymidine incorporation by untreated cellε waε expreεεed as 100%. All determinations were made in triplicate. The variation between the triplicate was lesε then 10%.
Table IX indicateε the antiproliferative effectε of aromatic alcoholε on promyelocytic (HL-60) tumor cell line. In addition, aromatic ketocarboxylic acidε inhibited T cell lymphoma tumor cells and retinal pigment (D407) cells.
TABLE IX Effect of an Aromatic Alcohol (Compound 20) Cell Lines Relative Cell Viability (%) Myeloid Cells:
Promyelocytic (HL-60) 77
Promonocytic (THP-1) 93
Hiεtiocytic Lymphoma (U-937) 93
Promonocytic (ML-1) 104 Myelogenous (KG-1) 106
Myelogenous (KG-la) 124
B Cell Lymphoma:
Burkitt Lymphoma (Daudi) 158
Burkitt Lymphoma (Raji) 223 T Cell Lymphoma : (Jurkat) 60
Epithelial Cells:
Retinal Pigment (D407) 79
Breast (BT-20) 89
Breast (MCF-7) 103 Epithelioid (HeLa) 94
Hepatocellular (Hep G2) 97
Embryonal Kidney (A293 LT) 157
Lung (A549) 284
EXAMPLE 10 Antiproliferative Effects of Flavanoidε
To examine the effects of flavanoids on tumor cell lines, 5xl03 cells were plated overnight at 37°C and then incubated with flavanoids (lug/ml). After 68 hours at 37°C, cells were pulsed with tritiated thymidine for 6 hours prior to harvesting. Thymidine incorporation by untreated cells was expressed as 100%. All determinations were made in triplicate. The variation between the triplicate was less than 10%.
Table X showε that flavanoidε exhibited antiproliferative effectε on promyelocytic (HL-60 and ML-1) cellε, myelogenouε (KG-1 and KG-la) cellε, hepatocellular (Hep G2) cells, breast tumor cell lines (BT-20, T-47D and MCF-7) and embryonal kidney (A293 LT) cells.
TABLE X Antiproliferative Effect of Flavanoids on Tumor Cell Lines
Cell Lines Relative Cell Viability (%)
Compounds 21 22
Myeloid Cells:
Promyelocytic (HL-60) 37 34 Myelogenous (KG-la) 58 73 Myelogenous (KG-1) 59 100 Promonocytic (ML1) 70 84 Histiocytic Lymphoma (U-937) 103 89 Promonocytic (THP-1) 106 103 B Cell Lymphoma : Burkitt Lymphoma (Raji) 124 115 Burkitt Lymphoma (Daudi) 148 175 T ceil Lymphoma : (Jurkat) 88 100 Epithelial Cells : Hepatocellular (Hep G2 ) 67 83 Epithelioid (HeLa) 104 108 Breast (BT-20) 24 33 Breast (T-47 D) 63 69 Breast (MCF-7) 57 117 Breast (SK-BR3) 121 106 Embryonal Kidney (A293 LT) 62 81 Retinal Pigment(D407 ) 138 93 Lung (A549) 207 126 Effects of Curcumin and Curcumin analogues on Kinase Activitv
EXAMPLE 11 Protein Kinase Asεayε
Cytosolic protamine kinase and autophosphorylation- activated protein kinaεe were obtained from Dr. Z. Damuni, Department of Biological Scienceε, Columbia, South Carolina. These protein kinaεe preparations were judged to be homogeneous based on SDS-PAGE and gel permeation chromatography. Phosphorylase kinase (170 units/mg) , phosphorylase b, catalytic subunit of protein kinase A (41 units/mg) , Hiεtone H-l and H-2B, protamine sulfate (εalmine) , myelin basic protein, curcumin and phosphatidyl-L-serine were obtained from Sigma Chemical Co. Protein kinase C (1200 units/mg) was obtained from Calbiochem Corp. gamma [32 P] ATP was obtained from ICN Biomedicalε, Inc.
