JPH10114663A - Pharmaceutical composition promoting cancer cell differentiation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject composition comprising a cell differentiating agent extracted and purified from human urine, capable of improving an abnormal methylated enzyme conjugate of cancer cell and having high treating and preventing effect on cancer. SOLUTION: This pharmaceutical composition comprises an effective amount of cell differentiation agent extracted and purified from human urine and its base and preferably uses together with thymidine. Furthermore, the differentiating agent is obtained by collecting human urine having 1.2-3.7g/l creatine concentration, passing the human urine through the absorbent XAD-16 or an absorbent having function similar thereto, extracting the eluate with ethanol or an organic solvent having a structure similar thereto to recover a purified cell differentiation agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a pharmaceutical composition extracted from human urine and purified for promoting cell differentiation and a method for producing the same. The present invention relates to a cell differentiation agent CDA-II.
Ameliorates abnormalities in the methylenzyme complex of cancer cells, and as a result, achieves a medical effect of leading cancer cells to terminal differentiation. This is a new method of anti-cancer drug and has a very high therapeutic effect because it directly eliminates the etiology. CDA-II has the effect of eliminating the effect of the protein bound to the methylation enzyme complex on cancer cells, has specific therapeutic selectivity, and has no side effects. The active ingredients of CDA-II are differentiation promoters, differentiation aids, anti-pathogenic agents (Anticachexia agents)
These active ingredients can obtain the highest therapeutic effect by a synergistic effect. The abbreviations used in this specification are described later as “Description of Abbreviations”.

[0002]

2. Description of the Related Art Cancer is a disease that is difficult to treat. The composition of cancer cells themselves is complicated, and the cancer cells continually divide, thereby invading normal organs and tissues and eventually dying. . The medical community in the past has considered the division of cancer cells as the most fundamental obstacle to cancer, and has adopted a method of stopping DNA synthesis with a cytotoxin to stop cell division.

[0003]

SUMMARY OF THE INVENTION As a result of the research, the present inventors have found that a methylase complex promotes the sustained division of cancer cells and is the most fundamental obstacle in cancer. . Methylase complexes play a very important role in cell division and differentiation. When the enzyme activity is high, the cell divides, and when the activity is reduced, the cell synthesizes a nucleic acid lacking a methyl group, and the differentiation of the cell is promoted to become a terminally differentiated cell that does not divide again. All cancer cells have unusual methylation enzyme complexes, which are highly active, fixed in a stable state, and cells continually divide. Thus, abnormal methylase complexes are a cause of cancer, and sustained cell division is a symptom of cancer.

The inventor of the present invention found that in 1977, methionine adenosyltransferase (MAT) of Novikov liver cancer cells was different from that of normal cells (see Reference (19)). I found that it was not only qualitative but also qualitatively different. At that time, the most important function of DNA methyl transfer for methyl transfer had not been discovered yet. After DNA methyltransfer was confirmed in the function of regulatory genes (see references (13) and (15)), the importance of abnormal MATs grew at a stretch. Because of the qualitative differences between enzymes in cancer cells and those in normal cells, we were able to use this difference to find the ideal specific-selective anticancer drug.

[Function of Methylase Complex] MAT is one of the three enzymes constituting the methylase complex. The other two enzymes are methyltransferase and s-adenosylhomocysteine hydrolase (hereinafter referred to as SAHH). There are many types of methyltransferases, and each enzyme has specific substrate selectivity,
Do not interfere with each other. However, as with MAT and SAHH, they form complex enzymes, but their methyltransferases whose functions are specific have their activities regulated by the same mechanism. These three constituent enzymes can exhibit a methyl transfer function only when they are combined.
In normal cells, complex enzyme binding is dependent on exogenous facilitators. For example, steroid hormones are promoters of complex enzymes in hormone target tissues. When a steroid hormone is present, these three constituent enzymes combine to form a stable and active complex enzyme. In the absence of the steroid hormone, the complex enzyme separates into a single enzyme, and the stability of the single constituent enzyme is low, and it is quickly destroyed and loses its activity (see Reference (23)). In cells of non-steroidal hormone target tissues, the formation of methylenzyme complexes is dependent on growth hormone stimulation, causing the cells to produce steroid hormone-like facilitators. That is, the methylase complex activity of normal cells is completely controlled by the foreign promoter. The nucleic acid methylase complex plays a very important role in cell division and differentiation. Its importance corresponds to growth hormone. There are numerous types of methyltransfer of nucleic acids, two of which are involved in cell division and differentiation. This methyl transfer is based on DNA 5-methylcytosine (hereinafter, referred to as “methylcytosine”).
Described as 5-mC. ) And 2′-methylribose of RNA. 5-mC is located symmetrically in the two sequences of DNA.

