Biological activity of a new Soviet natural inducer, double-stranded RNA

Antiviral activity of a two-spiral RNA (ts RNA), a new natural interferon inductor was studied. It was shown that ts RNA extracted from a phage infected E. coli culture was an active inductor of interferon and resistance to infection with the forestspring encephalitis virus experimental animals. In experiments on 10-12 g mice ts RNA administered in a dose of 50 micrograms/mouse 6 hours after the infection induced up to 1280 units/ml of the serum interferon. When the inductor was administered repeatedly, the experimental animals developed hyporeactivity resulting in a marked decrease in interferon production after the 3rd subsequent injection. The most pronounced effect with respect to the forest-spring encephalitis virus was observed when the inductor was administered intraperitoneally in a dose of 20 micrograms/mouse 4 hours before the infection. The protective effect was less pronounced when the inductor was administered 24 and 48 hours before the infection. A two-fold administration of the inductor did not increase the antiviral effect. When the inductor was administered in a dose of 100 micrograms 14 days before the infection, the animals showed an increase in the nonspecific resistance to the infection resulting in a marked antiviral effect.     

Relation between interferon-inducing activity of natural interferon inducers and their chemical structure]

The interferon-inducing activity of new derivatives of gossypol, a low molecular weight natural polyphenol, and its model compounds was studied in regard to their chemical structure. The active groups required for development of optimal interferon inductors on the basis of the natural polyphenols were defined. As a result of the studies the new interferon inductor AC-1 which is a condensation product of gossypol, a natural polyphenol, and a cellulose derivative was isolated. The interferon-inducing activity of AC-1 was investigated with respect to its dose and the administration route. High interferon-inducing activity of the interferon inductor after its administration by any route in doses of 10 to 100 mg/kg was demonstrated. The serum interferon titers reached 2560 units/ml.     

Optimization of the schemes for using interferon inducers

The results of study on 2 Soviet interferon inductors, i.e. the synthetic polyguacyl polynucleotide and natural double-strand phage RNA or dsRNA were studied. It was shown that the time course of accumulation and period of circulation of interferon depended on the route of the inductor administration. The antiviral activity of polyguacyl and dsRNA in experimental influenza and tick-borne encephalitis is described. The maximum protective effect with respect to experimental influenza was observed with intranasal administration of the drugs 4 hours before inoculation. A pronounced protective effect with respect to tick-borne encephalitis was observed with intraperitoneal administration of the inductors or their use in the form of aerosols. Direct correlation between interferon production and the final protective effect was found.     

Interferonogenic and antiviral activity of the double-stranded replicative form of phage f2 RNA in tissue culture and on animals]

Biological activity of a new natural interferon inductor, the replicative RNA form of phage f2 (RFf2) was studied. A possibility of using RFf2 for production of highly active interferon under conditions of superinduction providing an increase in the interferon yield by to 256--512 times as compared to the control samples was shown. The protective interferonogenic and antiviral effect of RFf2 in mice infected with Semliki forest virus (SFV) and tickborne encephalitis virus (TBEV) was studied on administration of the inductor by various routes. It was found that intraperitoneal administration of RFf2 in a dose of 10 gamma per a mouse protected the infected animals from death. It was accompanied by production of up to 1280 units/ml of interferon in the blood serum of the animals. Maximum protection of the animals from death under conditions of the experiment (80 per cent on infection with SFV and 65 per cent on infection with TBEV) was observed when the preparation was administered twice: 4 hours after the infection. Combined use of RFf2 with chemotherapeutics (rimantadine) increased the protective effect both in the tissue culture and in vivo.     

