Method of preparing a mixture of ribonucleotides

Details Method of preparing a mixture of ribonucleotides Method of preparing a mixture of ribonucleotides

1990-02-01

1991-11-12

Lilling, Herbert J.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435 90, 435 91, C12P 1930

Patent

active

050647580

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION



FIELD OF THE INVENTION

This invention relates to medicine and more particularly it relates to the method of preparing a mixture of ribonucleotides.


DESCRIPTION OF THE RELATED ART

Tapetoretinal abiotrophy is a group of hereditary diseases of man that are characterized by progressive loss of vision and blindness. The disease is rather common and its incidence varies from 0.2 to 5 per 1000 of population. Despite this high occurrence of taperoretinal abiotrophy, there are no effective means to treat it.
Known in the prior art is a method for preparing a mixture of ribonucleotides having medicating properties (Bulleten eksperim. biologii i meditsyny, 1989, Moscow, No. 9, p. 23-26). According to this method, ribonucleic acid with the concentration of 5-10 mg/ml is treated with pancreatic ribonuclease at a temperature of 45.degree. C. for an hour.
The method is not very efficient. The obtained mixture of ribonucleotides contains proteins, high-molecular pyrogenic substances and can therefore only be used for external application. Oral administration of the preparation causes deep degradation of its nucleotides and the medicating effect of the preparation is thus lost.
Known in the prior art is another method for preparing a mixture of ribonucleotides (Vestnik Akad. Med. Nauk SSSR, 1971, Meditsina, No. 7, p. 63-69) by hydrolyzing ribonucleic acid with pancreatic ribonuclease and dialyzing the obtained hydrolysate with subsequent isolation and concentration of the end product. The yield of thus prepared mixture is 40-43 per cent by weight.
The method has low yield, the purity of the end product is insufficiently high due to its contamination with low- and high-molecular pyrogenic compounds. Intramuscular administration of this preparation is very painful, it evokes elevation of body temperature in most patients and allergic reactions. Subconjunctival administration causes hyperaemia and oedema of the conjunctive of the eye-ball with enlargement and tenderness of the parotid lymph nodes.


SUMMARY OF THE INVENTION

The invention is based on the problem of providing a modified method by which the yield of the end product might be increased, the quality improved, and the pyrogenic properties ruled out.
The problem is solved by providing a method for preparing a mixture of ribonucleotides by hydrolysis of ribonucleic acid with pancreatic nuclease, isolation of the ribonucleotide fraction from the hydrolyzate with subsequent isolation of the end product in which, according to the invention, ribonucleic acid is hydrolyzed at pH 4.5-5.6 with subsequent separation of the ribonucleotide fraction from the hydrolysate on membranes with pores sized 50-150 .ANG..
In order to increase the yield of the end product, it is recommended that hydrolysis be carried out at a temperature of 62.degree.-65.degree. C.
If the temperature is below 62.degree.60 C., the enzymatic hydrolysis with pancreatic ribonuclease is slow; if the temperature is above 65.degree. C. the enzyme is partly denatured and the resultant hydrolysis is incomplete.
In order to prevent propagation of microflora, it is recommended that before separation of the ribonucleotide fraction, ethanol be added to the hydrolysate in the quantity of 15-30 per cent by volume. If the amount of ethanol added to the hydrolysate is below 15 per cent by volume, the growth of the microflora is now slowed. If the ethanol proportion exceeds the 30 per cent limit, the ribonucleotides are partly precipitated.
The ribonucleotide fraction should preferably be separated on the membranes at a temperature of 20.degree.-30.degree. C. and a pressure of 0.15-0.4 MPa. Filtration of the mixture under a pressure below 0.15 MPa and a temperature below 20.degree. C. increases substantially the time of the process without increasing the yield of the product; contamination with microflora is possible. If the pressure is above 0.4 MPa and the temperature is above 30.degree. C., the composition of the end product can be altered and the membrane destroyed. The end product i

REFERENCES:
patent: 3785926 (1974-01-01), Jachertz
patent: 3920519 (1975-11-01), Norimoto et al.
patent: 4190649 (1980-02-01), Beljanski
patent: 4335239 (1982-06-01), Beljanski
patent: 4758553 (1988-07-01), Ogoshi
Vestnik Akad. Med. Nauk. SSSR 1971, Meditsina, No. 7, pp. 63-69.

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