WO2000037091A1 - Pharmaceutical composition inducing cancer cell differentiation and the use for treatment and prevention of cancer thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treatment and prevention of cancer and preparation method thereof, especially to a cell differentiation agent which is named CDA-II prepared by extracting and purifying fresh human urine. The pharmaceutical composition is superior in effective for treatment and prevention of cancer. These effective components contain a differentiation inducer, a differentiation adjuvant and an anticachexia agent in CDA-II, and cooperate each other to achieve the best medical effect.

Description

 Drugs that induce cancer cell differentiation

 And its use in the treatment and prevention of cancer

 The invention relates to a medicine for treating and preventing cancer and a preparation method thereof, namely, a cell differentiation agent CDA-II (Cell differentiation agent) extracted and purified from fresh human urine. The preparation has good effects on the treatment and prevention of cancer.

 The invention discloses that CD A-II can release the abnormal state of cancer cell methylation complex enzymes, effectively promote the terminal differentiation of cancer cells, and achieve medical effects. This is a novel anti-cancer method aimed at eliminating the cause of the disease. It has been confirmed through experiments that the medical effect is quite significant. C D A-Π is a protein specific to cancer cells that binds to methyltransferase and eliminates its effect, so it has specific selectivity in treatment and has no adverse side effects. The active ingredients of C D A-II include differentiation inducers, differentiation aids, and anti- cachexia agents. These active ingredients work together to achieve the most effective medical effect.

 The combination of CDA-II and other anticancer drugs such as thymidine has better anticancer effects, and the synergistic anticancer effect is very obvious.

CDA-II is used in combination with Vitamin C and Vitamin B ₁₇ and has a very good anti-cancer effect.

Cancer is a fairly complex series of diseases, but cancer has one thing in common, that is, all cancer cells have the ability to divide continuously and fail to undergo terminal differentiation. The inventors found that the abnormal methyl transfer complex is the most important cause that constitutes a common feature of cancer. The present invention aims at this cause to seek medical treatment and prevention of cancer. A purified cell differentiation agent can be extracted from the urine of normal people. The preparation can restore abnormal methyl transfer complex alcohols of cancer cells to normal. As a result, cancer cells are induced to undergo terminal differentiation and no longer continue to divide or even die. To achieve the medical and preventive effects of cancer. This is a selective therapy, so the therapy is safe; and because healthy people also rely on these chemicals to fight cancer, this therapy can be said to be a natural anti-cancer method without adverse side effects. The active ingredients of the cell differentiation agent are a differentiation inducer and a differentiation assistant. Differentiation inducer can eliminate abnormal protein factors unique to cancer cells that bind to methyltransferase

(Ie, abnormal tumor specific protein factor), the differentiation aid is an inhibitor of each member enzyme of the complex enzyme, and has the function of enhancing the differentiation inducer. Cell differentiation agent in the present invention, wherein the differentiation inducing agent is PP- 0 and OA- 0. 7 9, comprising at least differentiation aids acid, hippuric acid, indole acetic acid, red urine and vitamin B _2. In addition, cell differentiation agents contain anti-excessive ingredients such as phenylacetylglutamine. Since most cancer patients have symptoms of excessive excretion, this ingredient is also quite helpful for the treatment of cancer. All in all, the cell differentiation agent has a variety of effective ingredients to assist each other to produce excellent medical effects. The inventors have also found that the use of cell differentiation agents in combination with other cytotoxic drugs such as thymidine will produce better anti-cancer effects. It has also been found that the combination of cell differentiation agents with vitamin C and vitamin B ₁₇ can effectively promote anti-cancer effects.

Background technique

 Oncogenes are part of the genes of human cells, so humans have cancer. There is still no good way to overcome this disease. The reason why cancer is difficult to treat is that the composition of the cancer cells themselves is complicated. However, it is more important to find appropriate prevention and treatment measures. Cancer cells continue to divide, invade normal organs and tissues, and eventually cause serious illness and death. The traditional medical community always regards the continuous division of cancer cells as the most fundamental crux of cancer, and then seeks cytotoxicity to stop D N A synthesis to prevent cell division, which is why cytotoxic therapy. The guidelines for treating cancer for the past four decades have focused on the development of cytotoxic therapies. The cancer medical market is also monopolized by this drug. Yet humans are constantly seeking other breakthrough new therapies.

The well-known vitamin A acid (retinoic acid) is used as an anticancer agent. Although the effect is very good (Huang et al., 1988; Warrell et al., 1991 references 8 and 28), the cancer cells relapse soon (Muindi et al. al, 1992 Adamson et al., 1993 References 1 and ^25). Relapse is not completely related to the differentiation induced by the differentiation inducer alone, because the differentiation inducer alone can cause damage and prevent cells from completing the differentiation procedure. The references cited and referred to in the present invention are as follows:

 1. Adamson PC, Boylan JF, Balis FM, Murfy RF Godwin KA, gudas LT, Poplack DG induces the time course of total trans-retinoic acid metabolism and the positive regulation of cellular retinoic acid (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 SL, Kastendieck JG Detection of appendicitis by measurement of uroer thrin. U.S. Patent 5, 053, 389, 1991.

 3. Borek E. et al. Altered excretion of modified nucleosides and β-aminoisobutyric acid in subjects with acquired immunodeficiency syndrome or at risk for acquired immunodeficiency 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, 1998.

 5. Clark PMS, Kricka LJ, Whitehead TP Use iso-Dalt technology to study the 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 (Annnu. Rev. Biochem.), 52: 93-124, 1983.

