RU2316320C1 - Anti-viral agent - Google Patents

Abstract

FIELD: pharmaceutical industry, in particular agents for viral disease treatment including HIV infection (AIDS and HIV-associated diseases).

SUBSTANCE: claimed agent in form of suppository contains per one 2 g suppository Fullevir (fullerenopolyaminocaproic acid sodium salt) 20 mg as active ingredient and ancillary substances such as propylene glycol 200 mg and balance: Vitepsol.

EFFECT: effective agent having no adverse influence on peripheral blood and body systems.

Description

The invention relates to the pharmaceutical industry and relates to the creation of agents for the treatment of viral diseases, including HIV infection (AIDS and HIV associated diseases).

The development and search for effective drugs for the prevention and treatment of viral infections is one of the urgent tasks of the modern pharmaceutical industry. If the problem of combating diseases of bacterial etiology is successfully solved by creating new antibiotics and other effective agents with protective activity, then the possibilities of combating viral diseases are limited.

The biogenic antiviral agent interferon is used to prevent influenza. The above analogues show that the creation of antiviral agents is far from an unsolved problem. Therefore, the search for new means of protection against viral infections is very relevant. Antiviral substances isolated from marine animals are known. These are polyfucose sulfates effective against HIV [EP 0495116 A1, 07.22.92)] and trisulfated triterpene glycosides from the Antarctic holothuria Staurocucumis liouvillei effective against herpes virus [J. Nat. Prod. 2001 Jun; 64 (6): 732-6].

Long-term intensive searches for antiviral substances have led to the creation of a limited set of pharmacologically active drugs. Many of them have a narrow spectrum of action and in most cases are toxic, which greatly limits their use as medicines. A few synthetic antiviral agents - midintan, methisazone - have limited preventive use, idoxyuridine, oxolin are used topically for viral skin diseases, and the toxicity of the former is very high [Pharmacology. M., Medicine, 1981, p. 399-401].

The present invention is the creation of funds with antiviral activity, especially effective in the treatment of HIV infection.

The technical result consists in creating a dosage form of fullevir for rectal administration. Rectal administration of fullevir does not affect the hematological parameters of peripheral blood, as well as the damaging effect on organs and systems of the body.

To solve this problem, an antiviral agent in the form of a suppository is proposed and contains the active substance fullevir (sodium salt of fullerenpolyaminocaproic acid) and excipients propylene glycol and Witepsol W-35 and / or H-15 in the following ratio of ingredients per 1 candle weighing 2 g :

fullvir 20 mg propylene glycol 200 mg vitepsol rest

Fullevir® is a water-soluble derivative of fullerene (75 mg / ml) - the sodium salt of fullerenaminocaproic acid with the number of attached groups of at least 2, the general formula

C ₆₀ H _n [NH (CH ₂ ) ₅ C (O) O ^- ] _n ,

where C ₆₀ is the fullerene core,

NH (CH ₂ ) ₅ C (O) O ^- - aminocaproic anion,

n is an integer of at least 2.

Molecular weight depends on the number of attached amino acids:

for the sodium salt of fullerene-tetrahydro-tetraaminocaproic acid C ₆₀ H ₄ [NH (CH ₂ ) ₅ C (O) O ^- ] ₄ 4Na ⁺ is 1333.2 g / mol.

Most chemical reactions with fullerenes are not selective, which complicates the synthesis of individual compounds. Therefore, both positional isomers and compounds differing in the number of attached amino acids are present in the product.

Production Method:

10 suppositories - rubbing 200 mg of the substance in 2 g of polyethylene glycol at 40 ° C until completely dissolved. Vitepsol (17.8 g) is melted and cooled to 35 ° C. Fullevir is added to the base with constant stirring. Candles form a mass of 2 g ± 5%.

Experimental studies have been conducted on the antiviral activity of fullevir.

The most appropriate way to evaluate the antiviral effect of HIV drugs is to model human cells infected with the virus. The criterion for the effectiveness of the drug is the absence or decrease in the amount of infectious virus in cells infected with this virus.

Used transplantable human lymphoblastoid cells MT-4. Cells were cultured at a concentration of 3.0-5.0 · 10 ⁵ cells in 1 ml of RPMI 1640 medium (produced by the Research Institute of Poliomyelitis and Viral Encephalitis RAMS) with 10% serum of cow embryos (produced by the Research Institute of Polio and Viral Encephalitis RAMS), 100 μg / ml gentamicin (Farmahim, Bulgaria). Cell viability was determined by staining with a 0.4% trypan blue solution (Sigma, USA).

Strains of HIV-1 _BRU , HIV-1 ₈₉₉ A from the collection of human immunodeficiency virus strains of the Research Institute of Virology named after D.I. Ivanovo RAMS.

