NZ522665A - Pharmaceutical composition inducing cancer cell differentiation and the use for treatment and prevention of cancer thereof - Google Patents

Abstract

A cell differentiation agent-II (CDA-II) purified from fresh human urine, that is effective for the therapy and prevention of cancer is disclosed. A common feature of cancer is that all cancer cells keep on dividing, and are unable to undergo terminal differentiation. Abnormal methylation enzymes are a major factor contributing to this unique feature. CDA-II is effective to convert abnormal methylation enzymes into normal enzymes, thus enabling cancer cells to undergo terminal differentiation, thereby eliminating the cause of the disease. Since the target is a cancer specific protein factor associated with methylation enzymes, the therapy is selective and without adverse side effects. Active components of CDA-II consist of differentiation inducers, helper inducers, and anticachexia agents. In addition, when CDA-II is employed in conjunction with thymidine a much better anticancer effect is achieved.

Description

5226 6 5 post-dated under sect 12(4) NEW ZEALAND PATENTS ACT, 1953 No: Date: COMPLETE SPECIFICATION PHARMACEUTICAL COMPOSITION INDUCING CANCER CELL DIFFERENTIATION AND THE USE FOR TREATMENT AND PREVENTION OF CANCER THEREOF We, BIO GRAND CO., LTD., a company incorporated under the laws of Taiwan, Republic of China, of Bl, No.90, Chien Kuo North Road, Sec.2, Taipei, Taiwan, Republic of China, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page la) Pharmaceutical composition inducing cancer cell differentiation and the use for treatment and prevention of cancer thereof Field of the Invention The present invention relates to a pharmaceutical composition used for treating and preventing cancers and the preparation thereof. More specifically, the present 10 invention relates to a cell differentiation agent (CDA-II) obtained from fresh urine, and the method for the preparation thereof. The pharmaceutical composition according to the present invention is effective in treating and preventing cancers.

According to the present invention, CDA-II can correct the ternary methylation enzymes in cancer cells from their abnormal state, thereby promote terminal 15 differentiation of the cancer cells to achieve desired therapeutic effects. This method for treating cancer aimed at eliminating the cause of the disease is effective as demonstrated by clinical trails. CDA-II antagonizes the function of a cancer specific protein bonded to the ternary methylation enzymes of cancer cells, therefore, the method of the present invention is highly selective and has no adverse effect. The 20 active components of CDA-II comprise differentiation inducers, differentiation helper inducers and an anti-cachexia agent. These active components act synergistically to achieve therapeutic effect.

CDA-II has a better anticancer effect when combined with other anticancer drugs, such as thymidine.

Further, CDA-II has a very good anticancer effect when combined with vitamin C and vitamin B17.

Cancer includes a variety of very complicated diseases; nevertheless, they all share a common feature that all cancer cells are able to keep on dividing, and can not undergo terminal differentiation. The present inventor has found that the abnormality 30 of ternary methylation enzymes were the major cause of cancer disease, which la provides a new strategy for cancer therapy. Cell differentiation agent, which is purified from the urine of normal persons, can transform abnormal ternary methylation enzymes in cancer cells to their normal forms. Thus cancer cells can be induced to undergo terminal differentiation, resulting in the termination of cell 5 replication and /or apoptosis. In this way cancer can be cured. This treatment is safe to patients as the cell differentiation agent acts selectively on cancer cells. Furthermore, healthy persons also rely on this mechanism to fight against cancers. Accordingly, such a treatment of cancer is thus called "natural anticancer method".

The active components in cell differentiation agent include differentiation 10 inducers and differentiation helper inducers. The differentiation inducer can eliminate the abnormal protein factor (i.e. abnormal tumor specific protein factor) bonded to the ternary methylation enzyme, which is present specifically in cancer cells. The differentiation helper inducer is the inhibitor of the component enzymes of the ternary methylation enzymes, which can potentiate the action of the differentiation inducer. In 15 the cell differentiation agent according to the present invention, the differentiation inducers are PP-0 and OA-O.79, and the differentiation helper inducers comprise at least phenylacetic acid, hippuric acid, indole acetic acid, uroerythrin and vitamin B2. In addition, the cell differentiation agent comprises an anti-cachexia component such as phenylacetylglutamine. Because most cancer patients develop the symptom of 20 cachexia, anticachexia agent is helpful to the treatment of cancer. Briefly, the combination of the active components in the cell differentiation agent can provide excellent therapeutic effect. The present inventor further found that the cell differentiation agent of the present invention could give a better anticancer effect when used with the other cytotoxic drugs, such as thymidine. The present inventor 25 also found that the cell differentiation agent of the present invention could also give a much better therapeutic effect when used together with vitamin C and vitamin B17.

Background of the Invention 2 Oncogenes are parts of human genome; thus, there were human being, there were cancers. However, up to now cancer is still a problem to be overcome by human. Because of the complexity of cancer, it is very difficulty to eradicate cancer cells from the patient. Cancer cells keep on dividing, invade into normal organs and tissues, and 5 finally cause serious diseases to result in death of the patient. Traditionally, the ability to keep on dividing is regarded as the major cause of cancer, and the therapy based thereon is the administration of cytotoxic drugs, which can inhibit the synthesis of DNA and the division of cells. For the last five decades, therapy of cancer has been developed mainly on the technology of using cytotoxic drugs. New therapy of cancer 10 is thus desirable.