7. Epifanova, OI, Abuladze, MK, and Zoniovska, AI Effect of low concentrations of actinomycin D on the rapid proliferation and stimulated cell culture initiation of DNA synthesis in rapidly proliferating and stimulated cell cultures.) Exp. Cell Res., 92: 25-30, 1975.

 8. Huang, ME, Ye, YC, Chen, SR, Chai, JR, Lu, JX, Zhoa, L., Gu, LJ, and Wang, ZY, all-reflective yellow in the treatment of acute promyelocytic leukemia 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 (Cell), 40: 484-486, 1985.

 10. Kampalath, B.N., Liau, M.C, Burzynski, Beta, and Burzynski, S.R. Prevention of benzo (a) pyrene-induced lung cancer with antitumor agent A10 chemotherapy

(Chemoprevention by Antineoplaston A10 of Benzo (a) pyrene-induced Pulmonary Neoplasia.) Clinical Research on Drug Development (Drugs Exptl. Clin. Res.), 13 (Suppl.): 51-56, 1987.

 11. Liau, M.c, Smith. D.W., and Huriibert, R.B., lhomoptyribo Preferred inhibition of riboside methylation and destruction of ribosome production by riboside

(Preferential in inhibition by lhomopty ribonucleotides of the methylation of ribosomal ribonucleic acid and disruption of the production of ribosomes in a rat tumor.) Cancer Res. 35: 2340-2349 1975b.

 12 丄 iau, Mc, Huno. ME, and Huriibert, Role of ribosomal RNA methylases in the regulation of ribosome production in mammalian cells. Biochemistry (Biochem.), 15: 3158-3164, 1976.

13. Liau, Mc Lin. GW, and Huriibert, RB, Partial purification and characterzation of tumor and liver S-adenosyimethionine synthetases. Cancer Res., 37: 427-435, 1977a. 14. Liau, Mc, Lin GW, Kmight, CA, and Huriibert, RB, Inhibitor of RNA Methylation by Intercalating Agents. Cancer Res., 37 ·, 4202- 4210. 1977b.

 15 丄 iau, MC, Chang, CF and Becker, FF Alteration of S-adenosylmethionine synthetase during chemical hepatocarcinogenesis and in resulting carcinomas. ) Research (Res.), 39: 2113-2119, 1979.

 16 丄 iau MC, and Burzynsk ^ SR Altered methylation complex isozymes as selective targets for cancer chemotherapy. Alters methylation complex isozymes as selective targets for cancer chemotherapy. Drugs Exptl. Clin. Res ), 12 (Supplement): 77-86, 1986.

 17. Liau., MC, Szopa, M., Burzynski, B., and Burzynski, SR Chemical surveillance: a new concept of the natural defense mechanism against cancer. ) Drugs Exptl. Clin. Res., 12 (Supplement) 71-76, 1987b.

 18 丄 iau, MC, Lee, SS, and Burzynski, SR Differentiation inducing components of antineoplaston A5. Advances in clinical chemotherapeutic experiments (Adv. Exptl. Clin. Chemother.), 6/88 : 9-25, 1988.

 19 丄 iau, MC Lee, SS, and Burzynski, SR Insufficient methylation of nucleic acids: Hypomethylation of nucleic acids: a key to the induction of terminal differentiation. International Journal of Clinical Chemotherapy Experiments ( Intl. J. Exptl. Clin. Chemother.), 2: 187-199, 1989.

20 丄 Iau, MC and Burzynski, Separation of active anticancer components of antitumor agents A ₂ , A ₃ and A ₅ (Separation of active anticancer components of antineoplaston A2, A3 and A5.) International Journal of Histological Response (Intl. J. Tiss. React.), 12 (Supplement): 1-18 1990a.

 21. Modulation of cancer methylation complex isozynski, SR Modulation of Liau, MC Lee, SS, and Burzynski, SR as determinants in the induction of antitumor agent AB-mediated terminal differentiation cancer methylation complex isozymes as a decisive factor in the induction of terminal differentiation mediated by antineoplaston AB.) International Journal of Histological Response (In «. J. Tiss. React.) 12 (Supplement): 27-36, 1990b.

22. Liau, MC, Asohraf A., Lee, SS, Hendry, LB and Burzynski, SR riboflavin, a low activity antitumor component of antitumor agents A ₂ and A ₅

(Riboflavin as a minor active anticancer component of Antineoplaston A2 and A5.) Intl. J. Tiss. React. 12 (Suppl.): 18-26, 1990c.

 23 丄 iau, MC, Liau, CD., Burzynski, SR Potanilation of induced terminal differenation by phenylacetic acid and related chemicals, Intl. J. Exptl Clin. Chemother.) 8: 9-17, 1992a.

 24. Liau, MC, Luang, Y "Liaum CP., And Burzynski, SR Prevention of drug-induced DNA hypermethylation by antineoplastin components. Intl. J. Exptl. Clin. Themother.), 5, 19-23, 1992b.

25. Muind: Clinical pharmacology of JRF, Frankd, SR "Huselton, D., Degrazia, F., Garland, WA, young GW, and Warrell, RP in patients with acute promyelocytic leukemia. Clinical pharmacology of oral all-trans retinoic acid in patients with acute promyelocytic leukemia.) Cancer Res., 52: 2138-2142, 1992. 26. MuIdoon TG, Copland JA, Hendry LB, Antineoplaston A10 activity on carcinogene-induced rat mammary tumorsm induced by carcinogens, Intl. J. Tiss. React .) 12 (Supplement): 51-56, 1990.

