RU2002755C1 - Cellulose and gossypol derivative showing interferon-inducing and anti-viral action - Google Patents

Description

On the PC spectra of the preparation there are intense absorption bands at 1610, characteristic of the carboxylate anion of the carboxymethyl group, and at 1460 and 1385 characteristic of the benzene ring and methyl group, respectively, of polyphenol - gossypol.

In the UV spectra of the preparation, there is an intense absorption band at A 385 nm characteristic of the naphthalene cycle. Based on the change in the intensity of this band, the polyphenol content in the polymer chain was calculated. The empirical formula is:

 - (С4оНзаСч4) а - (CeHioOsJe - (CsHnO а), where

a - 3%. b - 63%. at 34% (May.) and n 530-532.

Test of the proposed compound (K-37) for cytotoxicity, interferon-inducing and antiviral activity in vitro and in vivo ..

The cytotoxicity of the proposed K 37 preparation was determined in 24-48 h cultures of transplanted cell line L 929. On a monolayer of cells grown on the bottom of 96-well panels at 37 ° C in a thermostat with a constant carbon dioxide content of 3.5%. various concentrations of the study drug are applied. After 24 hours of cell contact with the drug, the cytotoxic effect is evaluated. As shown in Table 1, an increase in the concentration of the drug up to 3000 μg / ml does not lead to the cytopathic effect of the drug on the cells, while the initial gossypol exhibits pronounced toxicity to the cells even at a concentration of 125 μg / ml.

Study of the toxic effects of the intended compound in vivo (in mice).

The toxic dose of K-37 is determined on outbred white mice weighing 10-12 g with a single intraperitoneal injection of various concentrations of the drug and the volume of 0.2 ml per mouse calculate the percentage of survival of animals 2 weeks after its administration (Table 2). As can be seen from Table 2, the proposed K-37 preparation when administered with the most soluble amounts (2000 mg / kg) turned out to be non-toxic, while the initial gossypol preparation was already toxic at a concentration of 100 mg / kg body weight.

A comparative study of the interferon-inducing activity of gossypol and the proposed drug K-37 in vitro. Various concentrations of the proposed preparation K-37 and gossypol in combination with DEAE-lextran are added to the monolayer of L-929 cells. After 4 hours of contact with the preparations, the cells are washed twice with Hanks solution and 199 are added.

medium with 2% bovine serum. Interferon titers in the culture fluid were determined after 24 hours by suppressing the cytopathic effect (CPP) of the mouse encephalomyocarditis test virus (EMC) by the micromethod on homologous cultures. K-37 has a pronounced interferon-inducing activity when used at a concentration of 250 μg / ml (Table 3). The titers of interferon (IFN), synthesized in response to the introduction of K-37 into the cell culture 199, are 12-13 times higher than the level of IFN, formed in response to the introduction of the original drug gossypol.

The interferon-inducing activity of the proposed compound is in vivo (in mice).

The interferon-inducing activity of the proposed compound K-37 and the parent gossypol in mice was determined by the oral, rectal and subcutaneous route of administration. The results of the tests are shown in table.4. With the K-37 oral route of administration, the maximum production of serum interferon reaches 640 u / ml and is observed at a dose of 25 mg / kg. With rectal administration of K-37 at a dose of 10 mg / kg of body weight, interferon titers in blood serum reach 1280-2560 units / ml, with subcutaneous administration in the same dose - 1280 units / ml. The titers of IFN synthesized in response to the introduction of K-37, 16-32 times higher than the level of IFN induced by gossypol.

Antiviral activity of K-37 in vivo (in mice) ..

The protective effect of K-37 is studied in infections caused by mouse encephalomyocarditis viruses (Columbi strain i) and influenza (strain R-94) of mouse hepatitis (strain Mishcherin) on outbred mice weighing 12-14 g. The drugs are administered in an effective dose in volume 0.2 ml 4 hours after infection with the above viruses. The dose of infection of mouse encephalomyocarditis virus is 5.0-6.0 Ig LDU / 0.2 ml and hepatitis of mice 6.0 Ig LD50 / 0.2 ml. The hemagglutinating titer of influenza virus is 1: 1024 units / ml. The results are summarized in table 5. The data obtained suggest that the proposed drug, when applied according to the treatment regimen, has a pronounced antiviral activity against a wide range of experimental viral infections. (56) Sadykov A.S. et al. Interferon Inductors, Tashkent. 1978, p. 304.

USSR copyright certificate No. 886989, class. B 02 C 21/00. 1980.

USSR copyright certificate No. 721103, cl. A 61 K 45/02. 1979.

Table Cytotoxic effect of gossypol and K-37 in vitro (on cell culture L-929)

Note. / + / 25. / ++ / 50%, / +++ / 75%, / ++++ / 10% cell death; / - / - lack of toxic effect.

Table 2 The study of the toxicity of the proposed drug K-37 in vivo (in mice)

Note. For each dose of drugs, 20 mice were taken. x Literature data.

Table3

Comparative study of the interferon-inducing activity of gossypol and K-37 in vitro

(on cell culture L-929)

Ta b l and tsa4

Interferon-inducing activity of the proposed drug K-37 and gossypol im vivo (in the blood serum of experimental animals)

Table Ica5 Antiviral activity of the proposed drug K-37 in vivo (in mice)

Virus

Protection of animals,%

Influenza (strain R -94) Hepatitis in mice (Mishcherin strain)

Encephalomyocarditis in Mouse Mice (Columbi strain)

Continuation of Table 3

Credibility. R

75

 0.001

40

0.01

65

 0.001

112002755

Formulas a of the invention wherein 3 wt.%;

Derivative of cellulose and gossypol

formulas

h sien. N 1-

SY.OS.SSPM

b May 63 .; % in 34mass.%; n 530 - 532,

5 anti-viral

12

where 3 wt.%;

b May 63 .; % in 34mass.%; n 530 - 532,

5 possessing an interferon-inducing antiviral effect.