Retinoic acid is a differentiation inducer successfully used in the treatment of acute promyelocytic leukemia (Huang et al., 1988; Warrell et al., 1991, ref. 8 and 28). Although it has an excellent anti-cancer effect, cancer cells recur soon (Muindi et al., 1992; Adamson, et al., 1993, ref. 1 and 25). The recurrence is caused by the 15 incompleteness of differentiation associated with the use of differentiation inducer alone. The use of differentiation inducer alone results in the damage of the cell, making it impossible to complete the differentiation processes.

References Cited The references cited in and/or relevant to the present invention are listed below: 1. Adamson P.C., Boylan J.F., Balis F. M., Murfy R. F., Godwin K.A., Gudas L.T., Poplack D.G, Time course of induction of metabolism of all-trans retinoic acid and the up-regulation of cellular retinoic acid binding protein, Cancer Res., 53: 472-476,1993. 2. Bar-Or D., Greisman S.L., Kastendieck J.G, Detection of appendicitis by measurement of uroerythrin, U.S. Patent, 5,053,389, 1991. 3. Borek E., et al., Altered excretion of modified nucleosides and 30 P-aminoisobutyric acid in subjects with acquired immunodeficiency syndrome 3 or at risk for acquired immunodefociency syndrome, Cancer Res., 46: 2557, 1986. 4. Burzynski, S.R., Treatment of Malignant Brain tumors with Antineoplastons, Adv. Exp. Clin. Chemother, 6/88:45-46, 1988.

. Clark P.M.S., Kricka L.J., Whitehead T.P., Pattern of urinary proteins and peptides with rheumatioid arthritis investigated with the iso-Dalt technique, Clin. Chem., 26:201,1980. 6. Doerfler, W., DNA methylation and gene activity, Annu. Rev. Biochem., 52: 92-124,1983. 7. Epifanova, O.I., Abuladze, M.K., and Zoniovska, A.I., Effect of low concentrations of actinomycin D on the initiation of DNA synthesis in rapidly proliferating and stimulated cell culture, Exp. Cell Res., 92:25-30,1975. 8. Huang, M.E., Ye, Y.C., Chen, S.R., Chai, J.R., Lu, J.X., Zhao, L., Gu, L.J. and Wang, Z.Y., Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia, Blood, 72:567-572, 1988. 9. Jones, P. A., Altering gene expression with 5-azacytidine, Cell, 40, 484-486, 1985.

. Kampalath, B.N., Liau, M. C., Burzynski, B., and Burzynski, S.R., Chemoprevention by Antineoplaston A10 of Benzo (a) pyrene-induced Pulmonaiy Neoplasia, Drugs Exptl. Clin. Res., 13(supplement): 51-56,1987. 11. Liau, M.C., who is the same inventor of this application case, Smith, D. W., and Hurlbert, R.B., Preferential inhibition by homopoly ribonucleotides of the methylation of ribosomal ribonucleic acid and disruption of the production of ribosomes in a rat tumor, Cancer Res., 35:2340-2349,1975. 12. Liau, M.C., Hunt, M.E. and Hurlbert, R.B., Role of ribosomal RNA methylases in the regulation of ribosome production in mammalian ceils, Biochem., 15: 3158-3164,1976. 13. Liau, M.C., Lin, GW., and Hurlbert, R.B., Partial purification and characterization of tumor and liver S-adenosylmethionine synthetase, Cancer Res., 37:427-435, 1977. 4 14. Liau, NIC., Lin GW., Knight, C.A., and Hurlbert, R.B., Inhibition of RNA Methylation by Intercalating Agents, Cancer Res., 37:4202-4210,1977.

. Liau, M.C., Chang, C.F., and Becker, F.F., Alteration of S-adenosylmethionine synthetase during chemical hepatocarcinogenesis and in resulting carcinomas,cancer Res., 39:2113-2119,1979. 16. Liau, M.C., and Burzynski, S.R., Altered methylation complex isozymes as selective targets for cancer chemotherapy, Drugs Exptl. Clin. Res., 12(supplement): 77-86,1986. 17. Liau, M.C., Szopa, M., Burzynski, B., and Burzynski, S.R., Chemosurveillance; a novel concept of the natural defense mechanism against cancer, Drugs Exptl. Clin. Res., 12(supplement): 71-76, 1987. 18. Liau, M.C., Lee S.S., and Burzynski, S.R., Differentiation inducing components of antineoplaston A5, Adv. Exptl. Clin. Chemother., 6/88:9-25, 1988. 19. Liau, M.C., Lee, S.S., and Burzynski, S.R., Hypomethylation of nucleic acids: a key to the induction of terminal differentiation, Intl. J. Exptl. Clin. Chemother., 2: 187-199,1989.

. Liau, M.C., and Burzynski, S.R., Separation of active anticancer components of antineoplaston A2, A3 and A5, Intl. J. Uss. React., 12(supplement): 1-18, 1990. 21. Liau, M.C., Lee, S.S., and Burzynski, S.R., Modulation of cancer methylation complex isozymes as a decisive factor in the induction of terminal differentiation mediated by antineoplaston A5. Intl. J. Tiss. React., 12(supplement): 1-18,1990. 22. Liau, M.C., Ashraf A., Lee, S.S., Hendry, L.B. and Burzynski, S.R., Riboflavin as a minor active anticancer component of Antineoplaston A2 and A5, Intl. J. Tiss. React., 12(supplement): 18-26,1990. 23. Liau, M.C., Liau, C.R, Burzynski, S.R., Potantilation of induced terminal differetiation by phenylacetic acid and related chemicals, Intl. J. Exptl. Clin.