 27. TonioaI, D "Weiss, HK, and Basilio, CA temperature sensitive mutation affecting 28S ribosomal RNA production in mammalian cells (Pric. Natl. Acad. Sci. USA), 70: 1273- 1277. 1973.

 28. WarreIl, RP Jr., Frankel, SR, Miller, WH Jr. Sheinberg, DA, Itri, LM, Hettelman, WN, Vyas, R "Andreeff M., Tafuri, A., Jakubowski, A, Gabrilove., J ., Gordon, Ms "and Dmitrovsky, E. Differentiation therapy of acute promyelocytic leukemia with tretinoin (alI-trans-retinoic acid).) New England Journal of Medicine (N Engl. J. Med.), 324: 1385-1393, 1991.

 29. Spielhoiz. C., Golde. DW, Houghton, AN, Nualart, F., and Vera JC do not alter sodium-dependent ascorbic acid transport and increase dehydroascorbic acid transport in human melanoma (lncicased Facilityted Transport of Dehydroaseorbic Acid without Changes in Sodium-dependent Ascorbate Transport in Humen Melanoma Cells.) Cancer Res. 57: 2529-2537, 1997.

 30. Kamanoro Sano, "Eliminating Cancer", Shimao Publishing House, Taipei, Taiwan 1997.PP202,203

 Summary of the Invention

The object of the present invention is to provide a medicament for inducing differentiation of cancer cells and its use in the treatment and prevention of cancer. As mentioned above, cancer is a series of rather complicated diseases, but cancer has one feature in common, that is, all cancers. Cells have a constant division Ability, and terminal differentiation. The present invention unexpectedly found that abnormal methyltransferase is the most important cause of the common feature of cancer. Therefore, the present invention seeks the medical treatment and prevention of cancer based on this cause.

 The inventors have accumulated more than thirty years of intensive research and found that the abnormal methyltransferase is the most fundamental crux of cancer, because the abnormality of this enzyme leads to the continuous division of cancer cells. Methyltransfer complexes play an important leading role in cell division and differentiation. When these enzymes have high activity, they divide. When the activity changes from high to low, the cells synthesize methyl-deficient nucleic acids, and as a result, the cells are induced. Differentiate into terminally differentiated cells that no longer divide. All cancer cells have abnormal methyltransferase enzymes. These enzymes are immobilized in a highly active and stable state, so the cells continue to divide. Therefore, abnormal methyltransferase enzymes are the cause of cancer and continuous cell division. It is a symptom of cancer. Because cancer cells are caused by abnormal methyl transfer complexes, if effective drugs can be found to inhibit this abnormal enzyme, it will have a therapeutic effect on the existing disease and a preventive effect on the non-existing disease. In fact, there are enough chemicals in the normal human body to inhibit the formation of these abnormal methyltransferases. Therefore, the process of carcinogenesis takes a long time to promote, during which the patient accelerates the excretion of chemicals with anticancer effects. When these anticancer substances are reduced enough to inhibit abnormal methyltransferases, cancer cells begin to take root and multiply, and finally symptoms appear. Normal people also excrete a small amount of anti-cancer substances from the urine. After extraction and purification, these substances can selectively inhibit abnormal methyltransferases. As a result, cancer cells are induced to differentiate like normal cells. Because normal methyltransferases do not have anomalous factors, normal cell growth and function are not affected by these anticancer substances. Among anticancer drugs, this is the only chemical that is specifically selective, so patients do not suffer from adverse side effects. This kind of therapy, differentiation therapy, can be said to be a selective and natural cure, which is the inherent anti-cancer method of human beings. I am afraid it is difficult to find drugs that can be used among anticancer chemicals.

In general, although normal healthy people excrete some of these chemicals that can cause cancer cell differentiation from the urine, they can maintain a balance between gains and losses, so there are always enough differentiation-inducing chemicals in the body to inhibit the formation of cancer cells. Instead, cancer patients Excessive excretion loses the ability to control the proliferation of cancer cells. Measured from this perspective, differentiation therapy is not only a cure for the root cause, but also a natural anti-cancer recipe. Since there are chemicals in urine that can induce cancer cells to undergo terminal differentiation, the present invention extracts and refines cell differentiation agents from normal human urine. The preparation can restore abnormal methyl transfer complex enzymes of cancer cells to normal. It is induced to undergo terminal differentiation without continuing to divide and even die, achieving the medical and preventive effects of cancer.

 The present invention is implemented in this way. Since humans have adopted relatively inadequate measures to treat cancer for many years, the effect is not significant. The goal of treatment should be on the radical therapy of "methyl transfer complex alcohol". Cancer cells are caused by abnormal methyltransferases. Inhibiting such abnormal enzymes with drugs has a therapeutic effect on the existing disease and a preventive effect on the non-symptomatic disease. This therapy is an excellent treatment for both Western and Chinese medicine. Due to the excretion of a small amount of chemicals that can induce cancer cells to undergo terminal differentiation in normal human urine, the present invention extracts and refines the abnormal methyl transfer complex enzymes of cancer cells to normal, Cell differentiation agent and preparation capable of inducing cancer cells to undergo terminal differentiation without continuing to divide, so as to achieve medical and preventive purposes. The preparation of the present invention can selectively inhibit abnormal methyl transfer complex enzymes. As a result, cancer cells are induced to differentiate like normal cells. Because normal methyl transfer complex enzymes do not have abnormal factors, the growth and function of normal cells are not stable. Will be affected by these anti-cancer substances. Among the anti-cancer chemotherapies, this is the only one with specific selectivity. It is a natural cure for the disease. Healthy people also rely on these chemicals to fight cancer. In other words, this is an inherent anti-cancer method in humans. Anticancer drugs are hard to find.