[0006]

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When one gene has such a methyl group in the promoter sequence, this gene is regulated and cannot function. The methyl group of DNA plays the role of a selection gene (see references (12) and (15)). Each organ and tissue has its special basal cells, which have already differentiated to some extent, but have not reached the final stage and therefore still have the ability to divide. However, differentiation genes with special functions are present in the basal cells and are still in an unregulated state by the introduction of methyl groups. When basal cells divide upon growth hormone stimulation, the activity of the methylase complex increases with growth hormone. Since the distribution of DNA methyl groups is due to replication, the divided cells are identical to the mother cells and become basal cells. When growth hormone is no longer present, the activity of the methylase complex is immediately reduced, but DN
The A synthase is fairly stable, at which time a DNA lacking a methyl group is synthesized. During the two cell division cycles, DNA lacking a methyl group is synthesized, the functions of special and differentiation-related genes are exhibited, and the cells are transformed into terminally differentiated cells having special functions. Such cells no longer have the ability to divide. The synthetic action of DNA lacking a methyl group is an important mechanism of cell differentiation (see reference (28)).

[0008] Most of the methyl groups of the rRNA occur at the 2 'oxygen of the nucleotide. This type of methyl group introduction has an important regulatory effect on nucleotide production (see reference (18)). Cells produce RNA whose molecular weight is twice as large as the final product during nucleotide production.
The precursor is synthesized and the unwanted sequences are cleaved by RNase to form nucleotides, leaving useful sequences.
The distribution of methyl groups is all in the useful sequence, and if a methyl group is missing in the sequence of this part, the useful sequence in this part will be degraded as well as the unuseful sequence. Thus, if the methylation progress is complete, useful nucleotides can be generated; otherwise, no nucleotides are generated. Increasing nucleotide production is a prerequisite for cells to synthesize DNA. Only cells that increase nucleotide production can provide proteins necessary for DNA replication, such as nucleic acid synthetases, chromosomal proteins, and cellular constituent proteins.If nucleotides are not increased, these necessary proteins are sufficiently produced. In addition, cells cannot perform DNA synthesis. The importance of increased nucleotide production for cell division can be demonstrated from two simple experiments.

[0009] The first experiment utilizes the special sensitivity of radiation bacterium D. After stimulating cells with growth hormone for 8 hours, the production of nucleotides increases. It has been observed that adding D causes details to stop at the G1 stage.
(See (13)). Another experiment utilized cells that were sensitive to specific temperatures, where the rRNA processing and degrading enzyme of these cells completely lost activity at 39 ° C. It has been shown that increasing the temperature from 37 ° C. to 39 ° C. after stimulation with growth hormone for 8 hours stops nucleotide production and also stops cells at the G1 stage (see reference (41)). These two experiments clearly demonstrate that increased nucleotide production is a necessary component of cell division. The factor that determines nucleotide production regulation lies in the methylase complex,
Of course, it will also affect cell growth. Once growth hormone is absent, cells stop dividing,
Large amounts of nucleotides are not required. After the methylase complex first loses its activity, the cells still synthesize rRNA precursors that are short but lacking methyl groups.
These products fail to produce useful nucleotides, reduce the required protein, and the cells go through a process of differentiation that ultimately becomes terminally differentiated cells. It can be seen that the methylase complex plays a very important role in cell growth and differentiation.

[Role of Abnormal Methylase Complex in Cancer] Abnormal MAT is a special protein factor produced by cancer cells, and this element binds to normal MAT to produce abnormal cancer cell enzymes (see References). (See (21)). This binding completely changes the activity and regulatory mechanisms of the originally normal enzyme. Special protein factors in cancer cells also act on normal complex enzymes, such as steroid hormones. The three constituent enzymes combine to form a very stable and active complex enzyme. This is because it has this kind of special protein element,
The stability and activity of cancer cell complex enzymes are far superior to those of normal cells (see Reference (21)). Binding of the normal enzyme to the special protein elements of cancer cells not only alters the regulatory function of the complex enzyme, but also alters the kinetic function of the single constituent enzyme. Methionine (hereinafter referred to as Km) values of normal MAT and abnormal MAT are different. The former was 3 μM and the latter was 20 μM. For distinction, the former is referred to as MAT ^L and the latter as MAT ^LT . T represents a specific cancer factor. The difference between MAT and normal enzymes in cancer cells has already been demonstrated by other researchers (see reference (40)). Similar or similar factors alter the kinetic function of SAHH in cancer cells (Ref.
4)). Since cancer cells themselves produce a promoter of the methylating enzyme complex, this kind of enzymatic activity of cancer cells can maintain its own activity without depending on foreign factors. This high activity promotes the repetitive mitotic phase of cancer cells, so abnormal methylase complexes are the underlying cause of cancer.

[0011] All cancer cells have abnormal methylenzyme complexes. Abnormal methylase complexes have specific differences between normal growth and abnormal cancer growth, and we use those differences to look for drugs that specifically suppress abnormal methylase complexes. be able to. In fact, early experiments have shown that compounds such as oligonucleotides, which can suppress cancer cell methylase complexes, have very weak effects on normal enzymes and may not act at all. Conversely, compounds that promote normal complex enzyme activity cannot exert the same effect on abnormal cancer complex enzymes (see references (18) and (20)). These experiments indicate that aberrant methylase complexes of cancer cells are the most suitable targets for cancer chemotherapy.

