DE2026712A1 - Dna polyamine compositions for treating - lesions caused by radiation - Google Patents

Abstract

DNA-polyamine composition for treating lesions caused by radiation. Compns. are prepared by dissolving dioxyri bonucleic acid (DNA) and a basic amine or amino-acid separately in dilute sodium chloride solution opt. containing sodium citrate, mixing the two solutions at 37 degrees C for 6-36 hrs. and at pH 7, adding ethanol and recovering the precipitate formed by centrifuging. Suitable amines and amino-acids are spermine, spermidine, cadaverine, polylysine and polyarginine. The compositions may be used to cure lesions caused by radiation e.g. white corpuscle destruction and can be used to treat secondary effects in the X-ray and gamma-ray treatments for cancer.

Description

Connection to Radiation Damage Healing and Procedures for Its Manufacturing.

The invention relates to a method for the preparation of compounds to heal and prevent radiation damage in the human body that consists of basic polyamine and deoxyribonucleic acid (DNA).

It is well known that the biosynthesis of DNA in cells of humans and mammals is seriously harmed by ionizing radiation. It is also known that this biosynthesis of DNA is essential for cell division so that the prevention of DNA biosynthesis is the cause of the non-occurrence cell division and ultimately leads to cell death.

In more recent times the diagnosis and treatment of cancer In the early stages in medicine received a lot of attention and the application of various Types of treatment, such as the lit ionizing rays, pharmacological preparations or expanded surgical interventions.

Treatment with radiation or pharmacological agents is meanwhile associated with various harsh of side effects, especially in the hematological Organ where cell division is particularly active It is therefore often impossible to locate the Continue erectile therapy because of serious side effects, such as leukopenia, an abnormal one Decrease in white blood cells occurs.

Hence is a substance that is used to heal or prevent radiation damage leave, extremely desirable; such a substance is expected to have the side effects Cancer Treatment Can Fix.

ohl there are some protective remedies, such as sulfhydryl-containing ones Compounds with the radiation-induced free hadicals that form in the human body through ionizing radiation, they react, but they only work if they are given before the Z-rays take effect.

There are few remedies that can reduce radiation damage when used can heal after exposure to X-rays. These remedies are named "Restorators" in contrast to the first-mentioned remedies, the "l-rotectors".

Nowadays there is mainly interest in a restorator for healing the serious side effects of cancer treatment with ionizing radiation, like X-rays or Cobalt60-gamma-rays.

At the moment, however, are the substances that oppose the prevention of the DNA biosynthesis can help little-known; is certain only that high molecular weight DNA from certain species in healing radiation damage in irradiated cells or ornamental is effective.

However, it is very difficult to use DNA preparations which Obtained from human beings for the healing of radiation sheaths in i'ienschen because it is very difficult to consider human tissue materials SOURCE TO FIND DMA. Hence one must conclude that for the practical Use of DMA preparations remove the NA from any other heterologous species than must be obtained from human beings.

There may also be some potential for genetic damage in humans insist on using high molecular weight heterologous DNA as a pharmacological remedy applies, and therefore it is necessary to have lower molecular weight DNA with a molecular weight in the range of 200,000 to 500,000 to use for genetic damage such as this Rule out mutation, because it is well known that the lower molecular weight DNA cannot induce genetic conversion even in bacterial systems It is known that in higher living beings DMA is not in the free state, but in Combination with histone, a special protein, occurs.

There is also the possibility of inhibiting DMA biosynthesis or the cleavage of the strands of the bNA double helix attributed to the radiation is secondary to damage to the basic protein combined with the DNA caused.

It is therefore believed that not only the elimination of the inhibition DNA biosynthesis or harmful effects on the DNA molecule, but rather also eliminating the harmful effects of sin on histone, a basic protein, which is combined with the DNA molecule and modifies the biological point ion of the DNA, is quite necessary when looking for more effective help against the radiation damage in X-irradiated living beings.

It is, however, practically impossible to separate heterologous histones from nonhuman ones Living beings to remedy the harmful effects of radiation on the histone part to use as one could cause severe allergic reactions in people when using heterologous protein (antigen-antibody reaction).

On the other hand, it is clear that lower molecular weight amino acids or polyamines with a molecular weight of a few hundred cause an antigen-antibody reaction cannot trigger in mammals.

Based on the above facts, it was recognized that the administration of synthetic compounds from a nombinatin of DNA with antigen-innocuous ones basic polyamines or basic polyamino acids, which are the natural compound complex made of DNA and basic protein, histone, have better effects than that mere application of DNA against radiation damage alone. So the attempt was made various compounds from lower molecular weight DNA and individual types of basic To produce polyamines or basic polyamino acids.