Pharmacological analysis of the effect of natural double-helical nucleic acids on the detoxifying function of the liver

The effect of interferon inductors i.e. double stranded RNAs from S. cerevisiae and phage F6 on the liver detoxicating function was studied on noninbred albino mice. The liver detoxicating function was tested by duration of hexenal sleep. It was shown that intraperitoneal administration of the yeast and phage RNAs in doses of 1/5 LD50 for three times led to increasing of the narcotic sleep duration in the animals by 65 and 207 per cent, respectively. The effect was of the dose-dependent nature. The doses not inducing reliable inhibition of hexenal metabolism were equal to 1/10 LD50 for the yeast dsRNA and 1/27 LD50 for the phage dsRNA. The inhibitory effect of the dsRNAs was retained for 2-3 days after discontinuation of the drug use. When the dsRNAs were administered simultaneously with nembutal, an inductor of the liver microsomal enzymes, the dsRNAs eliminated its inducing effect. Simultaneous administration of alpha-tocopherol lowered the dsRNA effect on hexenal metabolism. The findings suggested that the dsRNA inhibitory effect on the liver detoxicating function was grounded on the mechanisms associated with inhibition of syntheses and activation of lipid peroxidation specific of the monooxygenase system under the action of the dsRNAs.     

Effect of T-activin on the production of immune interferon

A study was made of the effect of T-activin on the biosynthesis of immune gamma-interferon. It was shown that in 27% of patients with chronic nonspecific pulmonary diseases, production of gamma-interferon by lymphocytes was substantially reduced during exacerbation of inflammatory process in the lungs. It was discovered that T-activin was not an interferon inductor but enhanced its synthesis in patients with a low capacity of producing immune interferon even at small doses of interferon inductor. The preparation does not produce any effect on this process in normal subjects and in patients showing the normal level of gamma-interferon. Thus T-activin can be used for stimulation of interferonogenesis.     

Study of the antiviral activity of a poly I : poly-C complex with poly-L-lysine in monkeys]

Antiviral activity of poly-I-poly-C complex with poly-L-lysine was studied on macaco rhesus. The complex bifilamentous polyribonucleotide induced active production of serum interferon and provided pronounced protection of the monkeys infected intracutaneously with the variolovaccine virus (10 LD50 for the monkeys in intracutaneous infection). The effectiveness of the protective effect depended on the scheme and route of the drug administration. The highest prophylactic and therapeutic effect was provided by local administration of the complex in a dose of I mg per I kg of the body weight, the incubation period being increased 2--3 times and the period of the skin affections being decreased approximately 2 times. The results of the studies on the effect of poly-I-poly-C complex with poly-L-lysine were evident of definite prophylactic activity of the drug against experimental vernal encephalitis in the monkeys. The animals not treated with the inductor died on the 16th or 17th day after infection because of the paralysis of the trunc and extremities muscles. The clinical evidences of the disease in the animals treated with the drug were not uniform: from complete health to death.     

Comparative antiviral and interferonogenic activity of natural and synthetic interferon inducers in vitro and in vivo]

Biological activities of the RNA replicative form of phage f2, a natural interferon inductor and poly-I -- poly-C, a synthetic polyribonucleotide complex were studied comparatively. Differences in the comparative interferonogenic and antiviral activity of the inductors were as dependent on the type of the cell system. It was shown that DEAE-dextran increased the interferon-inducing activity of RFf2 in the cell culture by 4 to 8 times. The dynamics of the interferonogenic and antiviral activity of RFf2 in the L-929 cell culture was studied. Interferon appeared in the culture fluid in 6--8 hours and reached its maximum titers (128 IU50/ml) by the 24th hour, the maximum protection of the cells being also developed by the 12th--24th hour, reaching on an average 51 g PFU/ml. It was shown in the experiments with green marmosets that administration of RFf2 in the form of aerosol in a dose of 2.3 mg/kg induced interferon production in the blood serum the titers of which amounted to 80--160 IU50/ml 24 hours after the administration.     

Pharmacokinetics of the interferon inducer PHL-6 and interferon synthesis in target organs under various methods of drug administration]

The pharmacokinetics of PHL-6 and interferon synthesis dynamic in the target organs (tissues) of mice were studied during its and intraperitoneal administration. In the experimental setting, there was a direct correlation between the interferon production in the murine organs with single PHL-6 and distribution of 14C PHL-6. The highest radioactivity with its oral administration was detected in the liver and intestine. Interferon was actively synthesized in the intestine, liver and serum, showing the levels of 20000, 1024-2048 and 512-1024 IU/ml, respectively. The prolonged action of the drug was in good agreement with the low PHL-6 excretion from the body. It was also shown that almost the whole radiation dose 1 (greater than 98%) was excreted with feces and urine after single and chronic administrations of uniformly labeled PHL-6 which proved important clinical drug use.     