The active ingredients of the cell differentiation agent are a differentiation inducer and a differentiation assistant. Differentiation inducers can eliminate abnormal protein factors that are unique to cancer cells and bind to methyltransfer complex enzymes. Differentiation assistants are inhibitors of the enzymes of each member of the complex enzyme, and have the function of promoting differentiation inducers. An essential component of differentiation therapy, although it may not be as important as a differentiation inducer. Cell differentiation agent in the present invention, wherein the differentiation inducing agent is PP- 0 and OA- 0. 7 9, comprising at least differentiation aids acid, hippuric acid, indole acetic acid, red urine and vitamin B _2. In addition, another component contained in the cell differentiation agent of the present invention is also quite helpful for cancer treatment, that is, an anti-cachexia agent. The inventors of the present case found that stupid glutamine can combat the excessive excretion caused by cachexia symptoms of cancer patients (Liau et al., 1987; Muldoon et al., 1990 references 10 and 26), which is the main part of Figure 4 in Part 4 ingredient. Because most cancer patients have symptoms of excessive excretion, this ingredient is also quite helpful in the treatment of cancer. All in all, the cell differentiation agent has a variety of effective ingredients to assist each other to produce excellent medical effects.

 The present invention has also found that the use of the cell differentiation agent CA D-II in combination with other cytotoxic agents such as thymidine will produce a better anti-cancer effect.

In addition, the use of the cell differentiation agent CAD-II of the present invention in combination with vitamin C and vitamin B ₂ will produce a better anti-cancer effect.

 The cell differentiation agent of the present invention is prepared by first collecting normal human urine with a certain creatinine concentration, and then preparing the preparation through adsorption with an adsorbent and extraction, recovery, and purification with an organic solvent. The preparation of the present invention is an injection. Can be made into capsules or other pharmaceutical dosage forms.

 Compared with the prior art, the present invention has the advantage that the present invention uses the body's own anti-cancer substance to manufacture drugs for treating and preventing cancer, which fundamentally differentiates cancer cells without continuing to divide to achieve the purpose of treatment. Therefore, the present invention It is an effective method to cure the root cause, and the medicine of the present invention does not have any adverse side effects.

Overview of the drawings

 Please refer to the following detailed description of the preferred embodiments of the present invention and the accompanying drawings to further understand the technical content of the present invention and its purpose and efficacy. The drawings of the related embodiments are: Figure 1: Production of the cell differentiation agent of the present invention flow chart.

Figure 2: Gel filtration analysis diagram of the cell differentiation agent of the present invention; CDA-II injection is freeze-dried and concentrated, and then subjected to column chromatography, which is a chromatography column using B i ₀ -G e 1 P 2 ( 4 · 1 cm X 4 4 cm). The collected fractions were measured for the absorbance of A 225 (0-0), the determination of inhibition of MAT ^LT activity (0… 0), weight percentage, and HL- ⁶ 0. Differentiation activity of cancer cells (represented by% NBT +).

Figure 3: Urine globulin (0-0) and vitamin B ₂ (0… 0) as differentiation aids Graph of concentration versus reduction index.

Figure 4: A graph showing the relationship between the inhibitory activity of urin (0-0) and vitamin B ₂ (0… 0) on t RNA methyltransferase concentration and its concentration;

Figure 5: Promoting effect of urogranin and vitamin B ₂ on differentiation inducers, the horizontal axis represents the concentration of vitamin A acid (μM), and the vertical axis represents NBT (nitroblue tetrazolium) + cells Percentage. 0—0 means the control group with different concentrations of vitamin A acid; 0… 0 means the experimental group with 4 μM vitamin B ₂ ;

 0—. 1. A 0 represents the experimental group with 4 μM urocalcin added.

 Figure 6: A synergistic anti-cancer effect of the combined use of CD Α-II and thymidine according to the present invention. The horizontal axis represents the dose (mg / ml), and the vertical axis represents the relative activity (%) of cancer cell group formation. 0—0 means the group using thymidine alone; 0… 0 means the group using CD A—II alone; 0—. A. — 0 means the group using thymidine in combination with CD A—Π (1: 1).

 The activity of cancer cell colony formation was measured according to Example 4 using H B L-100 breast cancer cells, which is expressed as the relative percentage of cancer cell group formation in the control group without the addition of drugs. C D A-II and thymidine were used alone and 1: 1 in combination. Among them, three experimental groups are used, and their anti-cancer effects are shown in Fig. 6, respectively, which proves that the combined treatment has stronger anti-cancer effect than CD A-II or thymidine alone, and its synergistic enhancement effect is very significant.

Figure 7: The anti-cancer effect of CD A-II and antioxidants Vitamin C and Vitamin B ₁₇ , the activity of cancer cell colony formation was measured according to Example 4 using HBL-100 breast cancer cells. After 5 ml of each culture flask medium containing 3000 th HBL-1 0 0 cells, and various doses of CD A- II, vitamin C or vitamin B _17, cultured in 375 days, the cell population stained Giomsa Under the dissection microscope, count the population of more than 8 cells.

AdoHcy is S-adenosylhomocysteine and SAHH is S-adenosylhomocysteine (S- adenosylhomocystei ne hydrolase) the best embodiment of the present invention

 The production flow of the cell differentiation agent of the present invention is shown in Fig. 1. The following specific examples are used to further illustrate the present invention, and the scope of the present invention should not be limited accordingly. Example 1: Preparation of a cell differentiation agent

 The present invention includes collection, filtration, adsorption, solvent extraction and drying of urine, and dissolving with pyrogen-free water as the raw material of CDA-II.