The invention is now a method for Manufacturing of compounds from Deoxyribonukleinsäu.re and basic polyamine or basic Polyamino acids, characterized in that one deoxyribonucleic acid from one Molecular weight of 200,000 to 500 GOO, obtained from the sperm of fish such as salmon or trout, or from internal organs such as the thymus, liver or kidney, of mammals, like calves or young piglets, and basic polyamines or basic ones Polyamino acids in a weight proportion of 1/25 to 1/50 of the deoxyribonucleic acid separately in dilute sodium chloride solution (0.0O15 N) or a solution of a Mixtures of sodium chloride and sodium citrate (0.0015 N and 0.00015 M) dissolve each Solution.

by adding 0.1 N sodium hydroxide solution or Q, 1 N hydrochloric acid solution adjusts the pH to exactly 7.0, mixes the two solutions and reacts brings the mixture to stand for 6 to 18 hours at 37 ° C, about five times the volume ethanol (99β) is added to the reaction mixture, the mixture is left to stand for 30 minutes and the resulting white precipitate by centrifugation (5000 rpm, 10 minutes) separates.

To the recorded precipitation. give twice the volume of ethanol (99C, 6) and stirred, centrifuged again (5000 rpm, 10 minutes) and crystallized the resulting precipitate from ethanol or ethyl ether. You get the connection from deoxyribonucleic acid and basic polyamine or basic polyamino acid. The physical and chemical properties of the compound; which according to the Process according to the invention from deoxyribonucleic acid and basic polyamine or basic polyamino acid are: (1) molecular weight 200,000 to 500 000 (2) Constant sedimentation 12 S or more (3) Specific Weight 1,650 - 1,700 (4) viscosity n sp / c = 3 x 10 4 - 6 x 10-4 (5) spec. optical Rotation tan #, = 5 3 - (6) absorption spectra Emax = 2600 A ° - 2657 A ° (7) appearance colorless crystalline powder (8) Solubility: soluble in water, dilute saline solution (0.015 N), insoluble in ethanol, acetone and ethyl ether.

Likewise, the healing effect of the radiation damage by the invention Products checked by two methods.

The first method using the cultivation of colonies Tissues of cultured cells, consists of - Changs' liver cells of human origin with Eagles MEM (Minimum Essential IGedia) supplemented with 10% calf serum, Grow in i'-etri glass dishes for two weeks so that colonies are visible form, with the healing of X-irradiated cells from the radiation damage being evaluated will. Since dead cells are unable to produce visible colonies, will the healing effects of the compounds prepared according to the invention on the survival ratio of irradiated cells measured by counting the visible colonies. Changs' liver cells are exposed to radiation of 206, 400, 600 and 800 R with X-rays of 300 KVP; then the number of cells surviving two weeks is determined by counting the colonies determined.

Compared to the control group, which did not receive DNA, the Groups spiked with DNA had a slight increase, about that 1.5 times the number of colonies for each disturbance dose.

On the other hand, the complex compound of DNA and spermidine, one basic polyamine, after addition to the culture medium immediately after the X-irradiation of the cells show pronounced effectiveness.

The number of colonies increased by about four times that of the control group.

According to the second method, the healing effects of the invention Animal compounds in which the whole body of mice is exposed to X-rays will be checked.

The mice used belonged to the breed ddn and were female and had a body weight of 20 to 25 g.

The radiation factors were 550 R with total irradiation of 300 KVP, as a control group, was administered intraperitoneally to mice immediately after X-irradiation diluted saline solution (0.015 K IiaCl and 0.0015 M sodium citrate solution) injected, which is the solvent for DNA or the complex compound DNA: basic polyamine forms. The injection was repeated three times a week.

The second group was herring sperm DNA of liolecular weight 200,000 with a dose of 25 peg / g of mouse body weight intraperitoneally immediately injected after X-irradiation.

The injection was repeated three times a week for 4 weeks.

The third group received injections with the complex compound Herring sperm DNA with a molecular weight of 200,000 and spermidine, a basic one Polyamine. The weight ratio in the complex compound was DWA: spermidine = 50: 1.

The intraperitoneally injected immediately after the X-irradiation The dose was 25 µg DNA as the equivalent amount per gram of body weight; the injections were over three times a week Repeated for 4 weeks.

The fourth group was made with the complex compound of the same DNA and spermidine injected; here, however, the weight ratio was DNA: spermidine 25: 1. Injections were given in the same way as for the second group.

The fifth: group became the complex compound of the same DNA and spermidine injected, but with a weight ratio of 10: 1 = DNA: Spermldin0 Injections were given in the same way as for the second group.