Chemotherapy Targeting Aberrant Methylase Complex As described above, aberrant methylase complex is a fundamental cause of cancer. If the abnormal enzyme can be removed, the cancer cells become differentiated similarly to normal cells, and become terminally differentiated cells that do not divide further. This hypothesis has been proven to be correct. Novikov ascites liver cancer is poly (I)
After treatment in (C), the abnormal methyl-enzyme complex is changed to a normal enzyme, the synthesis rate of nucleic acid is reduced with a slight delay, and cell division is finally stopped (references (17), ( Two
4)). poly (I) (C) does not enter cells,
The effect of poly (I) (C) is very indirect. First, poly (I) (C) stimulates cancer cells to produce oligoisoadenylate synthase (see Reference (9)).
Through the product of this enzyme, oligoisoadenylate turns abnormal methylation enzyme complexes into normal enzymes.
Oligoisoadenylate is a product of differentiated cells. Oligopeptides and some organic acids are similar to oligoisoadenylate and can convert abnormal methylation enzyme complexes into normal enzymes (see references (22), (29)) ). Burzynski names these natural anti-cancer products in humans "antineoplastons" (see reference (4)). Many of these anticancer substances are low molecular weight metabolites that are further absorbed after being filtered by the kidneys and return to the blood to maintain a constant concentration.
Reabsorption is often incomplete, and small amounts of useful anticancer substances are excreted from the urine of healthy individuals. In the body, sufficient anticancer substances suppress the formation of cancer cells and maintain a balance. The inventors have named this type of natural chemical anticancer effect "Chemo-Surveillance" (see references).
(See (26)). After extraction and purification of the urinary anticancer substance from healthy subjects, it was found that two main components effectively suppressed abnormal MAT. One of these is an acidic peptide that binds to the dye, and one is an organic acid (see reference (29)). Normal enzymes have no abnormal factors and are not affected by anticancer substances at all. This is also the specific selectivity of natural chemical anticancer substances. Due to this selectivity, does not affect health. After being treated with these purified active ingredients, cancer cells are promoted to differentiate and become terminally differentiated cells (see Reference (28)). Enzyme studies have shown that the part where the abnormal methylating enzyme complex turns into the normal enzyme is the bottleneck that leads cancer cells to terminal differentiation. Intracellular S-adenosylmethionine (hereinafter referred to as AdoMet) and S-adenosylhomocysteine (hereinafter AdoMet)
Notated as Hcy. ) Has been demonstrated to demonstrate that enzyme changes play an important role in cancer cell differentiation. The AdoMet level of cancer cells is higher than that of normal cells (see Reference (11)). MAT ^LT of cancer cells is MA of normal cells
It is considerably higher than the ^TL Km value. Once a cancer cell differentiates and changes to a terminally differentiated cell, AdoMet and AdoHc
The amount of y is greatly reduced at the same time (Chiba et al., 198
8). MAT ^LT and SAHH ^LT lose carcinogens at the same time,
The MAT ^L and SAHH ^L. These are evidences taken from different angles and are consistent with the present invention.

[0013]

[Means for Solving the Problems]

[Natural Chemistry Inspection and Formation of Cancer] Why does cancer occur even though there are natural anticancer substances in the human body? Most cancer patients excrete low molecular weight metabolites from urine more than healthy people,
Long-term spills can result in a lack of anticancer substances in the body,
It cannot suppress the proliferation of cancer cells, leading to the appearance of clinical symptoms. The situation is more severe in severely ill patients (see references (25) and (26)). This is a kind of vicious cycle, and for some reason, the sick person excretes anticancer substances excessively, giving cancer cells an opportunity to develop and reproduce, and the proliferation of cancer cells results in the discharge of a larger amount of anticancer substances. Has been done,
Eventually, you lose your chemical inspection capabilities completely. At this time, the cancer cells can propagate without any restraint. Abnormal methylase complexes are important factors in cancer diseases, but disruption of the ability to monitor natural chemistry is also a very important factor. In order to effectively overcome cancer, it is necessary to consider both factors at the same time, to suppress aberrant methylase complex and to reduce excessive excretion of natural anticancer substances in the sick. An anticancer drug or cell differentiation agent (Ce
ll Diffirentiation Agent, CDA. ) Can act on these two factors simultaneously. The patient has gradually reduced the excessive excretion of low-molecular-weight metabolites and recovered to a healthy state when the condition improved (see Reference (25)). It is possible that the sick person excretes low molecular weight metabolites as a result of inflammation, and in the presence of inflammatory symptoms, excessive excretion is seen (see references (10), (3), (2)). Effective control of excretion is the most important issue in cancer treatment. As is well known, cytotoxic drugs themselves also promote excessive excretion (see reference (26)), have a negative effect on natural chemistry inspections, and have a destructive effect, rather than a protective effect. Although cytotoxic therapy removes all cancer cells cleanly, the sick person also loses the self-defense ability to control the remaining cancer cells with long-term cytotoxic destruction. Conversely, the components of the anticancer drug purified from urine can improve the excessive excretion of the sick person, restore the sick person's ability to monitor natural chemistry, and then control the remaining cancer cells with their own self-defense ability. Phenylglutamide contained in this urine-purified anticancer agent has the effect of improving the excessive excretion of cancer patients. Although it has no direct control on cancer cells, it protects humans and animals from monitoring natural chemistry by preventing excessive excretion of anticancer substances, and is effective in treating mild cancer patients. (See reference (25)). Furthermore, a remarkable preventive effect was observed on animal cancer formation (see references (16) and (35)).