Protein kinaεe C, protein kinase A, and cytosolic protamine kinaεe were aεsayed as described by Damuni et al., 1989, Purification and properties of a distinct protamine kinase from the cytosol of bovine kidney cortex. J. Biol . Chem . 264, 6412-6416 with modifications. Briefly, the assayε were performed in 0.05 ml mixtureε containing 25 mM Triε-HCl, pH 7.3, 10% glycerol, 1 mM Benzamidine, 14 mM B- mercaptoethanol, 0.2 mM phenylmethyl sulfonyl fluoride, 100 ug/ml leupeptin, 4 uM microcystin LR, 2 ug/ml aprotinin, protein kinase, 50 ug histone H-l (PKC) or histone H-2B or 100 ug protamine sulfate, 10 mM MgCl2, and 0.2 mM [gamma-32P] ATP (200-500 cpm/pmol) . The reaction was initiated by adding MgCl2 and ATP. After 10 minutes of incubation at 37°C, the reaction was terminated by the addition of 1 ml of 10% trichloroacetic acid (TCA) . Protein in the TCA terminated mixtures was pelleted by centrifugation for 2 minutes in a Beckman centrifuge at 15,000 x g. The pellet waε washed five times with TCA, added one ml scintillant and counted for radioactivity in a Packcard liquid scintillation counter. Control tubes were treated in an identical manner except that protein kinase was excluded from the mixture. Protein kinase C was assayed as described above except that the incubation mixture also included 0.5 mM CaCl2 and u40 ug/ml phosphatidyl- L-serine. Phosphorylase kinase was similarly assayed with the following modifications. The assay mixture contained 25 mM Tris-HCl, pH 7.3, 10% glycerol, 1 mM Benzamidine, 14 mM B- ercaptoethanol, 0.2 mM phenylmethyl sulfonyl fluoride, 100 ug/ml leupeptin, 4 uM microcystin LR, 2 ug/ml aprotinin and a protein kinase containing 0.5 mM CaCl2. Following incubation for 10 minutes at 37°C, the reaction was terminated with 1 ml of 10% TCA and treated as described above. The autophosphorylation-activated kinase waε firεt preactivated and then assayed with myelin basic protein as substrate. One unit of protein kinase activity was defined as the amount of enzyme that incorporated 1 nmol of phosphoryl groups into substrate/min. To ensure linearity the extent of incorporation of phosphoryl groupε waε limited to <1 nmol. Sodium-Dodecyl Gel Electrophoresis: Polyacrylamide slab gels (12%) were run in a Biorad protein ® II apparatus at 200 volts conεtant voltage. Protein bandε were detected by staining with coomassie brilliant blue, dried and autoradiographed. The activity of cellular tyrosine kinase having a molecular weight of 60 kDa was determined as described by Budde et al., J. Biol. Chem., 268:24868-24872 (1993), with the following modifications. Briefly, the incubation mixture contained 25 M tris-HCl, pH 7.3, 10% glycerol, 1 mM benzamidine, 14 mM β- mercaptoethanol, 0.2 mM phenylmethyl εulphonyl fluoride, 100 μg/ml leupeptin, 2 μg/ml aprotinin, protein kinaεe, 50 μg polyglutamic acid-tyrosine (4:1) , 10 mM MgCl2 and 0.2 mM[gamma-32P]-ATP (200-500 cpm/pmol) . Following incubation at 37°C for 10 minutes, the 0.05 ml of the mixture was blotted onto filter paper and immediately immerεed in 10% TCA. The paper was then washed with 10% TCA before counting for radioactivity in the presence of scintillant. EXAMPLE 12
Curcumin Inhibition of Kinaεe Activitv
The effect of different concentrations of curcumin on the activity of six different protein kinases is shown in the present invention. Figure 9 illustrates that curcumin inhibited all the kinases examined but to different degrees. At 1 mM curcumin, PhK, pp60s'src' PkC, PkA, AK and cPK were inhibited by 98%, 40%, 15%, 10%, 1% and 0.5%, respectively. However, higher concentrations of curcumin inhibited 98%, 95%, 46%, 49%, 17% and 2% of the activity of these kinases, respectively. The inhibitory effect was dose-dependent.