 When collecting urine, a sufficient amount of 1 N hydrochloric acid is placed in the collection bucket, which is about 20 liters of urine for one liter of hydrochloric acid. This promotes the activity of the differentiation agent and can be maintained for at least one month without being damaged.

 After PH is adjusted to 2, urine is first coarsely filtered with nylon cloth, and then finely filtered to remove substances having a molecular weight greater than 10,000 Daltons (Amicon filter fibers or other conventional methods can be used). The adsorbent XAD—16 (Sigma factory) is packed in a cloth bag and placed in a stainless steel filter bucket. Before use, it must be washed with twice the volume / weight (V / W) of ethanol, and then twice (V / W). Rinse off the ethanol with deionized water, and repeat this twice. Each time the urine passed through the adsorbent X A D-16 was washed with 4 times the capacity (V / W) of deionized water, and then dissolved with 2 times the capacity (V / W) of ethanol. After neutralizing the ethanol extract, evaporate the ethanol with a desiccator, and keep the temperature in the drying bucket below 5 ° C. The dried material is dissolved in pyrogen-free water as the drug substance for CD A-II.

 After the ethanol-extracted adsorbent is washed with 2 times the capacity (V / W) of deionized water, it can be reused until the adsorption capacity is reduced to about 70% of the new adsorbent, and then the new adsorbent is replaced. Generally, XAD— 16 can be reused about 200 times or more.

Since the human body excretes a certain amount of creatinine every day, the solid concentration in urine is approximately proportional to creatinine, so the quantification of chemical substances in urine is always more reliable based on creatinine. The urine collected as a raw material has a creatinine concentration between 1.2 and 3.7 g / L, with an average of 2.4 ± 0.6 g / L. The yield of CD A-II was about 0.5 1 ± 0.1 7 g / g of creatine S in the first hundred passes of the adsorbent. The solid content of urine is about 4 6.7 g / g of creatine gf, so the yield of the prepared CD A drug substance is about 1.1% of the solid content. Example 2: Preparation of cell differentiation agent injection

The final concentration of C DA-II injection is about 50 ± 2 g / ml, and the concentration of the drug substance is generally above 250 mg / ml, so the concentration of the diluted drug substance should be 1 3 About 0%. After the first hot raw water was diluted to a pharmaceutically acceptable material, through a series of filters, first with slow filter paper, and then followed by 1111 was filtered off 0. ⁴ 5 μ m filter membrane pores -, and finally with a special filter Membrane depyrogenation, the filtrate after depyrogenation, adjusted to the appropriate concentration with depyrogenized water, passed through a 0.2 2 μm filter in 8 hours, and completed in a sterile operation room (class 100 clean room) Filter sterilize. Immediately after sterilization, 100 ml or 250 liters of differentiation agent injection solution was prepared. Example 3: Preparation of cell differentiation agent capsule

 Part of C D A-II can be used to make capsule preparations. The above APIs are successively filtered through microfiltration membranes with slow filter paper, 1 μm and 0.4 5 μm filter holes. The filtrate was dried to a solid by freeze-drying. After the dried solids were ground into powder, the capsules were filled with 500 g of capsules using an automatic capsule machine, filled with aluminum foil, sealed, and finally sterilized by radiation. Example 4: Analysis of anticancer activity of cell differentiation agents

The anti-cancer effect of the cell differentiation agent is based on the active ingredient's inhibition of cancer cell abnormal methyl transfer complex enzymes, so the cancer cells are induced to undergo terminal differentiation to stop cell division, that is, the activity of the cell differentiation agent has the inhibition of the cancer abnormal enzyme MAT The effects of ^LT (methionine adenosyltransferase) can induce the differentiation of HL-60 cancer cells and inhibit the formation of human breast cancer cell populations, and these methods of measuring activity have been published in the literature (Liau et al. People, 1977a; 1988; 1990a References 13; 18 and 20). The present invention takes three batches of CD A-II preparations to analyze its anticancer activity, and details its operation. Proceed as follows.

The CD A-II injection formulation was used at a dose of 1 mg / ml. MA T ^LT is made from HL-60 cancer cells. The precipitated cells are first suspended in 0.0 5 MT ris, p H 7, 0.5 mM MgCl ₂ , and then the cell membrane is broken with a Dounce homogenizer.踌 The extract was separated by high-speed centrifugation (2 26, 0 0 0 X g, 0.5 hours). The extract liquid DEAE- cellulose chromatography in a gradient of solution of KC 1 MAT ^LT dissolved out and be purified (Liau et al., 1977a Reference 13). MAT ^LT activity was measured according to the previous method (Liau et al., 1977a reference 13), 0.0 5 ml reaction solution including 0.0 5 MT ris, p H 8.2, 15 MKC 1, 15 mM M g Cl ₂ , 5 mM DTT (dithiothreitol), 2 mM AT P and 1 μM [3 ^Η — CH ₃ ] methionine, 3 reacted for 30 minutes. The reaction solution was acidified, and then all were transferred to 1 square inch of phosphate fiber paper. These papers were washed with 5 mM phosphate buffer, p H 7 in the beaker several times, and the unreacted [ ³ H—CH ₃ ] was washed away. Methionine, and finally measured the absorbed [ ³ H—CH ₃ ] Adomet (S-adenosyl methionine) radiation to determine the activity of MAT ^LT (see Table 1 for results) .

