Polyribonucleotides duplex on the basis of polarisierte and polyborazylene

 

(57) Abstract:

The invention relates to biochemistry, namely to obtain biologically active substances with antiviral action. Describes polyribonucleotides duplex on the basis of polarisierte and polyborazylene containing compounds of bivalent platinum in the form or CIS-diamminedichloroplatinum or CIS-dihydroxyphenylalanine (II), and complex polyribonucleotides duplex has the formula [poly(A)poly (Y)]m[Pt(II)X2]nwhere Pt(II)X2- or CIS-diamminedichloroplatinum (II), X is - NH3Cl, or CIS-dihydroxyphenylalanine (II), X = NH2OCl, with m : n = 1 : (0,02 - 0,20 ). The connection provides increased antiviral and interferon-inducing activity, is not toxic and has a high stability, which makes possible its application in the clinical setting. 3 Il., table 4.

The invention relates to biochemistry, or rather to a group of biologically active substances with antiviral action.

Known drug - duplex of polyribosomes and polyribosylribitol, poly(I) poly(C). It has antiviral and interferon-inducing activity in the body replacr what about the increases in cells and organisms affected by viral infection [Field A. K., Tytell, A. A. , Lampson, G. P., and Hilleman, M. R. Inducers of interferon and host resistance. II. Multistranded synthetic polynucleotide complexes. //Proc. Natl. Acad. Sci. USA. - 1967. - V. 58, N 3. - P. 1004-1010. [1], De Clercq E. , Stewart W. E., De Somer P. Increased toxicity of double-stranded ribonucleic acid in virusinfected animals, //Infect, immunity. - 1973. - V. 7. - P. 167-172. [2]].

Also known duplex of polyribosomes and polyribosylribitol - poly(G)poly(C). He also has significant antiviral and interferon-inducing activity, but its disadvantage is the tendency to aggregation and gelation in aqueous solutions [Timkovsky A. L., Kogan, E. M., Aksenov O. A. and other thermal activation of poly(G)poly(C): description of the effect and the proposed mechanism. //Questions virusol. - 1982. - So 27. - S. 92-96. [3]].

Also known complex polyribonucleotides duplex poly(G) poly(C) with CIS-diamminedichloro-platinum (II) - [poly(G) poly(C)]m[Pt(II)X2]nwhere X is NH3Cl. Antiviral and interferon-inducing activity of this complex is higher than that of the original duplex. However, the increased activity occurs only in a narrow region at low concentrations of Pt(II)X2(the mass ratio of m: n = 1:0,02, and return the value of n:m = rb= 0,02 - molar degree of inclusion of Pt(II)X2), and do not overcome skloniste [V. Brabec, Vrana, O., Platonova G. A. et al. Biophysical studies of the modification of poly(ri)poly(rC) by cis-platin. Relation to the biological activity of the complex. //Chem.-Biol. Interact. - 1991. - V. 78. - P. 1-12. [4]].

Closest to the claimed is polyribonucleotides duplex of polarisierte and polyborazylene - poly (A) poly (U), which also has antiviral and interferon-inducing activity and little toxic and not prone to aggregation in solution. Because of these advantages poly(A) poly(U) is used in eye clinics as antiviral drugs, as immunomodulator cancer and is being tested against human immunodeficiency virus. However, its antiviral and interferon-inducing activity lower than that of other duplexes-analogues [1].

Because of low polyribonucleotides duplex poly(A) poly(U) is used in clinical practice, it is desirable to provide a higher activity.

It is provided that serves polyribonucleotides duplex on the basis of polarisierte and polyborazylene, characterized in that it additionally introduced compound of bivalent platinum or CIS-diamminedichloroplatinum (II), or CIS-dihydroxylated is)X2]n,

where Pt(II)X2- or CIS-diamminedichloroplatinum (II), X = NH3Cl

or CIS-dihydroxyphenylalanine (II), X = NH2OCl, with m:n= 1: (0,02-0,20).

The need to introduce in polyribonucleotides duplex on the basis of polarisierte and polyribosylribitol compounds of bivalent platinum is not obvious. Known results the introduction of bivalent platinum polyribonucleotides duplex on the basis of polyribosomes and polyribosylribitol - poly(I) poly(C). Meanwhile, there is increasing antiviral and interferon-inducing activity of the duplex [De Clercq E., Hermann D., W. Guschlbauer Interferon induction by platinum(II)-poly(I)poly(C) complexes. //Biochim. Biophys. Acta. - 1983. - V. 741. - P. 358-363. [5]], and the duplex is destabilized [Hermann D. , C. Houssier, Guschlbauer, W. Dissociation of double-stranded poly(I)poly(C) by cisdiammine-dichloro-Pt(II). //Biochim. Biophys. Acta. - 1979. - V. 564. - P. 456-472. [6]]. With the introduction of compounds of divalent platinum polyribonucleotides duplex on the basis of polyribosomes and polyribosylribitol - poly(G) poly(C) some of the increase in antiviral and interferon-inducing activity was observed, but only in a narrow interval of the content of compounds of bivalent platinum - with rbnot above 0.02. When increasing the degree of inclusion compounds of divalent PL, xenou O. A., E. murinus A., Kogan, E. M. and other Biological activity of a complex of poly(G) poly(C), modified compounds of bivalent platinum. //Questions virusol. - 1995. - So 40, N 2. - S. 56-59. [7]].

