RU2083221C1 - Ridostin as interferon inductor - Google Patents

Abstract

FIELD: medicine, veterinary science. SUBSTANCE: method suggests Ridostin formula for nonmutagenous preparation of lower toxicity, high tolerance and activity to interferon induction. Ridostin consists of 5-15% two-spiral ribonucleic acid derived from Saccharomyces serevisiae yeast killer strains and single-thread ribonucleic acid as the main components. EFFECT: higher efficiency. 7 tbl

Description

 The invention relates to medicine and veterinary medicine and can be used for the treatment and prevention of viral diseases.

 Interferon inducers that stimulate the production of endogenous interferon in humans and animals have several advantages over exogenous interferon preparations and are promising means of preventing and treating viral infections.

 The most active interferon inducers include synthetic polyribonucleotides and double-stranded (ds) PHK of natural origin.

 However, along with their high efficiency as interferon inducers, these drugs are highly toxic and exhibit mutagenic activity.

 Known interferon inducer double-stranded polyribonucleotide complex poly I: poly C, which has an inhibitory effect against a wide range of viruses. However, the drug poly I: poly C is also highly toxic and has mutagenic activity.

 Closest to the claimed preparation (prototype) is an inducer of interferon to PHK of killer strains of the yeast Saccharomyces cerevisiae, inducing high titers of interferon and exhibiting pronounced antiviral activity against a wide range of infections (influenza, herpes, HIV, tick-borne encephalitis, etc.), and on a number of models, he demonstrates advantages over other interferon inducers, such as larifan, poly G: poly Ts.

The disadvantages of the inducer of interferon dsRNA from yeast Saccharomeces cerevisiae are:
1. High toxicity, the drug belongs to the class of highly active toxic substances (LD ₅₀ when administered intraperitoneally to mice 5.1 9.7 mg / kg).

 2. The presence of a weak, but statistically significant mutagenic effect in the form of an increase in the number of chromosomal aberrations by 2-2.5 times compared with the control (intact animals).

 The aim of the invention is the creation of a new preparation of an interferon inducer based on dsRNA of killer strains of S. cerevisiae yeast - ridostin, which has high efficiency, reduced toxicity, good tolerance and mutagenic activity.

The goal is achieved by creating the optimal dosage form of an interferon inducer (ridostin), which includes dcRNA of yeast S. cerevisiae and single-stranded yeast PHK as the main components, while the content of dsRNA is limited to 5-15%
Ridostin is a sterile lyophilized preparation containing dcRNA in an amount of 5-15% and an additional single-stranded yeast PHK.

The batches of the drug have the following characteristics:
interferon-inducing activity of at least 640 IE ₅₀ / ml;
mean lethal doses for mice with an intraperitoneal route of administration of 45 to 120 mg / kg;
the number of metaphases with chromosomal aberrations did not exceed the values of this indicator in control animals.

The new significant distinguishing features of the proposed drug are the introduction of single-strand high-polymer PHK yeast into the composition of the preparation and a decrease in the content of the highly toxic component dsRNA to 5-15%
It was established by experimental data that yeast dsRNA exhibits a weak, but statistically significant mutagenic effect, increasing the frequency of chromosomal aberrations in mouse bone marrow cells after intraperitoneal administration of dsRNA in a dose of 1/4 LD ₅₀ (1.5 mg / kg) (Table 1). which significantly limits the possibility of using the drug as a therapeutic agent for medicine and veterinary medicine.

 As can be seen from the data table. 1, administration of only dsRNA results in a statistically significant increase in the number of chromosome aberrations in mouse bone marrow cells. In the case of the introduction of a complex preparation containing an additional single-stranded high polymer PHK, an increase in the frequency of chromosomal abnormalities is not detected.

 The introduction of a new biologically active component of single-stranded PHK could affect the target property of interferon-inducing activity. In this regard, the authors examine the interferon-inducing activity of drug samples containing single-stranded PHK in relation to dsRNA 1: 1 40: 1. Samples of drugs are administered intraperitoneally to white mongrel mice in doses containing the same amount of dsRNA (0.5 mg / kg).

 The results are shown in table. 2.

 As can be seen from the data table. 2, single-stranded PHK in any ratio, up to 40: 1, does not lead to a loss of interferon-inducing properties of dsRNA.

 Since the preservation of efficiency with a decrease in the content of the active principle was not obvious, we studied the dependence of interferon-inducing activity on the content of dsRNA in the preparation. Determination of interferon-inducing activity of drugs with different dsRNA concentrations is carried out on outbred mice. All drugs are administered intraperitoneally in the same dose of 5 mg / kg (table. 3).

As can be seen from the data table. 3, interferon titers that stably exceed the level of 640 IE ₅₀ / ml, allowing the drug to be classified as highly active interferon inducers, are obtained using samples with dsRNA more than 2.5%
At the same time, an increase in the percentage of dsRNA in the preparation above 10-15% does not give a significant increase in the titers of produced interferon, which makes it possible to limit the dsRNA in the preparation to 10-13%
It has been established by experimental data that preparations with a dsPHK content above 50% are highly toxic and highly active toxic substances (SDYA) of LD ₅₀ less than 50 mg / kg. A correlation analysis of the relationship between the composition of the drug and its toxicity shows that the level of toxicity is determined by the content of dsRNA there is a close inverse correlation between the average lethal dose of the drug and the content of dsRNA (Table 4).