[0014] From clinical experience, it is known that differentiation therapy is a highly valuable therapy. Antineplastone was used by Burzynski in patients with terminal cancer before 1985, with 60% of patients improving their symptoms, one-third of whom had a lifespan of more than 5 years. (See references (5), (6), (7)). This type of drug has no side effects. Initially, the therapeutic targets of all the therapeutic agents for differentiation were not the same, and the results reached that they promoted the differentiation of cancer cells through the modification of the aberrant methylase complex. For example, interferon or retinoic
Acid promotes the action of these drugs on cancer cells to produce antineplastone analogs and acts on abnormal methylase complexes to lead to terminal differentiation of cancer cells. This kind of anetine plastone analog is likely to be an oligoisoadenylate. Interferon is associated with hairy leukemia (ha
retinoic acid for Ary cell leukemia)
For eG-ranulocytic leukemia, it is recognized as the optimal drug (see references (14) and (42)). But,
The problem of recurrence has not been resolved (Refs. (34) and (1)
See).

[Differentiation Aid] A differentiation promoting agent can directly or indirectly remove abnormal factors of a cancer methylase complex and lead cancer cells to terminal differentiation. Methylase complex is a complex of three constituent enzymes, and we have found that inhibitors of each constituent enzyme enhance the action of differentiation promoters to remove abnormal factors. Although this inhibitor itself has no remarkable differentiation promoting action, the present inventors have named this compound a differentiation aid (Helper Inducer) (see Reference (3)).
2)). MAT inhibitors such as n-butyric acid, phenylbutyric acid and phenylacetic acid have remarkable differentiation assisting activities. In general, differentiation aids such as phenylacetic acid are readily available, have low toxicity, and can reduce the amount of differentiation promoter required. Particularly suitable for brain tumors. The brain is very special in structure, and the differentiation promoter does not disappear very quickly in protecting the blood-brain barrier.The use of phenylacetic acid, which easily passes through this barrier, provides a good therapeutic effect. (See reference (38)).

[0016]

DETAILED DESCRIPTION OF THE INVENTION In general, cancer patients have lost the ability to control the growth of cancer cells due to excessive excretion of anticancer substances. In this regard, differentiation therapy is not only a curative treatment, but also a natural anti-cancer prescription. In the present invention, a cell differentiating agent was extracted and purified from urine of a healthy person because urine contains a chemical substance that induces cancer cells to undergo terminal differentiation. This preparation can improve the abnormal methylation enzyme complex of cancer cells, and as a result, the cancer cells are led to terminal differentiation, cannot continue dividing, and die, and thus have a therapeutic and preventive effect on cancer. FIG. 1 shows a production follow chart of the cell differentiating agent in the present invention. The present invention will be described in further detail with reference to specific examples below.

[0017]

【Example】

[Example 1] Production of cell differentiation agent When urine is collected, sufficient 1N hydrochloric acid is put in a collection tank. Approximately 20 liters of urine use 1 liter of hydrochloric acid. pH
After adjusting to 2 the urine is filtered through a nylon cloth and material with a molecular weight greater than 10,000 daltons is filtered off. This is done using Amicon filter fibers or by other common methods. Adsorbent XAD-16
(Sigma) is packed in a bag and placed in a stainless steel funnel. Before use, it is first washed with twice (v / w) ethanol and again with twice (v / w) deionized water. This is repeated twice. The urine passed through the adsorbent XAD-16 is quadrupled (v / w). The substance adsorbed on XAD-16 is washed with 4 times (v / w) of deionized water and then eluted again with 2 times (v / w) of ethanol. After extraction with neutralized ethanol, the ethanol is evaporated to dryness in a dryer. The temperature in the drying tank is kept at 50 ° C. or less. The dry substance is dissolved in pyrogen-free water and CDA-
Ingredients for II. After the extraction with ethanol, the adsorbent is washed with twice the volume (v / w) of deionized water, used again, and replaced when the adsorption capacity is reduced to about 70% of that of a new product. In general, XAD-16 can be used more than 200 times.

The amount of creatinine excreted by the human body per day is constant, the concentration of solids in urine is directly proportional to creatinine, and it is highly reliable to quantify urine chemicals based on creatinine. The creatinine concentration of the collected urine was 1.2-3.7 g / l, average 2.4 ± 0.6 g / l.
Met. The production rate of CDA-II was 0.51 ± 0.17 g / g creatinine and the solid component of urine was 46.7 g / g creatinine from the first to the 100th passage of the adsorbent. That is, the production rate of the obtained CDA-II raw material drug was about 1.1% of the solid component.