Determination of the species specificity of interferons in the translation of the their mRNA from various cell cultures

Interferons obtained on induction of human lymphocytes with Newcastle viruses and staphylococcal enterotoxin A and diploid fibroblast cells of human embryos with poly (I).poly (C), as well as translation products of interferon mRNA obtained from these cells were analysed serologically. It was shown that the main type of interferon produced by the cells depended on the cell culture and inductor nature. It was defined at the level of the respective gene depression. Effective translation of mRNA of the interferons of the 3 types makes possible production of cDNA and creation of bacterial plasmids coding the genetic information for the synthesis of human interferon.     

Relation of the biological activity of double helical interferon inducers and their penetration into the cell and suppression of protein synthesis

The quantities of 125I-ds-inductors of interferon penetrating into the cells of transplantable cultures such as M19 (human fibroblast cells) and L-929 (mouse line) were not significant i.e. 10.5-4 per cent of the drug added. Under conditions of transfection with calcium phosphate and in complex with DEAE dextran the quantities of the inductors adhering to the cells and their contents in the cytoplasmic and nuclear fractions markedly increased. During the transfection with calcium phosphate up to 50 per cent of the applied inductor bound to the cells and its content in the cytoplasm and nuclei reached at least 10 per cent. After penetration into the cells poly I.poly C probably maintained its native structure and appeared to be firmly bound to the nuclear material. Preliminarily hydrolyzed inductors showed no such penetrating capacity. Contrary to the human fibroblast cells, in the mouse cells L-929 treated with the ds-inductors there was observed inhibition of the total protein synthesis which was probably due to activation of enzymes such as 2-5A-synthetase and proteinkinase. Increased penetration of the ds-inductors into the cells was accompanied by a marked (from 10- to 1000-fold) rise in their antiviral activity and a 2-4-fold rise in their interferon-inducing activity. It was concluded that there was immediate dependence of ds-inductor biological activity manifestation on the level of the inductor penetration into the cells.     

Interferon-inducing and immunostimulating activity of bonafton in an experiment

It was shown for the first time that the antiviral drug bonafton administered orally to nonlinear albino mice in single doses of 5, 12.5 and 25 mg/kg induced production of interferon in the animal blood serum. The maximum interferon titer of 160-320 IU/ml was observed 18 hours after the drug administration in a dose of 12.5 mg/kg. In low doses of 5 to 12.5 mg/kg bonafton increased the nonspecific resistance of the mice to experimental viral infections when administered orally in single doses not earlier than 2 weeks prior to the contamination. The ability of the drug to stimulate the host protective forces probably plays a certain role in the mechanism of its therapeutic action in severe viral infections of man such as severe recurring ophthalmic herpes, genital herpes, Beh?et's disease, Melkersson-Rosenthal syndrome and others.     

Comparative analysis of the action of an interferon inducer in in vitro and in vivo systems]

Investigation of interferon inductor (poly IC) in vitro (chick embryon fibroblasts) and in vivo (mice) showed that the main parameters of the preparation effect, i. e. induction of interferon and development of resistance to viruses are as a whole quite comparable. The phenomenon of hyporeactivity in the both systems was reproduced on repeated use of the inductor. At the same time significant differences in the stimulating effect of DEAE-dextran were registered. The merits and demerits of the in vitro system for using in studies on antiviral drugs of the above type are discussed.     

Immunomodulating action of a preparation of yeast dsRNA on cellular immunity in mice

The effect of a dsRNA preparation (an interferon inductor) on DTH induced by SRBC was studied. It was shown that at optimal antigenic load the dsRNA preparation inhibited DTH whereas at suboptimal and supraoptimal loads the preparation stimulated it. The findings indicated that the dsRNA preparation had an immunoregulatory effect. The immunoregulatory properties of the preparation must be associated with its action on the lymphocyte suppressor cells and macrophages.     