 The terminal differentiation of HL-60 cancer cells is based on the N B T + assay (Liau et al., 1988 reference 18). At the beginning of the experiment, HL-60 cells were diluted and cultured. Each culture bottle was filled with 10 liters of starting cells at a concentration of 1.5 X 10 cells / ml. The control and the culture bottle containing CDA-II were all passed through 9 6 After an hour of culture, remove a small portion and pellet the cells with a centrifuge. After these cells are treated with NBT reagent at 37 * C for half an hour, remove a small drop and place it in a cell counter and count the total number of cells and stain under a microscope. Number of cells that turned black (NBT +). N B T + the percentage is C

DA-II activity to guide differentiation (see Table 1 for results).

 CD Α— Π Another indicator of anticancer activity is the inhibition of HBL-1 0 0 breast cancer cell populations. The experiment started by washing the breast cancer cells with an increase in the proportional order once with physiological saline, and then adding about 2 ml of 0. 0 5% trypsin-0.5 3 mM ED

TA culture solution was incubated at 37 for 10 minutes. Measure cell concentration first, then take a portion of the dilute Release ³ x ¹⁰³ cells / ml. Take 0.5 ml of the dilution and add 4.5 ml of the culture bottle containing the control or CDA-II to culture at 37 V. After five days, the culture solution was decanted, washed with physiological saline once, and then fixed with methanol for 15 minutes, and the fixed cell population was soaked with Giemsa staining solution diluted 20 times for 30 minutes. Pour out the staining solution, wash and dry it, and count the number of cell populations under a dissecting microscope to determine the anti-cancer activity of CDA-II (see Table 1 for results). Example ⁵ : Analysis of effective components of cell differentiation agents

The CDA-II cell differentiation agent injection prepared in Example 2 was freeze-dried and concentrated to 200 mg / ml. 5 ml of the concentrated solution was added to a chromatographic column ( ^4.1 cm X 4 4 cm) of Bio-Ge 1 P 2 and then eluted with distilled water. One tube was collected automatically every 7 minutes. 0 ml. After the separation was completed, 25 microliters were taken from each tube, diluted with water to 1 liter, and the absorbance at A _{225 nm} was measured. In addition, 25 microliters were taken from each tube for the measurement of MA T ^LT activity inhibition. The measurement of MA T ^LT activity was performed according to the method described in Example 4. Then, the collection tube was divided into 8 parts as shown in FIG. 2 according to the absorbance of A ^^, and each part was completely evaporated to dryness by freeze-drying, and the drying amount was determined to determine the weight percentage distribution. The dried solid was re-dissolved in distilled water to determine the differentiation activity of HL-60 cancer cells. This activity was expressed as% NBT +. The differentiation activity was measured according to the method described in Example 4. The results are shown in Figure 2 .

Of the active ingredients of cell differentiation agents against cancer, the most important is of course an inducer that can induce cancer cell differentiation. The isolation, purification and mechanism of action of differentiation inducers have been published in the literature (Liau et al., 1988; 1989; 1990a; 1990b References 18, 19, 20 and 21). Differentiation inducers have two main chemical components; one is an acidic peptide that binds to pigments, which is called PP-0, and the other is an organic acid, referred to as OA-0.79. PP- 0 is the first part of the divided as shown in FIG. 2, Ο Α-0 · ⁷ 9 at portions 5 and 6. It should be particularly noted here that the performance of the differentiation activity of the cell differentiation agent is not caused by the separate action of the differentiation inducer, but often due to the synergistic effect with the differentiation assistant. Come out of activity. Instead, the differentiation aid is the main chemical component of the cell differentiation agent. The content of the differentiation inducer is very small, and the chemical structure is still unknown, because it has not been purified to a certain degree. As shown in Figure 2, the differentiation-inducing activity and the MAT ^LT inhibitory activity are in good agreement, but in some parts, as shown in Figures 2 and 3, although there is obvious MAT ^LT inhibitory activity, it does not induce cancer cell differentiation Of activity. This part may only have a differentiation aid and no differentiation inducer, so it does not have the activity to induce cancer cell differentiation.

 Differentiation auxiliaries are inhibitors of individual member enzymes of methyl transfer complex enzymes (Liau et al., 1992a reference 23), which can assist differentiation inducers to restore abnormal methyl transfer complex enzymes to normal enzymes, thus promoting differentiation inducers Differentiation function.

Among the CDA formulations known as MAT inhibitors are phenylacetic acid, indoleacetic acid, and hippuric acid. Phenylacetic acid is likely to be a hydrolyzate of phenylacetylglutamine during drying. It is distributed in parts ⁶ and 7 of Figure 2. It can be determined by C 1 8 HPLC (high performance liquid chromatography). Indole Acetic acid is also in this part. Hipporic acid is the main component of Part 5. The concentrations of these MAT inhibitors to achieve a 0.5 reduction index (ie, halving the effective dose of the differentiation inducer) were 4 mM phenylacetic acid, 8 mM uric acid, and 0.9 5 mM indoleacetic acid. Inhibitors of methyltransferases, which are differentiation aids, are much more effective than inhibitors of MAT. The former can often achieve the same effect as long as the latter is one thousandth or less.