Investigated the drug Fullevir® in powder form.

As an antiretroviral reference drug, the drug retrovir (azidothymidine) manufactured by GlaxoSmithKline (Belgium) was used.

Study Structure

1) Study of the cytotoxic effect of the drug

The test drug at various concentrations was added to the cells. Cells were incubated at 37 ° C in an atmosphere with 5% CO ₂ and 98% humidity for 3-6 days. Next, the viability and cell number were determined using a dye.

2) Study of the antiviral effect of the drug

a) Infection of cells * (* experimental design may be different) - human cells infect HIV.

b) The introduction of the drug - the drug was added to the cells infected with the virus. Incubation of infected cells at 37 ° C in an atmosphere with 5% CO ₂ and 98% humidity for 5-7 days. Accounting for results: light microscopy - the study of the cytopathic effect of the virus (CPP) and virus-induced syncytium formation (syncytium is a conglomerate of several cells with a common cell membrane formed as a result of the fusion of their membranes).

The degree of cytodestruction was evaluated under a microscope according to the generally accepted four-cross system with + or - signs, respectively, according to the number of dead cells in each of the four wells corresponding to one studied parameter.

++++ - 100% cell death in four wells used in the experiment on one breeding,

+++ - 75% cell death in each of the four wells,

++ - 50% cell death in each of the four wells,

+ - 25% cell death in each of the four wells,

+ - - the beginning of degeneration,

- - lack of cytodestruction.

An enzyme-linked immunosorbent assay using commercial enzyme-linked immunosorbent kits of Biorad, Organon Technika (Netherlands), determined the virus antigen in the culture fluid.

Determination of optimal conditions for the manifestation of the antiviral effect of the drug:

- the introduction of the drug before infection;

- the introduction of the drug simultaneously with the virus;

- the introduction of the drug after infection of the cells.

The data obtained showed that at a concentration of 1-100 μg / ml Fullevir® did not have a noticeable cytotoxic effect on human lymphoblastoid cells. The IC _{50 of the} drug (50% inhibitory concentration) was 400 μg / ml.

In the study of the antiviral activity of the drug against the human immunodeficiency virus, an antiviral effect was found.

The highest activity of the drug was observed at a concentration of 10 μg / ml - 95% cell protection. A dose of Fullevir® 1 μg / ml provided 80% resistance of the cells to the action of the virus. At these doses, the disappearance of virus-induced syncytia in the cells was noted.

An enzyme-linked immunosorbent assay confirmed the presence of a detected dose dependence: at a concentration of the drug of 0.5 μg / ml - a decrease in the virus antigen in the culture fluid by 25.4%, at 1 μg / ml - 63.7%, at 10 μg / ml - 95, 3% Thus, EC ₉₅ (95% effective concentration) is 10 μg / ml.

The study of the antiviral effect of Fullevir® with various schemes of its administration (prophylactic and therapeutic) showed its presence in all studied schemes.

Thus, a study of the drug Fullevir® on a model of human cell cultures revealed its pronounced antiviral activity against human immunodeficiency virus in doses of 1-10 μg / ml with a prophylactic and therapeutic administration schedule.

A study was conducted of the general toxic effect of the substance and dosage form for rectal use of the drug Fullevir®.

The acute toxicity of the substance Fullevir® has been studied in mice and rats. Indicators of LD ₅₀ when administered intravenously to mice were in the range of 198-234 mg / kg; with intravenous administration to rats, 312-383 mg / kg; when administered orally, it exceeded 8000 mg / kg for mice and 6250 mg / kg for rats; with a rectal route of administration exceeded 3125 mg / kg. The difference in LD ₅₀ is not significant depending on the sex of the animals. The death of animals occurred on the first day with clinical manifestations of a general nature: inhibition, refusal of food and water, cyanosis of visible mucous membranes. A pathological study revealed significant blood supply to the parenchymal organs, focal hemorrhages in the lungs and kidneys.

In a chronic experiment, the substance Fullevir® was tested on white outbred rats at doses of 0.3 mg / kg body weight (equitherapeutic), as well as 1.5 and 3.0 mg / kg for rectal administration. A total of 192 animals were used (96 females and 96 males). After 1, 3 and 6 months, as well as after 2 weeks of the recovery period, blood and urine samples were taken for equal groups of animals from the animals, after which they were sacrificed and pathological examination was performed. Throughout the entire period of administration of the substance Fullevir® to rats and during the recovery period, there were no signs of a change in the clinical condition of animals. The introduction of the drug did not affect the behavior, condition of the coat, visible mucous membranes of experimental animals.

