US3803116A - Compound for restoring radiation injury and process for preparation thereof - Google Patents

Abstract

COMPOUNDS FOR RESTORING RADIATION INJURY WHICH ARE THE REACTION PRODUCT OF A BISIC POLYAMINE OR BASIC POLYAMINO ACID AND DEXOYRIBONUCLEI ACID, AND A PROCESS FOR PREPARATION THEREOF.

Description

United States Patent Olfiee 3,803,] 16 Patented Apr. 9, 1974 3,803,116 COMPOUND FOR RESTORING RADIATION INJURY AND PROCESS FOR PREPARA- TION THEREOF Toyozo Sekiguchi and Tomio Fujii, Tokyo-t0, and Hldeo Kobayashi, Chiba, Japan, assignors to Kakenyaku Kako Kabushiki Kaisha, Tokyo-to, Japan No Drawing. Filed May 27, 1970, Ser. No. 41,045 Int. Cl. C07d 51/50 US. Cl. 260-1125 4 Claims ABSTRACT OF THE DISCLOSURE Compounds for restoring radiation injury which are the reaction product of a basic polyamine or basic polyamino acid and deoxyribonuclei acid, and a process for preparation thereof.

DETAILED EXPLANATION OF THE INVENTION This invention relates to compounds effective for restoring and protection against radiation injury composed of the reaction product of a basic polyamine and deoxyribonucleic acid -(-DNA) and to a process for preparing the same.

It is generally known that the biosynthesis DNA in the cells of human beings and mammals is severely damaged by ionizing radiation. Also it is recognized that such biosynthesis of DNA is indispensable for cell division, and thus the inhibition of DNA biosynthesis makes impossible cell division and finally leads to the death of the cells.

Recently diagnosis and treatment in the early stage of cancer have drawn strong attention in the medical field and have widened the use of various treatments such as those with ionizing radiation, pharmacological preparations or surgical operation.

However, radiation treatment or pharmacological treatment are generally accompanied by several kinds of side effects, particularly in hematologic organs where the cell division is most active. Thus, it is frequently impossible to continue the cancer treatment due to serious side effects such as leucopenia, the abnormal decrease of blood white cells.

Consequently a suitable substance capable of restoring and protecting against radiation injury is strongly desired, and such substance is expected to be able to restore the damage caused by the side elfects of cancer treatment.

However, there are some protective drugs such as sulphydryl containing compounds which are reactive with radiation induced free radicals which have been formed in the human body by ionizing radiation and are only effective when administered to the human beings before X-ray irradiation. But there are very few drugs which can restore the radiation injury when administered after the irradiation. Such drugs are called as restorator and differentiate with the former drugs, the protecters.

Today, it is a restorer that is strongly desired to restore the severe side efiects accompanying cancer treatment by ionizing radiation such as X-rays or cobalt gamma rays.

At present, however, substances capable of restoring the inhibition of the DNA biosynthesis are scarcely known, and only confirmed is the fact that high molecular weight DNA isolated from the same species is effective in restoring the radiation injury in irradiated cells of animals.

However, it is quite diflicult to use a DNA preparation which is isolated from human beings to restore human radiation injury because it is quite difiicult to obtain such human tissue materials as DNA source. In the practical use of a DNA preparation as a pharmacological drug it must be considered that the DNA must be isolated from a species other than human beings.

Furthermore, there may be some possibility of hereditary danger if there be used very high molecular weight foreign DNA derived from another species as a pharmacological drug, thus making it necessary to use lower molecular weight DNA ranging from 200,000 to 500,000 to avoid such hereditary danger of genetic mutation because it is well known that such lower molecular weight DNA cannot induce the genetic transformation even in the bacterial system.

It is well known that in the higher animals DNA is not present in the free state but combined with histone, a basic protein.

Also there is a possibility that the inhibition of DNA biosynthesis or strand scission of DNA double helices resulting from radiation may be caused secondarily by damage to basic protein combined with DNA.

Therefore, it is not only the inhibition of DNA biosynthesis or restoring the damage to the DNA molecule which must be considered but also the restoring of the damage to histone, a basic protein combined to DNA molecule and modified the biological function of DNA, are quite necessary to obtain the more effective restoration of the radiation injury of the irradiated mammals.

It is quite impossible to use foreign histone from foreign sources to restore the radiation injury to the histone moiety, however, because it cannot be avoided that severe allergic reaction would be induced when foreign protein was administered (antigen-antibody reaction).

On the other hand it is confirmed that lower molecular weight polyamino acids or polyamines having molecular weights of several hundreds cannot induce antigen-antibody reactions.