Chemother., 8: 9-17,1992. 24. Liau, M.C., Luong Y., Liau C.P., and Burzynski, S.R., Prevention of drug-induced DNA hypermethylation by antineoplaston components, Intl. J. Exptl. Clin. Chemother, 5:19-23,1992b.

. Muindi, J.R.F., Frankel, S.R., Huselton, C., Degrazia, F., Garland, W.A., 5 Young, C.W., and Warrell, R. P., Clinical pharmacology of oral all-trans retinoic acid in pateints with acute promyelocytic leukemia, Cancer Res., 52: 2138-2142,1992. 26. Muldoon, T.G, Copland, J.A., Hendry, L.B., Antineoplaston AlO activity on carcinogen-induced rat mammary tumors, Intl. J. Tiss. React., 12(supplement): 51-56,1990. 27. Toniola, D., Weiss, H. K., and Basilio, C.A., Temperature sensitive mutation affecting 28S ribosomal RNA production in mammalian cells, Proc. Natl. Acad. Sci. USA, 70: 1273-1277,1973. 28. Warrell, R.P. Jr., Frankel, S.R., Miller, W. H., Jr. Sheinberg, D.A., Itri, L.M., 15 Hettelman, W.N., Vyas, R., Andreeff, M., Tafuri, A., Jakubowski, A, Gabrilove, J., Gordon, M.S., and Dmitrovsky, E., Differentiation therapy of acute promyelocytic leukemia with tretinoin (all- trans- retinoic acid), N. Engl. J. Med., 324: 1385-1393, 1991. 29. Spielholz, C., Golde D.W., Houghton, A. N., Nualart, F., and Vera, J.C., 20 Incicased facilitated transport of dehydroascorbic acid without changes in sodium-dependent ascorbate transport in human melanoma cells, Cancer Res., 57: 2529-2537, 1997.

. Mr. Rentaro Sano, Annihilating Cancer, Shimao Publisher, Taipei, Taiwan, 1997, pp202-203.

Summary of the Invention The object of the invention is to provide a pharmaceutical composition that can 30 induce cancer cell to differentiate, and the use thereof in treating and preventing 6 cancer. As mentioned above, cancer includes a variety of very complicated diseases. However, a common feature of cancers is that all cancer cells are capable of perpetual cell division, and can not undergo terminal differentiation. Surprisingly, the inventor of the present invention found that abnormal ternary methylation enzymes were the 5 major cause of cancer, therefore a new strategy to treat and prevent cancer was developed based on this finding.

After extensive study over more than 30 years, the inventor of the present invention found that ternary methylation enzymes were the major cause of cancer. It is the abnormality of these enzymes that renders cancer cells immortal. The ternary 10 methylation enzymes play a very important role in cell division and differentiation. Cells are induced to divide when the activity of these enzymes is increased. On the contrary, cells are induced to synthesize methyl-deficient nucleic acids when the activity of these enzymes decrease, thereby are induced to differentiate into terminally differentiated cells, which are no longer capable of dividing. All cancer cells have 15 abnormal ternary methylation enzymes, and the activity of these enzymes is high in these cells, thus making these cells divide endlessly. Clearly, abnormal ternary methylation enzymes are the cause of cancer. Therefore, an inhibitor of these abnormal enzymes can effectively fight against cancer and can effectively prevent healthy cells to become cancer cells. As a matter of fact, there are enough natural 20 chemical substances in the body of a healthy person to inhibit the formation of abnormal ternary methylation enzymes. Therefore, calcinogenesis takes a very long period of time. In this period, the patient gradually accelerates the excretion of the anticancer chemicals. When the anticancer chemicals decrease to a concentration insufficient to inhibit the abnormal ternary methylation enzymes, the cancer cells take 25 root to develop. Healthy persons excrete a small amount of anticancer substances in the urine. Upon purification, the anticancer substances can selectively inhibit the abnormal ternary methylation enzymes, thus inducing cancer cells to differentiate as normal cells. These naturally occurring anticancer substances do not affect the growth and the function of normal cells, thus the patients will not suffer from adverse effect. 30 This strategy of therapy is thus called "differentiation therapy". 7 ,nteWEC]X-AL PROPERTY OFP»CF OP N.Z 19FEB20W received Generally, the excretion of the anticancer substances can be balanced by the production of such substances in the body of a healthy person, thus sufficient amount of such substances is maintained in the body to keep a check in the evolution of cancer cells. In contrast, a patient suffering from cancer excretes much more of the 5 anticancer substances, and gradually loses the ability to control the evolution of cancer cells. The inventor of the present invention has isolated and purified the anticancer substances, i.e. the cell differentiation agent, from the urine of healthy persons. The cell differentiation agent can transforpi the abnormal ternary methylation enzymes to their normal forms, induce cancer cells to undergo terminal differentiation, 10 and/or apoptosis. In this way cancer can be treated or prevented.

The active components in cell differentiation agent include differentiation inducers and differentiation helper inducers. The differentiation inducer can antagonize the abnormal protein factor bonded to ternary methylation enzymes, which is present specifically in cancer cells. Differentiation helper inducer is the inhibitor of 15 the component enzymes of the ternary methylation enzymes, which can potentiate the action of the differentiation inducer. Accordingly, the differentiation helper inducer is essential in the "differentiation therapy", although to a lesser extend compared with the differentiation inducer. In the cell differentiation agent according to the present invention, the differentiation inducers are PP-0 and OA-O.79, and the differentiation 20 helper inducers comprise at least phenylacetic acid, hippuric acid, indoleacetic acid, uroerythrin and vitamin B2.