Example 2 Preparation of Cell Differentiating Agent Injection The final concentration of the cell differentiating agent (CDA-II) injection was 50 ± 2.
mg / ml, and the concentration of the drug substance is generally 250 mg / ml or more. For this reason, the concentration of the diluted drug substance is about 130% of the final formulation concentration. First, the raw material drug is diluted with pyrogen-free water, and then filtered. The solution is filtered through a filter paper having a low speed, filtered again through a filter membrane having a filter hole of 1 μm and 0.45 μm, and finally, a pyrogen is removed using a special filter membrane.
The filtrate from which pyrogen has been removed is adjusted to an appropriate concentration with pyrogen-free water, and then bacteria are filtered using a 0.22 μm filter in a sterile room (100 class) within 8 hours.
Immediately after disinfection, the cells are packaged in 100 ml or 250 ml of a cell differentiation agent injection solution preparation.

Example 3 Production of Cell Differentiating Agent Capsules A capsule preparation is produced with a portion of CDA-II. The raw material medicine is filter paper with slow speed, the filter hole is 1μm and 0.45μ
m. The filtrate is dried again by freeze-drying to a solid. After the dried solid is powdered, a capsule preparation of 500 mg per tablet is produced by an automatic capsule packaging machine, sealed with aluminum foil, and finally sterilized by radiation.

Example 4 Analysis of Anti-Cancer Activity of Cell Differentiating Agent The anti-cancer effect of the cell differentiating agent is that an active ingredient suppresses cancer cell methylase complex, leads to final differentiation of cancer cells, and stops cell division. It is based on letting. That cell differentiation agents have an action to suppress cancer abnormal enzyme MAT ^LT, lead to differentiation of HL-60 cancer cells, as well as having activity of suppressing the formation of human breast cancer cell populations. Literature has already been published on methods for measuring these activities (see references (19), (27) and (29)). In the present invention, the anticancer activity of each of the three batches of the CDA-II preparation is analyzed, and the operation procedure is as follows.

The CDA-II injection solution 1 mg / ml is used. MAT ^LT was collected from HL-60 cancer cells, and the precipitated cells were first purified from 0.05 M Tris, pH 7, 0.5 mM
Suspend in magnesium chloride. Thereafter, the cell membrane is broken with a Dounce homogenizer. The enzyme extract is separated by high-speed centrifugation (226,000 × g, 0.5 hours). The extract was subjected to calcium chloride gradient elution with DEAE- cellulose chromatography and eluting the MAT ^LT (see reference (19)). MAT ^LT activity is also measured by the method described above (see reference (19)). 0.
0.05M Tris, pH8.
2, 0.15 M potassium chloride, 15 mM magnesium chloride, 5 mM DTT, 2 mM ATP and 1 μM [ ³ H
—CH ₃ ] methionine, and reacted at 37 ° C. for 30 minutes. The reaction solution was oxidized, then all transferred to phosphocellulose of one square inch, 5mM phosphate buffer The paper is placed in a beaker together with pH 7, the unreacted ^[3 H-CH
₃ ] Wash off methionine. Finally adsorbed [ ³
H-CH _3] radiation was measured in AdoMet, MAT ^LT
(See Table 1 for results).

The terminal differentiation of HL-60 cancer cells is measured by the NBT + measuring method (see Reference (27)). First, HL
-60 cancer cells are diluted and cultured, and 10 ml of the starting cell solution is added to each culture bottle to a concentration of 1.5 × 10 ⁵ cells / ml. After culturing the culture bottle containing the control and CDA-II for 96 hours, a portion is taken out, and the cells are centrifuged to precipitate. These cells use the NBT reagent,
After reacting at 37 ° C. for 30 minutes, remove, put one drop into a cell counter, and count the total number of cells and the number of black-stained (NBT +) cells under a microscope. The percentage occupied by NBT + is the differentiation-inducing activity of CDA-II (see Table 1 for results).

Another indicator of CDA-II anticancer activity is H
This is the suppression of the BL-100 breast cancer cell group. The breast cancer cells growing at a geometric series are washed once with physiological saline, and
Add ml of 0.05% trypsin-0.53 mM EDTA culture and incubate at 37 ° C. for 10 minutes. First, the cell concentration is measured and further partially diluted to 3 × 10 ³ cells / ml. Take 0.5 ml of the diluent and incubate 4.5 ml of control or CDA-II-containing culture bottle at 37 ° C. for 5 days. The culture is removed and washed once with saline. After fixing with methanol for 15 minutes, the fixed cell group is immersed in a 20-fold diluted Giemsa staining solution for 30 minutes. After the staining solution is discarded, the cells are washed with water and dried, and the number of cell groups is counted under a dissecting microscope, from which the anticancer activity of CDA-II is determined (see Table 1 for the results).

[0025]

[Table 1] Note: The CDA-II preparation uses an injection solution. The concentration of the preparation used was 1 mg / ml. MAT ^LT is purified from HL-60 cancer cells through DEAD-cellulose as described above,
To manufacture. Activity measurement is also performed by the method described above (see Reference (19)). Terminal differentiation of HL-60 cancer cells is measured by the NBT + assay (see reference (27)). The inhibitory action of cancer cell group formation employs a method of culturing human HBL-100 breast cancer cells (see Reference (29)).