There are two known methyltransferase inhibitors in the CDA preparation, both of which have good differentiation aid activity. These two ingredients are vitamin B ₂ and urocalcin. Vitamin B _{2 is} distributed in the second half of Part 8 of Figure 2. As mentioned above (Liau et al., 1990C), this yellow component can be purified by C 1 8 HPLC to obtain a very pure vitamin B ₂ with a content of about 0.0 4 About 6%, urocalcin is shown in parts 6 and 7 of Fig. 2. The fraction containing urocalcin can be purified by Sephadex SH column chromatography and thin-layer silica gel chromatography. The content is about 0.5% of the CD A-II preparation. 5%。 Due to the inevitable loss of the purification process, and the CDA-II preparation has obvious red, so the urine erythrodin content may be more than 0.5%. Example 6: Determination of the activity of differentiation aid of urogranin and vitamin B ₂ The activity of the differentiation aid was determined according to the method designed by the inventor of the present invention, Mr. Liao Mingzheng and others (Liau et al., 1992a Reference 23). In the experiment, the cultured HL-60 white blood cancer cells were used to determine the degree of differentiation of cancer cells by the quantitative method of NBT. First start of the experiment HL- 6 0 cells were diluted culture flasks each containing 1 0 ml solution starting cells, a concentration of 1.5 x 1 0 ⁵ cells / ml of a set of four control flasks as its only Vitamin A acid differentiation inducer was added, the dose was adjusted to induce NBT + cells between 15% and 60%, and a control bottle containing only solvent was added. Vitamin A acid is soluble in methanol, but the total amount of methanol added must not exceed 2%, so it will not affect the differentiation of cancer cells. Each of the other groups also has four culture flasks, but with a relatively low dose of vitamin A acid, and a control bottle with only a solvent, each group is added with a different dose of differentiation aid. After 96 hours of incubation, the number of cells in each bottle was measured, and the NBT measurement was performed according to the method described in Example 4. Generally speaking, the natural differentiation of HL-60 cells, that is, without any additives, is always less than 4%. In the dose range of the experimental group with only differentiation adjuvants, cell differentiation will probably not exceed 10%. The NB T + value of each bottle needs to be subtracted from the base of these control values.

Draw the curve of the relative relationship between dose and NBT + to obtain the ED ₅₀ value, which is the dose of differentiation inducer required by NBT + 50%. From these ED ₅ o values, the reduction index of differentiation aid can be calculated. Reduction index = ED ₅₀ with differentiation aid / ED _{5 ()} alone. The lower the reduction index, the higher the function of the differentiation aid.

The results are shown in Fig. 3. The concentration of vitamin B ₂ and urothalin to reach 0.5 reduction index is 3.0 μM and 1.8 μM, respectively, which is much higher than the above-mentioned MA T inhibitor activity. Example 7: Inhibition of t RNA methyltransferase by urocalcin and vitamin B ₂

t RN A methyltransferase is prepared from HL-60 cancer cells and is a high-speed cell extract according to Example 4. The cell extract was adjusted to pH 5 first, and the precipitated protein was separated by a centrifuge, and then dissolved in 0.0 5 MT ris, p H 7.8, A mixture of 0.5 mM Mg Cl ₂ and 5 mM HS CH ₂ CH ₂ OH. This enzyme solution was further purified by DEAD-cellulose chromatography, elution of t RN A methyl transfer with KC 1 gradient eluent (Liau et al., 1977b reference

1 4). t RN A methyltransferase activity was measured in 0.2 5 ml reaction solution, which included: 0.0 5 MT ris, p H 7.8, 0.1 M NH ₄ C 1, 0.0

4 MNH ₄ F, 0.5 mM M g C 1 ₂ , 5 mM DTT, 20 μg E.coli Beta t R Ν A, 0.2 5 C i [ ³ H — CH ₃ ] AdoMet (5 -adenosine A Thionine), and 25 micrograms of enzyme. The reaction was performed at 37 for 30 minutes. t RNA was precipitated with cold 5% TCA (trichloroacetic acid), and then collected by filtration on a Millipore (membrane pore 0.45 micron) film. After the film was dried, the amount of radiation was measured to determine the t RNA methyltransferase. active. The results are shown in Figure 4.

However, the effective dose to inhibit methyl transfer activity is much higher than the effective dose of differentiation aids, which may be related to the physical and chemical conditions for measuring these different activities. Different physical and chemical conditions (such as salt concentration) will affect the chemical substances and enzymes. Effective contact. Although the sensitivities are quite different, the activity of different methyltransferase inhibitors and differentiation aids maintains a certain proportional relationship. The more effective the methyltransferase inhibitor, the more effective the differentiation aid. We have also found other inhibitors of methyltransferases, such as ethidium bromide and hycanthone (Liau et al., 1977b reference 1 4), which are similar to urin and vitamin B ₂ Good differentiation aid activity. The concentration of these two methyltransferase inhibitors to reach 0.5 reduction index is 0.95 μM ethidium bromide and 2 μM Heinone. So methyltransferase inhibitors should not be questioned as effective differentiation aids. Example 8: Analysis of Differentiation Aids Promoting Differentiation

Differentiation assistants can not only reduce the effective dose of differentiation inducers, but also promote complete differentiation. HL- 6 0 culturing cancer cells, cultured initial concentration of 1. ⁵ x 1 0 5 cells / ml of. The culture flasks were divided into three groups of 4 to 5 bottles, one of which served as an additive-free control. One group added vitamin A acid from 0. 2 5 to 0.1

5 μM was used as a control group; the other two groups were added with 4 μM urocalcin and vitamin B ₂ . After 96 hours of incubation, an NBT test was performed as described in Example 4. Each test value must be subtracted from the control base without additives.