In addition, the cell differentiation agent according to the present invention also comprises another component, i.e. anticachexia agent, which is also helpful to the cancer treatment. It is found that phenylacetyl glutamine has the ability to reverse 25 excessive excretion caused by the cachexia of cancer patient [Mr. Liau M.C., (Same invention of this patent application case), et al., 1987; Muldoon et al., 1990, ref. 10 and 26], which is the main component of fraction 4 in Fig. 2. This component is helpful to the treatment of cancer because most cancer patients have developed the symptom of cachexia. Briefly, the cell differentiation agent can provide an excellent 30 therapeutic effect when a variety of active components are combined to produce synergistic effect.

It is found that the cell differentiation agent can provide a better anticancer effect when used in combination with other cytotoxic drugs such as thymidine.

It is found that the cell differentiation agent can provide a much better anticancer 5 effect when used in combination with vitamin C and vitamin B2.

The cell differentiation agent according to the present invention is prepared by collecting normal human urine for the purification of anticancer components that includes ultrafiltration, reverse phase chromatography, evaporation and freeze drying. The product of the present invention can be prepared as a injection formulation, or 10 capsule.

Compared with prior art, the advantage of the present invention is utilizing anticancer substances that naturally exist in human bodies, and the treatment is directed to the root of the disease by making cancer cells differentiate and stop dividing. Therefore, the present invention is an effective therapy aimed at the 15 elimination of the cause of the disease, and the medicine according to the present invention has no adverse effect.

Brief Description of the Drawing The above object and the content of the present invention will become more apparent by describing in detail preferred examples thereof with reference to the attached drawings in which: Fig. 1 is a flow chart showing the process for the preparation of cell 25 differentiation agent according to the present invention.

Fig. 2 shows the cell differentiation agent of the present invention analyzed by the gel filtration. After concentration by lyophilization, the CDA-II injection formulation was separated through column chromatography, in which Bio-Gel P2 chromatographic column (4.1 cm x 44 cm) was used. The fractions collected were 30 measured the OD value at A255nm (O—O), the inhibition of MATlt activity (O...O), 9 weight percent, and differentiation activity on HL-60 cancer cells (represented by %NBT+).

Fig.3 shows the relationship between the concentration of uroerythrin (0—0) and vitamin B2 (O.. .0) as differentiation helper inducers and the decrease index.

Fig. 4 shows the relationship between the concentration of uroerythrin (O—O) and vitamin B2 (O.. .0) and the inhibition activity toward tRNA methyltransferases.

Fig.5 shows the enhancement of uroerythrin and vitamin B2 to the activity of differentiation inducer, the abscissa represents the concentration of retinoic acid (faM), the ordinate represents the percentage of NBT (nitroblue tetrazolium)+ cells. 10 O—O represents the control group in which different concentrations of retinoic acid are added; 0...0 represents the group in which 4 |aM vitamin B2 are added, O—•—•—O represents the group in which 4 (iM uroerythrin are added.

Fig. 6 shows the synergistic anticancer action between CDA-II of the present invention and thymidine, the abscissa represents dosage (mg/ml), the ordinate 15 represents the relative activity (%) of the cancer cell colony formation. O—O represents the group in which thymidine is used alone; 0...0 represents the group in which CDA-II is used alone; O—•—•—O represents the group in which thymidine and CDA-II are used together (1:1).

The activity of cancer cell colony formation is determined by using HBL-100 20 breast cancer cells according to the method described in Example 4, which is expressed by the percentage of the activity of cancer cell colony formation in a control group in which none of the chemicals is added. A group in which thymidine is used alone, a group in which CDA-II is used alone, and a group in which thymidine and CDA-II are used together (1:1) were tested. The result is shown in Fig. 6, which 25 demonstrates that the combined administration give a better therapeutic effect than single administration of CDA-II or thymidine. The synergistic effect is remarkable.

Fig. 7 shows the anticancer effect of CDA-II, antioxidant vitamin C, and vitamin B17. The activity of cancer cell colony formation is determined by using HBL-100 breast cancer cells according to the method described in Example 4. Each culture 30 flask contains 5ml medium, which contains 3000 HBL-100 cells and different amounts of CDA-II, vitamin C or vitamin B17. After incubation at 37 °C for 5 days, the cells were stained with Giemsa, and numbers of colonies with more than 8 cells were counted under a microscope.

In the present invention, AdoHcy represents S-adenosylhomocysteine, and StHH represents S-adenosylhomocysteine hydrolase.

Detailed Description of the Preferred Embodiment The process for the preparation of cell differentiation agent according to the present invention is illustrated in Fig. 1. While the present invention is described further referring to the examples below, it is not intended to limit the scope of the invention.

Example 1: Preparation of cell differentiation agent The process for the preparation of cell differentiation agent includes collection of urine, filtration, adsorption, solvent extraction and drying. An aqueous solution without pyrogen was used as the raw material of CDA-II.

When conducting the collection of urine, IN HC1 was added into the collection 20 container in a proportion of 1 liter of HC1 to 201iters of urine. The purpose for this is to maintain the activity of the differentiation agent, which in this way can be maintained at least one month. The urine was filtrated through a nylon cloth after the pH was adjusted to 2, then the substances with molecular weight over 10,000 Dalton were removed by ultra filtration (Millipore filter or the like can be used). Adsorbent 25 XAD-16 (Sigma) was placed in a hop-pocket, and then the hop-pocket was placed in a plastic steel (or stainless steel) funnel. Before the adsorbent was put into use, it was washed with 2 volume/weight (v/w) ethanol, then with 2 (v/w) deionized water to remove ethanol, and this step was repeated twice. After the urine had passed through the absorbent XAD-16, it was washed with 4 (v/w) deionized water, and then was 30 eluted with 2 (v/w) ethanol. The ethanol eluant was neutralized, and ethanol was 11 removed by evapiration under vacuum, while the temperature was kept below 50 °C. The dried substance was dissolved in distilled water and was used as the raw material of CDA-II.