Among the kinaseε examined, PhK was most completely inhibited by the lowest concentration of curcumin. Near complete inhibition of cellular tyroεine kinaεe was also seen. The inhibitory effectε of curcumin on PhK iε shown in Figure 10A. The effects of curcumin were seen at a dose of curcumin as low as 5 uM and the inhibitory effect plateued at about 3 mM. A similar effect was εeen when the reaction product of PhK, i.e., phosphorylase b was analyzed by SDS polyacrylamide gel electrophoresis. Figure 10B showε that the inhibitory effects of curcumin were seen at 5 uM and no phosphorylated product was obεerved at 1.36 uM.
In order to examine the inhibitory constant of curcumin, the effect of the inhibitor on PhK at different concentrations of the subεtrate was examined. Theεe results were then analyzed by Lineweaver-Burke plot analysis. Figure IIA illustrateε that the curves for the inhibition of curcumin at different εubεtrate concentrations were linear. Thus, curcumin is a non-competitive inhibitor and binds to the enzyme at a site different from the phosphorylase. Further, a plot of different curcumin concentrations against the slope indicated a Ki of 0.75 mM (Figure 11B) .
Natural structural analogues of curcumin also inhibit PhK activity. Phosphorylase kinase (134 units/ml) was aεsayed with curcumin and its analogueε aε described above. As shown in Table XI, these analogues were inhibitory to curcumin but none inhibited the enzyme to the same extent as curcumin.
TABLE XI Inhibitory Effects of Curcumin and Curcumin Analogues of Phosphorylase Kinase (PhK) Activity
Compounds PhK inhibition (% of control) curcumin 67 aromatic ketone (cmpd 7) 24 ferulic acid 15 cinnamic acid 15 aromatic ketone (cmpd 4) 14
All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.
WHAT IS CLAIMED IS:

Claims

Claims
1. A method for the treatment of pathological cell proliferative diseaseε compriεing administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof.
2. The method of claim 1, wherein said disease is selected from the group consisting of neoplastic diseaεeε and non-neoplaεtic diseases.
3. The method of claim 2, wherein said neoplastic disease is selected from the group conεiεting of ovarian cancer, bladder cancer, lung cancer, cervical cancer, breaεt cancer, proεtate cancer, gliomaε, fibrosarcomaε, retinoblaεtomaε, melanomaε, soft tissue sarcomas, osteoεarcomaε, leukemiaε, colon cancer, carcinoma of the kidney and pancreatic cancer.
4. The method of claim 2, wherein said non-neoplastic diεease is selected from the group consiεting of psoriasis, benign proliferative skin diseaεeε, ichthyoεis, papilloma, basal cell carcinoma, εquamouε cell carcinoma, reεtinosis, scleroderma and hemangioma.
5. The method of claim 1, wherein said animal is a human.
6. The method of claim 1, wherein said curcumin is administered in a dose of from about 1 microgram to about 100 milligrams.
7. The method of claim 1, wherein said analogue is selected from the group consiεting of 4- hydroxy-3-methoxycinnamic acid, 4-methylenedioxy cinnamic acid, 3, 4-dimethoxycinnamic acid, 4-(4- hydroxy-3-methoxyphenyl) -3-buten-2-one, zingerone, 4- (3, 4-methylenedioxyphenyl) -2-butanone, 4-(p- hydroxyphenyl) -3-buten-2-one, 4-hydroxyvalerophenone, 4-hydroxybenzylactone, 4-hydroxybenzophenone, 1, 5-biε (4-dimethylaminophenyl) -1, 4-pentadien-3-one, 6- hydroxydibenzoylmethane, 3, 4-dihydroxycinnamic acid, cinnamic acid, 3, 4-dihydroxyhydrocinnamic acid, 2- hydroxycinnamic acid, 3-hydroxycinnamic acid, 4- hydroxycinnamic acid, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenethyl alcohol.