Based on the facts mentioned above, the present in'ven tors conceived that the administration of artificial compounds of DNA combined with antigenetically safe basic polyamines or basic polyamino acids resembling the na tural complex compound of DNA and basic protein, histone, would be more effective than the simple administration of DNA alone against radiation injury and tried to produce various compounds from lower molecular weight DNA and several kinds of basic polyamine or basic polyamino acid,

Thus it is an object of this invention to provide a process of producing compounds from DNA and basic polyamines or basic polyamino acids comprising the steps of dissolving DNA having molecular weight ranging from 200,000 to 500,000 isolated from sperm of fish such as salmon or trout or from internal organs such as thymus, liver or kidney of mammals such as calf, or young pig and basic polyamines or basic polyamino acids, the weight of polyamine or polyamino acid being V to ,6 of the weight of, separately in dilute sodium chloride solution (0.0015 N) or in a mixed solution of sodium chloride and sodium citrate (0.0015 N and 0.00015 M). Then the pH of each solution is adjusted exactly to pH 7.0 'by adding 0.1 N sodium hydroxide solution or 0.1 N hydrochloric acid, then the solutions are mixed and reacted, allowing the mixture to stand at 37 C. for ca. 6 to 36 hours. Then there was added 99% ethanol to the reaction mixture in a volume about 5 times that of the mixture, and the mixture was allowed to stand for 30 minutes, the resulting white precipitate by eentrifugation of at 5,000 r.p.m. for 10 minutes. Twice the volume of 99% ethanol was added to the collected precipitate under agitation, the mixture was again subjected to centrifugation at 5,000 rpm. for 10 minutes and the collected precipitate was recrystallized from ethanol or ethyl ether to obtain the DNAzbasic polyamine or basic polyamino acid compound.

Physical and chemical properties of the compound constituted from DNA and basic polyamine or polyamino acid according to the process of this invention are as follows:

(1) Molecular weight, 200,000 to 500,000

(2) Sedimentation constant, 12s or higher (3) Specific gravity, 1.6501.700

(4) Viscosity, n =3 X 10 -6 X 10- (5 Specific optical rotation, tan a=5 l0- 10" (6) Absorption spectra, B =2600 A.-2657 A.

(7) Appearance, colorless crystalline powder (8) Solubility, soluble in water, dilute saline (0.015 N insoluble in ethanol, acetone and ethyl ether.

The restorative efieet on radiation injury by the product according to this invention was tested by two methods.

First, there was employed the colony forming method using tissue cultured cells which consists of cultivating the cells, human origin Changs liver cells, with Eagles MEM (minimum essential media) supplemented with 10% of calf serum in a glass petri dish for two weeks to form visible colonies thereby evaluating the restoration of the irradiated cells from radiation injury. Since dead cells are incapable of forming a visible colony, restorative effects of the compounds obtained according to this invention were evaluated by observing the survival ratio of the irradiated cells by counting the number of visible colonies. Changs liver cells were subjected to the radiation of 200, 400, 600 and 800 R. with X-ray of 300 kvp., and the number of cells surviving after two weeks was determined by the colony count.

In comparison with the control group to which DNA was not added, the groups to which DNA alone was added showed a slight increase, ca. 1.5 times, of the number of colonies at each radiation dose.

On the other hand the complex compound of DNA and spermidine which is one of the basic polyamines which may be used according to the invention showed. marked effectiveness when it was added to the culture media immediately after the X-ray irradiation of the cells.

Namely, the number of colonies was increased to about four times that in the control group.

Secondly, the restorative effect of the compounds of this invention was tested by animal experiment using mice the whole bodies of which were irradiated with X-rays.

The mice used were strain ddn, female, and the body weight ranged from to g. Radiation factors were 550 R. for the whole body irradiation by X-rays of 300 kvp.

In the control group the mice were intraperitoneally injected immediately after the X-ray irradiation with dilute saline (0.015 M NaCl and 0.0015 M sodium citrate solution) which was the solvent for DNA or the DNA basic polyamine complex compound. The injections were repeated three times a week.

In the second group were injected intraperitoneally immediately after irradiation with herring sperm DNA having a molecular weight of 200,000 at a dose of 25 ug./ g. body weight of the mouse. The injections were repeated three times a week for four weeks.

In the third group the mice were injected with the complex compound of herring sperm DNA having a molecular weight of 200,000 and spermidine, a basic polyamine. In the complex compound the weight ratio of DNA to spermidine was 50:1.

The dose of complex intraperitoneally injected immediately after the irradiation was 25 ,ug. of DNA equivalent for each gram of 'body weight and the injections were repeated three times a week for four weeks.

In the fourth group the mice were injected with the complex compound of the same DNA and spermidine, but the weight ratio of DNA to spermidine was 25:1. The injection was carried out for the second group.

In the fifth group the mice were injected with the complex compound of the same DNA and spermidine, but

4- the weight ratio of DNA to spermidine was 10:1. The injection was carried out as for the second group.

The results of the tests are shown by the following Table 1.

Injection with DNA alone was almost inefiective in the survival ratio as shown by the mice experiments in which the mice were subjected over their entire bodies to an exposure dose of X-rays greater than LD But, it was found that DNA and spermidine compounds in various weight ratios showed upon injection an increase of more than twice in the survival ratio and an extended average life span of about a week, this being shown in the animal experiments, in comparison with the control group.