After elution with ethanol, the adsorbent was washed with 2 (v/w) deionized 5 water, the absorbent can be used in a new cycle of absorption, until the adsorption capacity decreases to about 70% of that of a new adsorbent. Generally, XAD-16 can be reused about 200 times.

Because human body excretes daily a definite amount of creatinine, and the concentration of solid substances in urine is proportional to the amount of creatinine, 10 thus, the quantitation of the chemicals in urine is based on the amount of creatinine. In the urine collected and used in this example, the concentration of creatinine is in the range of 1.2 - 3.7 g/1, the average of it is 2.4±0.6 g/1. In the first 100 cycles of adsorption, the yield of CDA-II is about 0.51+0.17 g/g creatinine, and the solid substance in urine is about 46.7 g/g creatinine. Therefore, the yield of the CDA raw 15 material is about 1.1 % of the solid substances.

Example 2: The preparation of the injection formulation of cell differentiation agent The final concentration of CDA-II in the injection formulation is about 50±2 mg/ml or 40±2 mg/ml, while the concentration of CDA-II in the raw material is above 20 250 mg/ml. Therefore, the raw material is required to be diluted to a concentration which is about 130% or 107% of the final concentration of the injection formulation. After dilution with distilled water from which pyrogen was removed, the raw material was subjected to a series of filtration. First, the raw material was filtrated with a filter paper, then was filtrated through Millipore membrane filters membranes with pore 25 sizes of lpm and 0.45pm, respectively, and finally was filtrated with a Millpore Pellicone system to remove pyrogen. After adjusted to the desired concentration with deionized water without pyrogen, the solution was passed through a filter with a pore size of 0.22 jim in 8 hours in a sterilized operating room (class 100 decontaminated chamber) to accomplish sterilization filtration. Then, the filtrate was prepared as a 100 30 ml or a 250 ml differentiation agent injection formulation. 12 Example 3: The preparation of cell differentiation agent capsule After the raw material was filtrated sequentially through a paper filter, and Millipore membranes filters with pore size of lpxn and 0.45 jam, respectively, the 5 filtrate was dried by lyophilization. The dried material was ground, then packaged with an automatic capsule machine into capsules of 500 mg in weight Then the capsules were packaged with aluminum foil, and sterilized by radiation.

Example 4: Assay of the anticancer activity of cell differentiation agent 10 The anticancer effect of cell differentiation agent is assessed based on the inhibition of the abnormal ternary methylation enzymes of cancer cells, the inducer of cancer cells to undergo terminal differentiation, and the termination of cancer cells to divide. That is to say, cell differentiation agent can inhibit the abnormal enzyme MATlt (methionine adenosyltransferase) of cancer cells, induce the differentiation of 15 HL-60 cancer cells, and inhibit the formation of human breast cancer cell colony. These methods have been described [Liau, et al., 1977a, 1988, 1990a, ref. 13, 18, 20] Three aliquots of CDA-II were used to analyze its anticancer activity, as is described below: IT CDA-II injection formulation was used in a dosage of 1 mg/ml. MAT was 20 obtained from HL-60 cancer cell. First, the precipitated cells were suspended in a solution of 0.05M Tris, pH 7, 0.5 mM MgCl2, and then the cells were homogenized with Dounce homogenizer. The enzyme solution was separated by high-speed centrifugation (226,000xg, 0.5 hr). The enzyme was purified by DEAE-cellulose T T* chromatography, and MAT was eluted with a KC1 gradient solution and purified 25 [Liau, et al., 1977a, ref. 13]. The activity of MATlt was determined as previously described [Liau, et al., 1977a, ref. 13]. The 0.05 ml reaction solution containing 0.05 M Tris, pH 8.2,15 M KC1,15 mM MgCl2, 5 mM DTT (dithiothreitol), 2 mM ATP and ljoM [3H-CH3] methionine was incubated at 37°C for 30 minutes to allow reaction to proceed. The reaction was stopped with 0.4M PCA. The supernatant was then 30 transferred to cellulose phosphate paper of 1 square inch. The paper was put into a 13 beaker and washed with 5mM phosphate buffer, pH 7 to remove unreacted [3H-CH3]methionine. Finally, the radiation of adsorbed [3H-CH3]AdoMet (S-adenosyl methionine) was measured to determine the activity of MATlt (the result is shown in table 1).

The terminal differentiation of HL-60 cancer cells was analyzed by NBT+ method [Liau, et al., 1988a, ref. 18]. At the beginning, the HL-60 cells were diluted to 1,5 xlO5 cells/ml each-culture flask. After incubation for 96 hours, an aliquot was taken from each culture flask for the determination of cell concentration. Another aliquot was centrifuged to pellet the cells, then the cell pellet was suspended in NBT 10 solution and incubated at 37°C for 30 min. An aliquot was placed in a hemocytometer for the determination of differentiation. The total number of cells and the cells stained as black (NBT+) were counted under a microscope. The percentage of NBT+ indicates the activity of CDA-II to induce cell differentiation.