Example 5 Analysis of Active Ingredient of Cell Differentiating Agent The CDA-II cell differentiating agent injection solution obtained in Example 2 is freeze-dried and concentrated to 200 mg / ml. 5 ml of the concentrated solution was applied to a Bio-Gel P2 chromatography column (4.1 cm × 4).
4 cm), elute with distilled water, collect in one test tube every 7 minutes and make up to 10 ml per tube. After separation is complete, remove 25 μl from each test tube, dilute with water and add 1 ml
Is measured by the absorbance of A255. In addition, 25 μl is collected from one test tube and used for measurement of ^MATLT activity inhibition. MAT ^LT activity is measured according to Example 4. Thereafter, the test tube was moved to the position shown in FIG.
Divide into eight parts as shown. Freeze dry each part to remove water completely. The measured dry weight is determined by the distribution of weight%. The dried solid is redissolved in distilled water and HL-6
0 is used to measure the differentiation activity of cancer cells. This activity is NBT +
%, And the differentiation activity was measured by the method described in Example 4, and the results are as shown in FIG.

The most important anticancer active ingredient of the cell differentiation agent is a differentiation promoting agent for promoting the differentiation of cancer cells. The separation / purification and mechanism of action of the differentiation promoter have been published in the literature (see references (27), (28), (29), (30)). The differentiation promoter mainly has two kinds of chemical components. One is a dye-bound acidic peptide, hereinafter referred to as PP-O. Another one
One is an organic acid, hereinafter referred to as OA-0.79. PP-O is in the first part and OA-0.79 is in the fifth and sixth parts shown in FIG. Here, it should be particularly described that the differentiation activity of the cell differentiation agent is not performed solely by the differentiation promoter, but is generated in cooperation with the differentiation aid. The differentiation aid is a main component of the cell differentiation agent, and the differentiation promoting agent is in a very small amount and has not been purified to the purity that determines the structure, and thus the chemical structure is not known. Differentiation promoting activity to the activity of MAT ^LT inhibition shown in Figure 2 is very consistent. However, the ^MATLT inhibitory activity is clearly seen in the second and third parts of FIG. 2, but there is no cancer cell differentiation promoting activity. This part contains a small amount of a differentiation aid, but does not contain a differentiation promoting agent. Therefore, there is no activity of promoting the differentiation of cancer cells. A differentiation aid is an inhibitor of the methylating enzyme complex (see reference (31)), which helps the differentiation promoting agent to convert the methylating enzyme complex to a normal enzyme, To improve.

Already known MAT inhibitors in CDA formulations are phenylacetic acid, indoleacetic acid and hippuric acid. It is highly likely that phenylacetic acid was generated by hydrolysis during the drying of phenylglutamide. C18 HPL distributed in the sixth and seventh parts of FIG.
C is measured. Indole acetic acid is also distributed in this part. Hippuric acid is the main component of the fifth part. These M
The AT inhibitor has a reduction index of 0.5.
x, an index that halves the effective amount of the differentiation promoting agent), phenylacetic acid is 4 mM, hippuric acid is 8 mM, and indoleacetic acid is 0.95 mM, respectively.
Methyltransferase inhibitors among the differentiation aids are more effective than MAT inhibitors. The former can achieve the same effect in one-thousandth or less of the latter. Both of the already known methyltransferase inhibitors in the CDA preparation have excellent differentiation aid activity. These two components is vitamin B ₂ and Urorubin. Vitamin B ₂ is distributed in the latter half of the eighth part of FIG. As described above (references (3
See 1)), the yellow component through C18 HPLC, it is possible to obtain a very pure vitamin B _2. Content is around 0.04%. Urolbin is distributed in the sixth and seventh parts of FIG. Pure urolbin can be obtained from this part by Sepadox SH column chromatography and silica gel thin film chromatography. The content is around 0.5% of the CDA-II preparation. It seems that there is a loss in the purification process, and the CDA-II preparation also shows a considerable red color, so its content is estimated to be 0.5% or more.

Example 6 Measurement of Differentiation Aid Activity of Urolvin and Vitamin B ₂ Measurement of a differentiation aid activity employs a method previously designed by the inventor (see Reference (32)). The experiment was performed using cultured HL-
Using 60 leukemia cancer cells, the degree of differentiation of the cancer cells is measured by an NBT quantification method. First, HL-60 cells are diluted and cultured, and each culture bottle is filled with 10 ml of a starting cell solution, and the concentration is adjusted to 1.5 × 10 ⁵ cells / ml. A set of four culture bottles is used as a control, to which only the retinoic acid differentiation promoter is added. Adjust to an amount to induce 15-60% of NBT + cells. The other control is a solvent-only control. Retinoic acid is soluble in methanol, but the total amount of methanol does not affect the differentiation of cancer cells.
Should not exceed 2%. Each of the other sets is a culture bottle of 4 bottles, but using a relatively low amount of retinoic acid.
The other is a solvent-only control, and each set contains a different amount of differentiation aid. After culturing for 96 hours,
The number of cells in each bottle was measured, and NBT was obtained by the method described in Example 4.
Perform the measurement. Generally, it is 4% or less in the case of spontaneous differentiation of HL-60 cells, that is, without additives. Within the range of amounts used in the set of differentiation aids alone, cell differentiation hardly exceeds 10%. The NBT + value for each bottle needs to be deducted based on these control values.