The results are shown in FIG. 5, the degree of differentiation-inducing agent alone differentiation can only reach about 85%, if the urine red help biotin or vitamin B _2, degree of differentiation is up 100%. Promoting the degree of differentiation entirely medically may be more important than reducing the effective dose of a differentiation inducer. Example 9: Assistance of anti-cancer effects of cell differentiation agents and other anti-cancer chemotherapeutic cells Cancer cells have very active methyl transfer complex enzymes. If only chemical drugs are used to inhibit DNA synthesis, it will cause excessive methyl transfer, which is a gene Reason for repeated synthesis. Repeated gene synthesis leads to the formation of drug-resistant cells (Liau et al., 1992b Reference 24), which is an important factor in the failure of chemotherapy. The use of cell differentiation agents to inhibit abnormal methyl transfer complex enzymes can reduce the formation of drug-resistant cells and contribute to the treatment of cancer. Facts have also proved that cell differentiation agents do indeed help the treatment of certain anticancer drugs. For example, the present invention utilizes CDA-II and thymidine were used in combination (see Figure 6). Example 10: Combined anticancer effect of cell differentiation agent and antioxidant

Hypoxia can promote the differentiation and death of cancer cells. The use of vitamin C and vitamin B ₁₇ to achieve hypoxia is particularly suitable for anti-cancer purposes, because cancer cells have 10 times the capacity to absorb vitamin C than normal cells (References 2 9), while vitamin B ₁₇ is selectively Β-Glucosidese (β-Glucosidese), which is highly active in cancer cells, is broken down into toxic oxidase-inhibiting cyanide (Reference 30), which has a relatively low activity in normal cells. As shown in Figure 7, CDA-II, vitamin C, and vitamin B ₁₇ can inhibit the formation of HBL-10 human breast cancer cell populations, 1 ( _{5 ()} are 0.69, 0.53, and 0.1, respectively. 3 mg / ml. The results of the combination of CDA-II and vitamin C or vitamin B ₁₇ are shown in Table 2. The results clearly show that the combination of CDA-II and vitamin C or vitamin B ₁₇ has a synergistic anti-cancer effect. Table 1. Anti-cancer effects of cell differentiation agents (CDA-II)

 Preparation lot number% MAT inhibition% NBT +% breast cancer cell population inhibition

 01 49 58 100

 02 55 53 100

03 50 54 100 Note: The CDA-II preparation is an injection preparation, and the dosage concentration used is 1 g / ml. MAT ^LT was prepared from HL-60 cancer cells by DEAE-cellulose purification as described above, and the activity was measured according to the previous method (Liau et al., 1977a). The terminal differentiation of HL-60 cancer cells is determined according to the NBT + assay (Liau et al., 1988 reference 18). As for the inhibition of cancer cell colony formation, a human HBL-100 breast cancer cell culture method was adopted (Liau etal, m 1990a reference 20).

Table 2 Synergistic effect of CDA-II combined with vitamin C or vitamin B ₁₇

H B L— 1 0 0 Cell colony formation; Inhibition% Synergistic effect of combined medication Drug and dose, mg / ml Individual predicted value Measured value

CD A— II, 0.4 16 28 39

C, 0.2 12

CD A— II, 0.6 38 50 58

C, 0.2 12

CDA- II, 0.8 65 77 80

C, 0.2 12

CD A— II, 0.4 16 36 39 B ₁₇ 0.05 20

C D A— II, 0.6 38 58 58

B ₁₇ 0.05 20

C D A— II, 0.8 65 85 83

B ₁₇ 0.05 20

The experiment was performed according to FIG. 7. HBL— 100 cells were treated with different doses of CDA-II, vitamin C, and vitamin B ₁₇ alone, and CDA-II was used in combination with vitamin C and vitamin B ₁₇ .

 Industrial applicability

 Compared with the prior art, the present invention has the advantage that the present invention uses the body's own anti-cancer substance to manufacture drugs for treating and preventing cancer, which fundamentally differentiates cancer cells without continuing to divide to achieve the purpose of treatment. Therefore, the present invention It is an effective method to cure the root cause, and the medicine of the present invention does not have any adverse side effects.

Claims

Rights request 1. A pharmaceutical composition for treating and preventing cancer and inducing differentiation of cancer cells, comprising: a cell differentiation agent and a carrier thereof using an abnormal methyl transfer complex enzyme in a cancer cell as a drug target, the cell differentiation agent and Its carrier is extracted and refined from human urine.  2. The pharmaceutical composition according to claim 1, which is prepared into a dosage form such as an injection, a capsule.  3. The pharmaceutical composition according to claim 1, which is used in combination with thymidine to promote anti-cancer effects. 4. The pharmaceutical composition according to claim 1, which contains a differentiation inducing agent, PP-0 and OA-0.79; and a differentiation assistant, at least comprising phenylacetic acid, maleic acid, indoleacetic acid, uridine and Vitamin B ₂ .  5. The cell differentiation pharmaceutical composition according to claim 1, comprising a component for preventing excessive excretion by a cancer patient, such as phenylacetylglutamine. 6. The cell differentiation pharmaceutical composition according to claim 1, which is used in combination with vitamin C and vitamin B ₁₇ to produce a hypoxic state and promote anti-cancer effects.  7. The method for preparing a pharmaceutical composition for inducing cancer cell differentiation according to claim 1, comprising at least:  (1) Collect human urine, in which the creatine liver concentration is between 1.2 g / liter and 3.7 g / liter;  (2) Adsorbent X A D—16 or an adsorbent with similar function;  (3) extraction with ethanol or similar organic solvents; and  (4) Recovering and purifying the cell differentiation agent preparation.