Another indication of the anticancer activity of CDA-II is the inhibition of colony 15 formation of HBL-100 breast cancer cell. At the beginning, the breast cancer cells growing at the exponential phase were washed with HBS solution, then about 2 ml 0.05% trypsin-0.53 mM EDTA flask was added, and the solution was incubated at 37 °C for 10 minutes. Thereafter, the cell density was measured, then an aliquot was taken and diluted to 3xl03 cells/ml. 0.5 ml of the diluted solution was added into 4.5 20 ml culture medium with or without CDA-II and incubated at 37°C. Five days later, the medium was discarded, and the cells were washed with isotonic saline, then methanol was added to fix cells for 15 minutes. The fixed cells were stained for 30 minutes with Giemsa staining solution diluted 20 fold. After the staining solution was discarded, the cells were washed with water and dried. The colony number above 8 cells was 25 counted under a microscope to determine the anticancer activity of CDA-II (the result is shown in table 1).

Example 5: Characterization of the active components in cell differentiation agent The CDA-II cell differentiation agent injection formulation prepared in Example 30 2 was lyophilized and concentrated to 200 mg/ml. 5 ml of the concentrated solution 14 was added onto a Bio-Gel P2 chromatographic column (4.1 cm x 44 cm), then the column was eluted with distilled water. The fractions were collected every 7 minutes into a test tube, which is 10 ml in volume. After the completion of the elution, 25 (il of the eluant from each of the test tube was taken, and diluted with water to 1 ml to 5 determine OD at A255nm- Another 25 jxl of the eluant was taken from each tube to determine the inhibition of MATlt activity. The MATlt activity was measured according to the method described in Example 4. The eluant was separated into 8 fractions as shown in Fig.2 based on the OD at A255nm- Each fraction was lyophilized and weighed to determine the weight distribution. The dry solid was dissolved in 10 distilled water to determine the differentiation inducing activity of HL-60 cancer cells, which is represented by %NBT+. The differentiation inducing activity is measured according to the method described in example 4, and the result is shown in Fig.2.

Of the active anticancer components in cell differentiation agent, the most important one is the differentiation inducer that induces cell to differentiate. The 15 separation, purification and its action have been described [Liau, et al., 1988; 1989; 1990a; 1990b; ref. 18,19,20,21], The differentiation inducers comprise two major components; one of them is an acidic peptide conjugated with pigment, abbreviated as PP-O, the other one is an organic acid, abbreviated as OA-O.79. PP-O is in the first fraction shown in Fig. 2, OA-O.79 is in the fifth and sixth fractions. It should be noted 20 that the demonstrated differentiation activity of the cell differentiation agent does not reflect the activity of the cell differentiation inducer itself, but is contributed by the synergy with the differentiation helper inducers Surprisingly, differentiation helper inducers are the main components in the cell differentiation agent, the content of the differentiation inducers is very low. Because they have not been sufficiently purified 25 to be characterized, the chemical structure of which is still unknown. As is shown in Fig. 2, the cell differentiation inducing activity coincides with the activity of MATlt inhibition. However, in some fractions, such as fraction 2 and fraction 3 have noticeable MATlt inhibition activity, but have no activity to induce cancer cells to differentiate. Probably these fractions comprise only differentiation helper inducers, 30 and no differentiation inducer, therefore have no activity to induce cancer cells to differentiate.

The differentiation helper inducers are the inhibitors of the component enzymes of the ternary methylation enzymes [Liau, et al., 1992a, ref. 23], which assists the differentiation inducer in transforming the abnormal ternary methylation enzymes to 5 their normal forms, therefore potentiates the differentiation action of the differentiation inducer.

It is known that MAT inhibitors in CDA include phenylacetic acid, indole acetic acid and hippuric acid. Phenylacetic acid, which is found in fraction 6 and fraction 7 in Fig.2, is probably the product resulted from the hydrolysis during the drying 10 process of phenylacetyl glutamine, which can be identified with C18 HPLC (High Performance Liquid Chromatography). Indole acetic acid is also found in these fractions. Hippuric acid is the major component in fraction 5. To reach 0.5 reductive index (i.e., the effective dose of the differentiation inducer is reduced to 50% of it), the concentration of these MAT inhibitors are 4mM phenylacetic acid, 8mM hippuric 15 acid, and 0.95mM indole acetic acid. As the differentiation helper inducer, the inhibitor of methyltransferase is much more effective than the inhibitor of MAT. To achieve a certain effect, the amount required for the inhibitor of methyltransferase is only one thousand or less of the amount of the inhibitor of MAT. It is known that there are two inhibitors of methyltransferase in CDA, both of them have excellent activities 20 as the differentiation helper inducers. The two inhibitors are vitamin B2 and uroerythrin. Vitamin B2 is found in the latter half of fraction 8 in Fig.2, as is described above [Liau, et al., 1990C, ref. 22]. This component, which is yellow in colour, can be purified into vitamin B2 by CI 8 HPLC, and the content of which is about 0.04% of the CDA-II. Uroerythrin is found in fraction 6 and fraction 7 in Fig. 2. High purity of 25 uroerythrin can be obtained Sephadex SH chromatography and silica gel thin layer chromatography, and the content of which is about 0.5% of the CDA-II. Because it is hard to avoid loss during purification, and CDA-II preparation has a remarkable red colour, the content of uroerythrin may be more than 0.5%.

Example 6: The determination of the activity of uroerythrin and vitamin B2 as 16 differentiation helper inducers.