The relative relationship between the amount of drug and NBT + was plotted as a curve, from which the ED ₅₀ value was obtained. That is, NB
It is the amount of the differentiation promoter required for T + 50%. These E
Reduction index of differentiation auxiliaries from D ₅₀ values are obtained. Reduction index = ED ₅₀ value in the presence of a differentiation aid / E in the case of a differentiation promoter alone
D becomes ₅₀ value. The lower the reduction index, the higher the function of the differentiation aid. As shown in FIG. 3, vitamin B ₂
And urorubin have a concentration of 0.5 reduction index of 3.0μ.
M, 1.8 μM, greatly exceeds the activity of the MAT inhibitor described above.

Example 7 Inhibitory Activity of Urolvin and Vitamin B ₂ on tRNA Methyltransferase TRNA methyltransferase is extracted from HL-60 cancer cells. That is, it is the high-speed cell extract shown in Example 4. First, the cell extract was adjusted to pH 5, the precipitated protein was separated by a centrifugal separator, and the 0.05 M
Tris, pH 7.8, 0.5 mM magnesium chloride,
Dissolve in a mixture of 5 mM HSCH ₂ CH ₂ OH. This enzyme solution is again subjected to DEAD-cellulose chromatography, taking a potassium chloride gradient extract, and extracting and purifying tRNA methyltransferase (see Reference (20)). tRNA methyltransferase activity was 0.2
Measure in 5 ml reaction. For that, 0.05M Tri
s, pH 7.8, 0.1 M ammonium chloride, 0.04
M ammonium fluoride, 0.5 mM magnesium chloride, 5 mM DTT, 20 μg E. coli B tRNA,
0.25 μCi [ ³ H—CH ₃ ] AdoMet, 25 μC
g Contains enzymes. The reaction is performed at 37 ° C. for 30 minutes. tRNA
Used cold 5% TCA precipitation followed by millipo
Filter with a re (membrane pore 0.45 μm) thin film. After the thin film is dried, the radiation dose is measured, and the activity of tRNA methyltransferase is measured.

As shown in FIG. 4, urolbin and vitamin B ₂ have an activity of inhibiting tRNA methyltransferase. However, the effective amount of the methyltransferase inhibitory activity is much higher than the effective amount of the differentiation aid.
This may be relevant for measuring the physiochemical environment of these different activities. Different physiochemical environments (eg, salt concentrations) can affect the effective contact of chemicals with enzymes. There are considerable differences in sensitivity, with the activity of different inhibitors of methyltransferase and the activity of differentiation aids being in a certain proportional relationship. More effective methyltransferase inhibitors will be more effective adjuvants. We also have ethidium brom-ide and hycantho
Methyltransferase inhibitors such as ne (NSC 142982) have been discovered. They are urolbin and vitamin B
It has an effective differentiation aid activity similar to ₂ . These two methyltransferase inhibitors have a 0.5 reduction index concentration of 0.95 μM in eth-idium bromide and a hycant
It is necessary to reach 2 μM in the hone. There is no doubt that methyltransferase inhibitors are effective differentiation aids.

[Example 8] Analysis of differentiation promoting action of differentiation aid The differentiation aid not only can reduce the effective amount of the differentiation promoting agent, but can also completely promote the degree of differentiation.
Culture HL-60 cancer cells. Starting concentration of culture is 1.5
× 10 ⁵ cells / ml. Culture bottles are divided into 3 sets, 4 per set
~ 5 bottles. One bottle is a control to which no additive is added. One set contains retinoic acid 0.025 ~
Add 1.15 μM to make a control group. Outside 2
Add 4 μM urolvin and vitamin B ₂ to the set. 9
After culturing for 6 hours, the NBT measurement described in Example 4 is performed. Subtract control values without additive from each measurement.

The results are shown in FIG. 5. When the differentiation-inducing agent was used alone, the degree of differentiation reached only 85% at the maximum. The addition of Urorubin or vitamin B _2, differentiation degree reaches 100%. It is more important to completely promote the degree of differentiation than to reduce the effective amount of the differentiation promoting agent therapeutically. As is well known, retinoic acid is an anticancer drug and has a high therapeutic effect (see Reference (14)).
Cancer cells recur immediately (Refs. (34) and (1)
See). Incomplete differentiation and relapse are associated with the use of differentiation promoters alone. This is because the differentiation promoting agent damages the cells and the cells cannot completely differentiate. The differentiation procedure requires two slow cell division cycles (see references (43), (44) and (45)), and if cells are damaged, differentiation ceases and leads to terminal differentiation. Does not reach. Abnormal methyltransferase is improved with a differentiation promoter to remove oncoprotein factors, and the complex enzyme is decomposed into three constituent enzymes. Some methyltransferases, when used alone, have the activity of degrading nucleic acids and have been shown to damage cells (see Reference (18)). If the injury is severe, the cells will die. But if the injury is not severe, it will be stopped and repaired for a long time and reti
If the effective concentration of noic acid cannot be maintained, the revived cells will return to their original state and recur. Although the differentiation aid is not as important as the differentiation promoter, the differentiation aid is an essential component in the treatment of differentiation.