8. A method of inhibiting the activity of phoεphorylase kinase comprising administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof.
9. The method of claim 8, wherein said animal is a human.
10. The method of claim 8, wherein said curcumin is administered in a dose of from about 1 microgram to about 100 milligrams.
11. The method of claim 8, wherein said analogue is selected from the group consiεting of 4- hydroxy-3-methoxycinnamic acid, 4-methylenedioxy cinnamic acid, 3, 4-dimethoxycinnamic acid, 4-(4- hydroxy-3-methoxyphenyl) -3-buten-2-one, zingerone, 4- (3, 4-methylenedioxyphenyl) -2-butanone, 4-(p- hydroxyphenyl) -3-buten-2-one, 4-hydroxyvalerophenone, 4-hydroxybenzylactone, 4-hydroxybenzophenone, 1, 5-biε
(4-dimethylaminophenyl) -1, 4-pentadien-3-one, 6- hydroxydibenzoylmethane, 3, 4-dihydroxycinnamic acid, cinnamic acid, 3, 4-dihydroxyhydrocinnamic acid, 2- hydroxycinnamic acid, 3-hydroxycinnamic acid, 4- hydroxycinnamic acid, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenethyl alcohol.
12. A method for the treatment of pathological cell proliferative diseases comprising administration to an animal of a pharmacologically effective dose of a flavonoid or an analogue thereof.
13. A method of inhibiting the activity of tyrosine kinase comprising administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof.
14. The method of claim 13, wherein said animal is a human.
15. The method of claim 13, wherein said curcumin is administered in a dose of from about 1 microgram to about 100 milligrams.
16. The method of claim 13, wherein said analogue is selected from the group consisting of 4- hydroxy-3-methoxycinnamic acid, 4-methylenedioxy cinnamic acid, 3, 4-dimethoxycinnamic acid, 4-(4- hydroxy-3-methoxyphenyl) -3-buten-2-one, zingerone, 4- (3 , 4-methylenedioxyphenyl) -2-butanone, 4- (p- hydroxyphenyl) -3-buten-2-one, 4-hydroxyvalerophenone, 4-hydroxybenzylactone, 4-hydroxybenzophenone, 1, 5-bis (4-dimethylaminophenyl) -1, 4-pentadien-3-one, 6- hydroxydibenzoylmethane, 3, 4-dihydroxycinnamic acid, cinnamic acid, 3, 4-dihydroxyhydrocinnamic acid, 2- hydroxycinnamic acid, 3-hydroxycinnamic acid, 4- hydroxycinnamic acid, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenethyl alcohol.
AMENDED CLAIMS
[recei ved by the Internationa l Bureau on 20 June 1995 (20.06.95) ; origi nal cl aim 3 cancel led ; original cl aim 1 amended ; remaining claims unchanged ( 1 page ) ]
1. A method for the treatment of neoplast ic diseases comprising administration to an animal of a pharmacologically effective dose of curcumin or an analogue thereof , wherein said neoplastic disease is selected from the group consisting of lung cancer, breast cancer, and melanomas .
2. The method of claim 1 , wherein said disease is selected from the group consisting of neoplastic diseases and non-neoplastic diseases .
3. (Cancelled)
4. The method of claim 2, wherein said non- neoplastic disease is selected from the group consisting of psoriasis, benign proliferative skin diseases, ichthyosis, papilloma, basal cell carcinoma, squamous cell carcinoma, restinosis, scleroderma and hemangioma.
5. The method of claim 1, wherein said animal is a human.
6. The method of claim 1, wherein said curcumin is administered in a dose of from about 1 microgram to about 100 milligrams.
7. The method of claim 1, wherein said
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