The action of the differentiation helper inducer was determined according to the method designed by the present inventor of this application case, Dr. Ming C. Liau [Liau, et al., 1992a, ref. 23]. The differentiation of cancer cells was measured by using 5 leukemic cancer cells HL-60 to quantitate the NBT+cells. First, HL-60 cells were subcultured at an initial cell density 1.5x10s cells/ml, lOml/flask. A set of four flasks were used as the control, wherein only retinoic acid was added as differentiation inducer, the amount of which was adjusted to induce NBT+ cells to the range of 15% to 60%. Another flask was used as the blank which was added the solution only. The 10 total amount of methanol in which retinoic acid was dissolved should not exceed 2%, so as not to affect the differentiation of cancer cells. Each of other sets also comprises four flasks, which were added smaller amounts of retinoic acid, and a blank flask with only the solvent Different amounts of the differentiation helper inducers were added in each set. The cell numbers in every flask were counted after incubation for 15 96 hours, and NBT was assayed according to the method of Example 4. Generally, the natural differentiation of the HL-60 cells, i.e. without the action of any of the additives, is usually lower than 4%. In the sets containing only differentiation helper inducer, the differentiation of cells is less than 10%. The value of NBT+ in each flask is required to subtract the value of the blank control.

The value of ED50, i.e., the amount of the differentiation inducer required when NBT+ is 50%, can be obtained by plotting the amount against NBT+. The reductive index can be calculated from the ED50 value. Reductive index = ED50 in the presence of differentiation helper inducer/ ED50 with cell differentiation inducer only. The lower the reductive index, the higher the activity of the differentiation helper induced. 25 As can be seen from Fig.3, the concentrations of vitamin B2 and uroerythrin are 3.0 (J.M and 1.8 |iM, respectively, to reach the 0.5 reductive index, which are much lower than the MAT inhibitors mentioned above.

Example 7: The inhibition activity of uroerythrin and vitamin B2 on tRNA 30 methyltransferase. 17 tRNA methyltransferases were prepared from the high speed supernatant of HL-60 cancer cells shown in Example 4. First, the supernatant was adjusted to pH 5, and the proteins precipitated were separated by centrifugation, then dissolved in 0.05M Tris, pH 7.8, 0.5 mM MgCl2 and 5mM HSCH2CH2OH. After the solution 5 passed though DEAE-cellulose column, tRNA methyltransferase were purified by KC1 gradient [Liau, et al., 1977b, ref. 14]. The activity of tRNA methyltransferases were determined in 0.25 ml reaction solution comprising 0.05M Tris, pH 7.8, 0.1M NH4CI, 0.04M NH4F, 0.5mM MgCl2, 5mM DTT, 20(ig Escherichia coli Bp(micro) tRNA, 0.25 p.Ci [3H-CH3] AdoMet (S-adenosylmethionine) and 25 |ag enzymes. The 10 reaction is carried at 37 °C for 30 minutes. tRNA was precipitated with cold 5% TCA (trichloroacetic acid), then was collected on Millipore membrane(pore size is 0.45pm). After the membrane was dried, the radiation was assayed to determine the activity of tRNA methyltransferases, and the result is shown in Fig. 4.

The effective amount to inhibit tRNA methyltransferases is much lower than the 15 effective amount as the differentiation helper inducer. The reason for this is possibly related to the physical and chemical conditions for measuring these different activities. Different physical and chemical conditions (such as the concentration of salts) may affect differently the effective contact between chemicals and enzymes. Despite the differences in sensitivity, the activities of different tRNA methyltransferase inhibitors 20 ate proportional to the activities of differentiation helper inducers. The tRNA methyltranferase inhibitor with higher activity is a better differentiation helper inducer. We have also found that other tRNA methyltransferase inhibitors, such as ethidium bromide and hycanthone [Liau, et al., 1977b, ref. 14], are excellent differentiation helper inducers as uroerythrin and vitamin B2. The concentration of ethidium bromide 25 and hycanthone is 0.95 |jM and 2 jjM, respectively, to reach 0.5 reductive index. Therefore, it is no doubt that the inhibitors of methyltransferases can be used as effective differentiation helper inducers.

Example 8: Analysis of the differentiation helper inducers to promote differentiation 30 Differentiation helper inducers can not only decrease the effective amount of the 18 differentiation inducer, but also promote the completeness of differentiation. HL-60 cancer cells were subcultured at an initial cell density of 1.5x10s cells/ml. The culture flasks were grouped into three sets; each set includes 4-5 flasks. One flask was used as the blank without additives. One set was added 0.025- 0.15 fiM retinoic acid as the 5 control; the other two sets were added 4 |j. M of uroerythrin or vitamin B2. NBT test was conducted as described in Example 4 after incubation for 96 hours. The values thus obtained were subtracted with the value obtained by the blank without additives.

As can be seen from Fig. 5, the extent of differentiation only reached about 85% when the differentiation inducer is used alone. When combined with uroerythrin or 10 vitamin B2, the extent of differentiation reached 100%. It is much more important to achieve the completeness of differentiation than to decrease the effective amount of the differentiation inducer.

Example 9: Synergistic anticancer action of cell differentiation agent with other 15 chemical anticancer agent Ternary methylation enzyme is very active in cancer cells. If DNA synthesis is inhibited by chemicals, over-transfer of methyl is resulted, and genes are repeatedly synthesized. Repeated synthesis of genes result in the formation of anti-drug cells (Liau, et al., 1992b, ref. 24), and this the one of the important reasons causing the 20 failure of chemotherapy. The formation of anti-drug cells can be minimized through inhibition of the abnormal ternary methylation enzyme by using cell differential agent, which is helpful to the treatment of cancer. It has been demonstrated that cell differentiation agent can enhance the therapeutic effect of some anticancer drugs, for example, as is shown with the combination of CDA-11 and thymidine in the present 25 invention (Fig. 6).