EXAMPLE l 0.5 g. of spermine (C H N molecular weight 202.27) and 10 g. of DNA having molecular weight of ca. 500,000 extracted from salmon sperm were separately dissolved into the mixed solution of 0.01 M sodium chloride and 0.014 M of the sodium citrate and each of the resulting solutions was adjusted to pH 7.0 by adding 0.1 N sodium hydroxide or 0.1 N hydrochloric acid. Then these solutions were well mixed to effect reaction and allowed to stand at 37 C. for 18 hours. Then, 5 volumes of 99% ethyl alcohol was added to the mixture and allowed to stand for 30 minutes. The resulting white precipitate was collected by centrifugation at 5,000 r.p.m. for 10 minutes, then added with twice the volume of 99% ethyl alcohol under agitation and again subjected to centrifugation at 5,000 r.p.m. for 10 minutes. The precipitate thus collected is recrystallized from ethyl alcohol to obtain 10.4 g. of colorless crystalline powder.

EXAMPLE 2 0.2 g. of spermidine trihydrochloride (C H N molecuar weight 145.25) and 10 g. of DNA having a molecular weight of ca. 500,000 extracted from herring sperm were separately dissolved into a solution of a mixture of 0.015 M sodium chloride and 0.0015 M sodium citrate and the resulting solutions were adjusted to pH 7.0 by adding 0.1 N sodium hydroxide solution or 0.1 N hydrochloric acid. Then these solutions were well mixed to effect reaction and allowed to stand at 37 C. for 18 hours. And then, 5 volumes of 99% ethyl alcohol was added to the mixture and allowed to stand for 30 minutes.

The resulting white precipitate was collected by centrifugation at 5,000 r.p.m. for 10 minutes, then added with twice the volume of 99% ethyl alcohol under agitation and again subjected to centrifugation of 5,000 r.p.m. for 10 minutes. The precipitate thus collected is recrystallized from ethyl ether to obtain 10.1 g. of the desired product as a colorless crystalline powder.

EXAMPLE 3 0.5 g. of cadaverine (C H N molecular weight 102.18) and 5 g. of deoxyribonucleic acid having molecular weight of ca. 300,000 extracted from thymus gland of calf were separately dissolved into 0.01 M sodium chloride solutions, and the solutions thus obtained were mixed to react and then allowed to stand at 37 C. for 36 hours. The reaction mixture was treated similarly as in Example 1 to obtain 5.4 g. of the desired product as a colorless crystalline powder.

EXAMPLE 4 l g. of polylysine, i.e. a basic polyamino acid, and g. of deoxyribronucleic acid having molecular weight of ca. 200,000 extracted from thymus of calf were separately dissolved in 0.01 mol sodium chloride solutions, and the solutions thus obtained were mixed to react and allowed to stand at 37 C. for 18 hours. The reaction mixture was treated similarly as in Example 1 to obtain 10.1 g. of the desired product as a colorless crystalline powder.

EXAMPLE 5 0.1 g. of polyarginine, i.e. a basic polyamino acid, and 5 g. of deoxyribonucleic acid having molecular weight of ca. 500,000 extracted from salmon sperm were separately dissolved in 0.01 M sodium chloride solution, and the solutions thus obtained were mixed to react and then allowed to stand at 37 C. for 18 hours. The reaction mixture was treated similarly as in Example 1 to obtain 5.0 g. of the desired product as a colorless crystalline powder.

What is claimed is:

1. A compound comprising a complex formed between deoxyribonucleic acid and a basic polyamine selected from the group consisting of spermine, spermidine and cadaverine or a basic polyamino acid selected from the group consisting of polylysine and polyarginine.

2. A compound as claimed in claim 1 in which said deoxyribonucleic acid has a molecular weight within the range of 200,000 to 500,000.

3. A compound as claimed in claim 2 in which the weight ratio of acid to basic polyamine or basic polyamino acid in said complex is within the range of 10:1 to :1.

4. A process for the preparation of a compound as claimed in claim 1 consisting essentially of the steps of dissolving deoxyribonucleic acid in a first dilute sodium chloride solution or a dilute sodium chloride solution containing sodium citrate; dissolving a basic polyamine selected from the group consisting of spermine, spermidine and cadaverine or a basic polyamino acid selected from the group consisting of polylysine and polyarginine in a second dilute sodium chloride solution or a dilute sodium chloride solution containing sodium citrate; adjusting the pH value of each of the resulting solutions to 7.0; mixing the pH-adjusted solutions to effect reaction between the deoxyribonucleic acid and the basic polyamine or basic polyamino acid; allowing the reaction mixture to stand at 37 C. for 6 to 36 hours; adding ethanol to the pH- adjusted reaction mixtures and collecting the precipitate thus formed by centrifugation.

References Cited UNITED STATES PATENTS 2,710,860 6/1955 Ruskin 260211.5 R 3,089,869 5/ 1963 Mauvernay 26021l.5 R 3,155,647 11/1964 Dutcher et al. 260-2115 R 3,215,687 11/1965 Tsuchiya et al. 260211.5 R 3,287,351 11/1966 Cantineau et al. 260211.5 R 3,300,476 1/1967 Zahn et al. 260-2115 R 3,314,937 4/1967 Vendrely et al. 260--211.5 R

LEWIS GOTIS, Primary Examiner I. R. BROWN, Assistant Examiner US. Cl. X.R. 26021l.5 R; 424-