As a result, the number of mice, the number of 30-day surviving mice after the X-irradiation 550 R and the average life span of X-irradiated mice in each experiment are shown in Table 1 below Group number mice surviving 30 days survival after X-irradiation 550R time (days) Mice number% 1. Diluted Salt Solution 48 8 16.5 13.75 2. Low molecular weight DMA 48 9 19.5 15.88 25 µg / g 3. Low molecular weight 40 14 34.0 19.25 DNA: spermidine complex (50: 1), 25 µg DNA Equivalent / g 4. Lower molecular weight 48 15 31.0 16.17 DNA: spermidine complex (25: 1), 25 µg DNA Equivalent / G 5. Lower molecular weight 40 14 34.0 18.43 DNA: spermidine complex (10: 1), 25 µg DNA Equivalent / g Injection with DNA alone was as good as ineffective due to the survival ratio in the experiment with whole-body X-irradiated mice and an irradiation dose of more than LD50.

It was found, however, that the injection was initiated with DIiA and spermidine compounds in different weight ratios an increase in the survival ratio It more than doubled the average lifespan by about a week extended and, in animal experiments, extraordinary compared to the control group Effect.

Example 1 0.5 g of spermine (C10H26N4; molecular weight 202.27) and 10 g of DNA with the molecular weight of about 500,000 extracted from salmon sperm separately in the solution of a mixture of sodium chloride (0.01 M) and sodium citrate (0.014 M) solved.

Each solution is made by adding sodium hydroxide (0.1 N) or hydrochloric acid (0.1 N) adjusted to pH 7. These solutions are mixed well to make the implementation to effect, and left to stand at 370C for 18 hours. Then 5 volumes of ethyl alcohol (99 [beta]) is added to the mixture and it is left to stand for 30 minutes. The. Originated White precipitate is separated off by centrifugation (5000 rpm, 10 minutes), then double the volume of ethyl alcohol (99%) is added, stirred and centrifuged (5000 rpm, 10 minutes). The precipitate obtained is recrystallized from ethyl alcohol. 10.4 g of a colorless, white powder are obtained.

Example 2 0.2 g of spermidine trihydrochloride (C7H19N3; molecular weight 145.25) and 10 g of DNA with a molecular weight of about 500,000, extracted from herring sperm, are separated in a mixed solution of sodium chloride (0.015 M) and sodium citrate (0.0015 M) solved. The solutions are brought to pH 7 by adding sodium hydroxide solution (0.1 W) or hydrochloric acid solution (0.1 N). The solutions will be mixed well to effect reaction and allowed to stand at 37 ° C for 18 hours. Then 5 volumes of ethyl alcohol (99%) are added to the mixture and it becomes 30 Left to stand for minutes.

The resulting white precipitate is centrifuged (5000 rpm, 10 minutes) separated, then treated with twice the volume of ethyl alcohol (99%), stirred and centrifuged (5000 rpm, 10 minutes). The recorded precipitation will recrystallized in ethyl alcohol. 10.1 g of the desired product are obtained as colorless white powder.

Example 3 0.5 g of cadaverine (C5H14N2; molecular weight 102.18) and 5 g deoxyribonucleic acid with a molecular weight of about 300,000, extracted from the thymus of the calf, are separated in a sodium chloride solution (0.01 Mol) dissolved. The solutions thus obtained are mixed for reaction and then 36 Left to stand at 37 ° C for hours. The reaction mixture is treated as in Example 1. 5.3 g of the desired product are obtained as a colorless white powder.

Example 4 1 g of polylysine, d.i. basic l-olyamino acid, and 10 g Deoxyribonucleic acid with the molecular weight of about 200,000 extracted from of the calf thymus are dissolved separately in sodium chloride solution (0.01 mol). The solutions thus obtained are mixed for reaction and kept at 37 ° C for 18 hours ditched. The reaction mixture is treated as in Example 1. One obtains 10.1 g of the desired product as a colorless, white powder.

Example 5 0.1 g of polyarginine, d, i. basic. Polyamino acid, and 5 g deoxyribonucleic acid with the molecular weight of about 500,000 extracted from salmon sperm, are separately dissolved in sodium chloride solution (9.01 mol). the solutions thus obtained are mixed for reaction and then at 37 ° C. for 18 hours ditched. The reaction mixture is treated similarly to Example 1. tWan receives 5.0 g of the desired product as a colorless white powder

Claims (2)

Claims. 1. Compound of basic polyamine and deoxyribonucleic acid for Radiation Damage Healing. 2. Method of making a connection for healing or prevention of radiation damage from deoxyribonucleic acid and basic amine or basic amino acid, characterized in that deoxyribonucleic acid and basic acid are separated Amine or basic amino acid in dilute sodium chloride solution or sodium acetate containing sodium chloride solution dissolves, the SO obtained solutions for reaction mixed, the reaction mixture is allowed to stand for 6 to 36 hours at 370C, its pH value adjusts to 7, alcohol is added to the mixture and the precipitate thus formed is carried out Centrifugation takes up.