It was found experimentally:

1. With the introduction of compounds of divalent platinum polyribonucleotides duplex on the basis of polarisierte and polyborazylene increasing antiviral and interferon-inducing activity, and in a much wider range of the degree of inclusion of Pt(II)X2than for duplex poly(G) poly(C) - up to rb= 0,2.

2. Duplex poly(A) poly(U) with the introduction of compounds of bivalent platinum stabilized, as evidenced by increasing its melting point.

3. Toxic manifestations in introducing the compound of bivalent platinum duplex poly(A) poly(U) up to the level of rb= 0,2 missing that makes it possible to use it with a compound of bivalent platinum in the clinical setting.

In Fig. 1 shows the structural formula of the sector polyribonucleotides duplex poly(A) poly(U) with CIS-diaminedichlorplatinum (II). In Fig. 2 shows the structural formula of the link of polarisierte (IP-dihydroxylation - platinum(II) (b).

The inventive complexes polyribonucleotides duplex [poly(A) poly(U)]m[Pt(II)X2]nobtained as follows.

Duplex poly(A) poly(U) derived from poly(A) with molecular weight of 100-500 thousand daltons (Da) and poly (U) with molecular weight of 100-500 thousand Yes mixing them in equimolar amounts in a solution containing 0.005 M sodium phosphate buffer, pH 7.4 and 0.1 M sodium chloride and incubation of the mixture at 37ofor at least 1 hour [1]. After this duplex cialiswhat against 0.01 M solution of etilendiamintetraatsetata Na, then against 0.1 M NaClO4. To 1 ml of 0.1 M solution of NaClO4, pH 7.0, containing 1.18 µmol poly(A) poly(U), add necessary for obtaining the desired values of rbthe amount of solution containing 1 ml 0,923 µmol of Pt(II)X2and NaClO40.1 M, pH 7.0, and the necessary volume of 0.1 M solution of NaClO4, pH 7.0, supplementing the volume of the reaction mixture to 1,262 ml. of the resulting solution is heated at a temperature of 37oC for 48 hours in the dark. Get the solution that contains the complex polyribonucleotides duplex and Pt(II)X2when the concentration of duplex - 700 µg/ml of This method are the complexes with different content of compounds of Pt(II), as evidenced by the examples (table who can be found in table 2.

Proof of education declared ingredients:

a) change the elution profile in gel chromatography in comparison with the profile of the original duplex poly(A) poly(U), the molecular weight of the obtained complex - 1.5-2 million Yes;

b) the composition of the complex is characterized by the number of covalently linked atoms of Pt(II) one nucleotide residue by the method of differential pulse polarographic on a mercury drop electrode [6].

The melting temperature of the duplex poly(A) poly(U) was determined spectrophotometrically by recording the changes in optical density of the solutions at 260 nm on a spectrophotometer SF-26 heating solutions in sealed cuvettes placed in a thermostatted enclosure. The optical density measured at a given temperature (At), was attributed to the initial optical density, measured at 25o(A25). The melting temperature (TPL) took the temperature value corresponding to 50% growth At/A25[Michelson A. M. , Massoulie J., W. Guschlbauer Synthetic polynucleotides. //Progr. Nucl. Acids Res. Mol. Biol. - 1967. - v. 6. - P. 83-141. [8]].

All the obtained complexes with different content of Pt(II)X2tested for antiviral and interferon-inducing activity. In the table 3 about the conduct of the white mice. Antiviral activity of the complexes increases monotonically with increasing rb.

Table 4 displays the data on the induction of interferon in the brain of mice with intravenous complexes of poly(A) poly(U) with CIS-Pt(II)X2.

With increase in the content of inorganic compounds Pt(II)X2in combination with polyribonucleotides duplex poly(A) poly(U) the level of interferon in the brain of animals increases with rbits maximum value occurs when rb= 0,20. Complexes with higher rbtoxic to mice when administered intravenously [6, 7].

The results of the tests show that unlike other polyribonucleotide duplexes-analogues, polyribonucleotides duplex on the basis of polarisierte and polyborazylene after introducing compounds of bivalent platinum gets higher antiviral and interferon-inducing activity, increased stability and the ability to induce the formation of interferon in the brain in warm-blooded animals.

Thus, the claimed substance has significant advantages. Introduction compounds of divalent platinum polyribonucleotides Duo increases with increase in the content of compounds of Pt(II)X2in a wide range of values of rbup to rb= 0,20. Adding to the duplex poly(A) poly(U) compounds of Pt(II)X2increases its stability, which is reflected in the increase of the melting temperature of the duplex. With the introduction of the complex into the bloodstream of a warm-blooded animal a significant level of interferon was detected in the brain that serves as a testimony of overcoming the blood-brain barrier and allows the use of the claimed substance as an antiviral drug against nairovirus infections.

Polyribonucleotides duplex on the basis of polarisierte and polyborazylene, characterized in that it additionally introduced connection divalent plate in the form or CIS-diamminedichloroplatinum (II), or CIS-dihydroxyphenylalanine (II), and complex polyribonucleotides duplex has the formula

[poly(A)poly(U)]m[Pt(II)X2]n,

where Pt(II)X2- or CIS-diamminedichloroplatinum (II), X = NH3Cl, or CIS-dihydroxyphenylalanine (II), X = NH2l, with m:n = 1:(0,02 - 0,20).

 

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