Clinicoimmunological monitoring of therapy in patients with associated forms of yersiniosis

Characteristics of the clinical process and immunological profile in children with yersiniosis as a monoinfection or in association with acute intenstinal infections and virus hepatitis A are presented. The efficacy of the immunotropic therapy with cycloferon, an interferon inductor, and recombinant interferon in the patients with the viral and bacterial association of the disease (yersiniosis + hepatitis A) and initial disbalance of the serum cytokines was estimated. Dependence of the interferon clinicolaboratory efficacy on the initial levels of serum y-interferon, IL2 and IIA, promoting shorter terms of hyperthermia, diarrhea syndrome and cytolysis syndrome was shown. It allowed to optimize the scheme of the pathogenetic therapy of Yersinia mixed infection.     

Effect of a natural interferon inducer (RFf2) on the formation of vaccinal immunity to tick-borne encephalitis]

The effect of the interferon inductor of the natural origin (RFf2) on formation of vaccinal immunity to vernal encephalitis was studied. A single intraperitoneal administration of the preparation in a dose of 10 gamma per a mouse 2 hours after the first injection of the vaccine resulted in increased resistance of the mice to the lethal dose of the infecting virus which was introduced 14 days after the vaccination completion. The production dynamics of interferon induced by RFf2 and its level in the serum of the immunized mice were the same as those in the non-vaccinated animals. An increased number of the vaccine injections, up to 3 did not result in a significant increase in the immunity with respect to either the level of the antiviral resistance or the level of the virus-neutralizing antibody accumulation.     

Homogeneous interferon from E. coli depresses hepatic cytochrome P-450 and drug biotransformation

A highly purified homogeneous human interferon produced from cloned genes depressed the levels of hepatic cytochrome P-450 and related xenobiotic metabolism. Using another cloned human interferon and several impure preparations of human and mouse interferon, it appears that only interferons with antiviral activity in the mouse depress cytochrome P-450 in that species. This is the first direct evidence that interferon decreases hepatic drug biotransformation and likely explains the depression of drug elimination which occurs during viral infections or following the administration of interferon inducers.     

Para-aminobenzoic acid--an interferon inducer]

Para-aminobenzoic acid (PABA) was shown to be an early type interferon inductor. PABA (10 micrograms/ml) induced interferon production in vitro in the cells of human peripheral blood and in vivo in albino mice (10 mg/kg). The results of the study suggested that PABA was able to induce production of interferon-alpha/beta in various immunocyte populations. By its interferonogenic activity PABA was comparable with the known interferon inductors. One of the mechanisms of the previously described in vivo antiherpes action of PABA can be attributed to its interferon inducing activity.     

Characteristics of the factors that suppress and stimulate interferon production

A repressor of interferon production is contained in the hyaloplasm of the chick embryo fibroblast cells in the state of hyporeactivity. A factor stimulating interferon production (FSIP) was found in the hyaloplasm of the cells subjected to single or double induction with poly (pI) x poly (pC) followed by exposure to actinomycin D. Both preparations were of the protein nature and possessed high biological activity. They repressed or stimulatd interferon production at a dilution of 1:512-1:1024. The preparations had tissue and inductor specificity, did not affect the Venezuelan equine encephalomyelitis virus and interferon antiviral activity. Unlike the repressor, the FSIP proved to be thermolabile and pH-sensitive in acid media. The half lives of the repressor and stimulator were about 10 and 5 hours respectively.     

Tumor response to interferon inducer and radiation effect of serum interferon levels

The effect of interferon inducing complex of polyriboinosinic, polyribocytidylic acid with poly-L-lysine and carboxymethylcellulose - Poly (ICLC) - on the response of Lewis lung carcinoma in C57B1 mice to radiation treatments was studied. Improved tumor response was obtained in mice receiving 1.5 mg/m2 or higher of Poly (ICLC). Such doses have induced more than 1000 mu/ml of serum interferon. The same doses of Poly (ICLC) potentiated the epilation effect of radiation. The injection of 0.15 mg/m2 of Poly (ICLC) led to protection of the tumor and the stimulation of it's growth. It also did not potentiate the epilation effect. In this study, one weekly administration of Poly (ICLC) was as effective as three times per week treatment. The cellular mechanism of the increased radiosensitivity caused by Poly (ICLC) was reflected in the reduction of the width of the shoulder on the cell survival curve, which was dependent on the dose of the drug. In cell cultures, doses of 100 micrograms/ml synchronized the cell division, thus contributing to the increase in radiosensitivity.