Example 10: The combination of the anticancer action of cell differentiation agent and antioxidants Hypoxia can stimulate apoptosis and result in the death of cancer cells. The 30 utilization of vitamin C and vitamin B17 to create a hypoxia state is especially suitable 19 for the treatment of cancer. Cancer cells are capable of absorbing vitamin C 10-fold more than normal cells [ref. 29], while vitamin Bn can be selectively decomposed by p-glucosidase present only in cancer cells to yield toxic cyanide, which inhibits the activity of oxidase [ref. 30]. {3-glucosidase is highly active in cancer cells, but is much 5 less active in normal cells. As is shown in Fig. 7, CDA-II, vitamin C and vitamin Bn can inhibit the formation of colonies of HBL-100 human breast cancer cells, and the IC50 of which is 0.69, 0.53 and 0.13 mg/ml, respectively. The result of combined use of CDA-H and vitamin C or vitamin B17 is shown in table 2, which demonstrates that the combined use of CDA-II and vitamin C or vitamin B17 is additive.

Table 1 The anticancer action of cell differentiation agent (CDA-II) Lot No.

% MAT inhibition %NBT+ % Inhibition to the colony formation of breast cancer cells 01 49 58 100 02 55 53 100 03 50 54 100 Notes: CDA-II used is injection formulation, the dosage of which is 1 mg/ml. MATlt 15 was prepared from HL-60 cancer cells by purification through DEAE-cellulose chromatography and the activity was determined as previously described [Liau, et al., 1977a, ref. 13]. The terminal differentiation of HL-60 cancer cells was determined according to the NBT+ method [18]. The inhibition of colony formation of cancer cells was determined by cell culture using human HBL-100 breast cancer cells [Liau, 20 etal., 1990a, ref.20].

Table 2 The additive action of CDA-II and vitamin C or vitamin Bn The colony formation of HBL-100 cells: inhibition percentage Combined administration Drugs & dosages mg/ml Administered alone Predicted value Measured value CDA-II 0.4 16 28 39 C 0.2 12 CDA-II 0.6 38 50 58 C 0.2 12 CDA-II 0.8 65 77 80 C 0.2 12 CDA-II 0.4 16 36 39 Bj7 0.05 CDA-II 0.6 38 58 58 B17 0.05 CDA-II 0.8 65 85 83 Bn 0.05 The experiment was carried out according to Figure 6. HBL-100 cells were cultured with different dosages of CDA-II, vitamin C and vitamin Bn alone, and with the combination of CDA-II and vitamin C, or the combination of CDA-II and vitamin B17.

The present invention may be embodied in other specific forms without departing 10 from the spirit of the essential attributes thereof; therefore, the illustrated embodiment should be considered in all respects as illustrative and not restrictive, reference being made to the appended claims rather than to the foregoing description to indicate the scope of the invention. 21 Industrial applicability Compared with prior art, the advantage of the present invention is utilizing the anticancer substances naturally occurring in human bodies to treat and prevent cancer, 5 thereby making the cancer cells to differentiate and stop dividing. Therefore, the present invention is an effective treatment to eliminate the cause of the disease present only in cancer cells, and the medicine according to the present invention has no adverse effect. 22 INTcLLcGrUAL rrtUrfcrilT OFPJCE OP N2 19 FEB 2004 received

Claims (7)

WHAT WE CLAIM IS:

1. A pharmaceutical composition for use in treating and preventing cancer by inducing cancer cells to differentiate, which comprises cell differentiation agent targeting at 5 abnormal methylation enzymes of cancer cells and a carrier; wherein said cell differentiation agent and said carrier are extracted and purified from human urine.

2. A pharmaceutical composition according to claim 1, wherein said composition is made in the form of an injection formulation, a capsule, and a tablet 10

3. A pharmaceutical composition according to claim 1, wherein the composition is combined with thymidine.

4. A pharmaceutical composition according to claim 1, wherein said composition includes differentiation inducers and differentiation helper inducers; wherein said differentiation inducers are PP-O and OA-O.79, and the 15 differentiation helper inducers further comprise at least phenylacetic acid, hippuric acid, indole acetic acid, uroerythrin and vitamin B2.

5. A pharmaceutical composition according to claim 1, wherein said composition further comprises components for preventing cancer patient from excessive excretion. 20

6. A pharmaceutical composition according to claim 1, further comprises vitamin C and vitamin B17.

7. A method for the preparation of the composition according to claim 1, comprising the steps of; (1) collecting human urine, in which the concentration of creatinine is in the 25 range of 1.2 - 3.7 g/1; (2) passing the collected urine through an adsorbent such as XAD-16 or the like; (3) extracting with an organic solvent such as ethanol or the like; and (4) recovering and purifying the cell differentiation agent 23 Use of cell differentiation agent targeting at abnormal methylation enzymes of carrier cells in the manufacture of a medicament for treating and preventing cancer by inducing cancer cells to differentiate. A use as claimed in claim 8 wherein said cell differentiation agent is prepared by the method of claim 7. A pharmaceutical composition as claimed in claim 1 substantially as hereinbefore described with reference to the Examples. A use as claimed in claim 8 substantially as hereinbefore described with reference to the Examples. END OF CLAIMS 24 INTELLECTUAL PROPERTY OFFICE OF N2 19 FEB 2004 received