LD ₅₀ samples of the drug with dsRNA 5 15% and containing single-stranded PHK ranged from 45 to 120 mg / kg with an intraperitoneal route of administration, i.e. preparations belonged to highly or moderately toxic compounds.

 The results of an experimental study of the biological properties of the drug ridostin in comparison with its prototype dsRNA yeast, summarized in table. 1-4, indicate that the proposed change in the composition of the interferon inducer eliminates undesirable side effects, eliminates mutagenicity, reduces the toxicity of the drug, reduces the consumption of the expensive component of dsRNA while maintaining its target properties - interferon-inducing activity.

It is the use of distinctive essential features of the claimed composition of the drug: the content of dsRNA in an amount of 5-15% and the introduction of single-stranded PHK into the composition of the preparation allows to obtain a qualitatively new effect, namely:
1. High interferon-inducing activity.

 2. Reduced compared with the prototype toxicity.

 3. The lack of mutagenic activity.

 4. Reducing the cost of the dose of the drug, which together makes it promising for clinical use.

Example 1. The composition is prepared in the following ratio of components, wt. dsPHK 9% single-stranded PHK 30% NaCl the rest. The remaining composition examples are prepared in a similar manner. A series of the drug with the above composition has the following characteristics: interferon-inducing activity - 1088 ± 64 IE ₅₀ / ml; average lethal dose with intraperitoneal administration to mice 71 mg / kg; no mutagenic activity was detected (the number of chromosomal aberrations was 1.0% in the experiment compared with 1.8% in the control, which were animals that received 0.9% NaCl).

Example 2. Take the following ratio of components: dsPHK 8% single-stranded PHK 41% NaCl the rest. A series of the drug with the above composition has the following characteristics: intferferon-inducing activity - 1180 IE ₅₀ / ml; average lethal dose of 120 mg / kg; the number of chromosomal aberrations is 2.5% (compared with 2.0% in the control), which indicates the absence of mutagenic activity.

Example 3. Take the following ratio of components, wt. dsPHK 15% single-stranded PHK 45% NaCl the rest. A series of the drug with the above composition has the following characteristics: interferon-inducing activity - 1051 ± 392 IE ₅₀ / ml, average lethal dose of 80 mg / kg, the number of chromosomal aberrations is 1.8% (compared to 1.5% in intact animals), which the absence of mutagenic activity.

 Example 4. Take the following ratio of components, wt. dsPHK 5.0% single-stranded PHK 52% NaCl the rest. A series of drugs with this composition has the following characteristics: interferon-inducing activity - 1280 ± 0; medium lethal dose with intraperitoneal administration to mice 90 mg / kg; the number of chromosomal aberrations is 2.0% (compared with 1.5% in intact animals).

Example 5. The interferon-inducing activity of ridostin samples was studied by intraperitoneal administration of an ICR colony to white mice. Drugs are administered at a dose of 5 mg / kg. Blood was drawn from the retroorbital sinus of mice 6 hours after administration. The presence of interferon is judged by the suppression of the cytopathic effect (CPD ₅₀ ) of the test virus (vesicular stomatitis virus Indiana strain) in the culture of L-929 mouse fibroblasts by microtiter titration. The titer of serum interferon (in IE ₅₀ / ml) is taken as the reciprocal of its highest dilution, at which protection of 50% of the culture cells from the cytopathic effect of 100 CPD _{50 of the} test virus is observed. The results are presented in table. 5.

 From the data table. Figure 5 shows that drug samples containing 5-15% dsRNA and single-stranded PHK are active interferon inducers.

 Example 6. Determination of medium lethal doses of 3 series of ridostin and 4 series of dsRNA with different contents of the main components is carried out by the express method according to Prozorovsky on white random-bred mice with intraperitoneal administration.

 The results are presented in table. 6.

 The mean lethal doses of ridostin are 71% to 90%, which allows them to be classified as medium toxic compounds, in contrast to preparations containing dsRNA in an amount of more than 15% related to highly toxic and potent compounds.

Example 7. The mutagenic activity of the ridostin samples was evaluated by the method of analysis of chromosomal aberrations in the bone marrow cells of C 57 BL / 6 mice weighing 18-20 g of age 2-3 wt. The drugs are administered intraperitoneally at doses equal in content to dsRNA and corresponding to approximately 1/4 LD ₅₀ . The mutagenic effect is assessed by the presence of chromosomal aberrations in bone marrow cells, taking into account structural aberrations (fragments) 24 hours after administration. 4 males are taken for each analysis point, 100 metaphases are analyzed from each animal. The reliability of the difference between experience and control is determined by the Student criterion with Fisher's correction.

 The results are shown in table. 7.

 As can be seen from the data presented, preparations containing dsRNA in the selected interval (5-15%) and an additional single-stranded PHK do not exhibit mutagenic activity in contrast to the prototype.

 The drug Ridostin underwent a full cycle of preclinical trials.

Claims (1)

 An interferon inducer containing double-stranded RNA from Saccharomices cerevisiae yeast, characterized in that it additionally contains single-stranded high-polymer yeast RNA in the following ratio of components, wt. Double-stranded RNA 5 15
Single-strand high polymer RNA 30 52
NaCl Else

Images