In addition to the necessary differentiation promoting agent and auxiliary agent, the cell differentiation agent has another very effective component for treating cancer. It is an anticachexia agent. We have found that phenylglutamide counteracts cachexia in cancer patients to promote hyperexcretion (see reference (26)). In particular, the protective effect against cancer is remarkable (see References (16) and (35)). Phenylglutamide is shown in Fig. 2.
Is the main component of the fourth part. That is, the cell differentiating agent contains many necessary chemical components and has a synergistic effect. Although it contains unnecessary chemical components, they are not harmful to the body and there is no need to remove them.

Example 9 Synergistic Effect of Differentiation Aid and Other Anticancer Chemotherapeutic Agents Carcinogenesis is many, but aberrant methylase complex is an important factor. Other oncogenes are also important factors that cause symptoms. Although the resolution of the aberrant methyltransferase has already eliminated a major problem, we believe that attacking from more fronts will result in even greater therapeutic effects. Cancer cells contain a highly active methylase complex and can be used to DN
Inhibiting A synthesis promotes excessive methyl transfer. This triggers gene duplication synthesis, which forms drug resistant cells (see
(See (33)). This is a major cause of chemotherapy failure. Suppressing the abnormal methylase complex with a cell differentiating agent can reduce the formation of drug-resistant cells and is useful for cancer treatment.
This fact demonstrates that cell differentiation agents synergize with some anticancer drugs.

[0037]

[Description of abbreviations]

(1) AdoHcy: S-adenosylhomocysteine (2) AdoMet: S-adenosylmethionine (3) CDA: cell diffirentiation agent (cell differentiation agent) (4) DTT: dithiothreitol (5) HPLC: high performance liquid chromatograp
hy (high performance liquid chromatography) (6) MAT: methionine adenosyltransferase (7) NBT: nitroblue tetrazolium (8) SAHH: S-adenosylhomocysteine hydrolase (9) XAD: adsorbent (manufactured by Sigma)

[0038]

The effect of the present invention is to extract and purify a cell differentiating agent from the urine fluid of a healthy subject in order to treat and prevent a cancer symptom with respect to an abnormal methylase complex. This formulation can normalize the abnormal methylase complex of cancer cells, so that the cancer cells are promoted to terminal differentiation, do not divide, and die. Thus, a therapeutic and preventive effect is seen for cancer.

[0039]

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[Brief description of the drawings]

FIG. 1 is a production flowchart of a cell differentiation agent.

FIG. 2 is a graph showing the results of gel filtration analysis of a cell differentiating agent. After concentrating the CDA-II injection by freeze-drying, it is separated by a chromatography column. At that time, Bio-Gel P2
Column (41 cm x 44 cm). The collected effluent Each A255 absorbance (solid line), MAT ^LT activity inhibition (dotted line), wt%, HL-60 cancer cell differentiation activity (NB
(Expressed as T +%).

FIG. 3 is a graph showing the differentiation aid activity of urolvin (solid line) and vitamin B ₂ (dotted line). The horizontal axis shows the concentration (μM), and the vertical axis shows the reduction index.

FIG. 4 is a graph showing the inhibitory activities of urolvin (solid line) and vitamin B ₂ (dotted line) on tRNA methyltransferase. The horizontal axis is the concentration (mM), and the vertical axis is the relative activity%.

FIG. 5 is a graph showing the enhancing action of urolbin and vitamin B _{2 on} a differentiation promoter. The vertical axis NBT +% occupied by the cells, the solid line different added retinoic acid concentration controls set, the dotted line test set with the addition of 4μM vitamin B _2, dashed line shows the test set with the addition of 4μM Urorubin.

FIG. 6 is a graph showing the synergistic effect of CDA-II and thymidine on cancer. The horizontal axis is the drug amount (mg / ml), the vertical axis is the relative activity (%) of cancer cell group formation, the solid line is a test group using thymidine alone, the dotted line is a test group using CDA-II alone, and the dashed line is A set in which thymidine and CDA-II are used in a ratio of 1: 1 is shown. The anticancer effect is higher when used in combination than when used alone, and a synergistic effect is remarkably exhibited. The activity of cancer cell group formation is measured using the HBL-100 breast cancer cells described in Example 4. The ratio is shown as a ratio of the control group to the formation of the cancer cells.

Claims (5)

[Claims] 1. A pharmaceutical composition comprising a cell differentiation agent obtained by extracting and purifying a therapeutically effective amount from human urine and its base, and targeting an abnormal methylase complex. 2. The pharmaceutical composition according to claim 1, which is used for treating and preventing cancer. 3. The pharmaceutical composition according to claim 1, which is used in combination with thymidine. 4. The pharmaceutical composition according to claim 1, which has a dosage form such as an injection or a capsule. 5. A human urine having a creatine concentration of 1.2 to 3.7 g / l is collected and passed through an adsorbent XAD-16 or an adsorbent having a similar function, and ethanol or an organic solvent having a similar structure is collected. A method for producing a cell differentiating agent, comprising recovering a purified cell differentiating agent preparation extracted by the method.