Prolonged rectal administration of the substance Fullevir® to rats for 6 months did not affect the peripheral blood counts. Changes in the biochemical parameters of blood serum that occur during separate periods of the administration of the substance Fullevir® to rats do not have a dose, time or other dependence. The level of these changes is not significant, the values of the indicators do not go beyond the norm for this type of animal. As a result of the pathoanatomical study, no morphological signs of toxic effects of the substance Fullevir® on the organism of experimental rats were revealed with rectal administration for 6 months at doses of 0.3-3.0 mg / kg.

The acute toxicity of the Fullevir® dosage form was studied with a single rectal administration to rabbits and rats. The administered doses of the Fullevir® dosage form were: for rabbits - 10 mg / kg body weight (in terms of substance, the dosage form is 1 suppository weighing 2 g for a rabbit weighing 2 kg); for rats - 7.5 mg / kg body weight (by substance). When observing the animals, there were no signs of intoxication or local irritant effect after administration of the drug.

Dosage form Fullevir® was tested in a chronic experiment on rabbits with rectal administration in two doses (by substance): 0.3 mg / kg (equitherapeutic) and 3.0 mg / kg. For each dose, groups of rabbits of 24 individuals were formed. The same number of rabbits made up the control group. The total number of rabbits in the experiment is 72 heads.

Throughout the entire 6-month course of the administration of the fullevir® dosage form and during the recovery period, there were no signs of a change in the clinical condition of the animals. The body weight of the experimental and control animals at all periods of observation did not have statistically significant differences.

Rectal administration of the fullevir dosage form to rabbits does not affect the hematological parameters of peripheral blood. The pathological study did not establish morphological signs of the toxic effect of the substance Fullevir® on the organism of experimental rabbits with rectal administration for 6 months at an equi-therapeutic dose of 0.3 mg / kg (for the substance). When exposed to a dose of 3.0 mg / kg for 3 months, in one case, signs of earlier minor focal damage to the kidneys, presumably of a post-infarction nature, were established. No other pathology was found.

Thus, by laboratory studies of the substance of the new drug Fullevir® and its dosage form for rectal administration, the absence of a pronounced damaging effect on the organs and systems of the body in the dose range from equitherapeutic - 0.3 mg / kg body weight - up to ten times its . The drug may be recommended for clinical trials.

Fullevir® in laboratory studies on animals does not have allergenic activity. There were no cases of occurrence of immediate or delayed hypersensitivity to the drug with different methods of administration to the body in doses up to 300 mg / kg. In these doses, the immunotoxic effect of the drug has not been established. Fullevir® does not have mutagenic activity against mammalian cells in doses up to 15 mg / kg.

The pharmacokinetics and distribution of the drug Fullevir® in organs was studied after a single injection in doses of 0.25 and 2.5 mg / kg in two animal species: mice and rabbits.

The area under the kinetic curve "concentration (C) - time (t)" (AUC) was: for a dose of 0.25 mg / kg - 490 ng × h / ml, for 2.5 mg / kg - 4881 ng × h / ml .

The linearity of the change in the concentration (C) of the drug Fullevir® in plasma from the dose in the studied dose range (0.25 and 2.5 mg / kg) was established.

Analysis of tissue accessibility data (f _T ), which characterizes the intensity of drug penetration into peripheral tissues, shows that Fullevir® penetrates all organs. Fullevir® penetrates most intensely into the liver, spleen, kidneys, lungs, thymus and heart. Inaccessible to the drug brain and omentum.

The average retention time (MRT _t ) for all organs exceeds the average retention time in plasma.

Fullevir® in therapeutic doses binds well to plasma proteins. Fullevir® is eliminated unchanged. A study of the excretion of the drug Fullevir® showed that in three days 52-54% of the administered dose is excreted in the urine.

Thus, experimental studies of the drug fullevir have shown that it is effective against HIV infection.

The use of the drug fullevir in the treatment of patients with HIV infection leads to a decrease in the viral load in lymphocytes and blood serum, an increase in the number of CD-4 cells, an increase in the duration of remission, the prevention of opportunistic infections arising from immunodeficiency, and can be indicated for use in AIDS and HIV associated diseases.

Before starting therapy, it is necessary to conduct a complete clinical and laboratory examination of the patient with the obligatory determination of the level of viral load on the plasma and the number of T-4 lymphocytes. Before treatment and during treatment, it is also necessary to control the amount of virus in lymphocytes isolated from the blood of patients.

It is also necessary to regularly monitor renal function, determine the content of blood cells, and carry out a neurological examination.

It is not recommended during treatment with the claimed agent to combine with neuro- and neurotoxic drugs.

The course of treatment is selected individually and depends on the severity of the disease, as well as on the patient's response to treatment. An effective combination with a single infusion of the drug fullevir.

Recommended course of treatment:

- intensive care: 1 suppository rectally daily for 3 months. Repeated courses of treatment after consulting a doctor are possible;

- maintenance therapy: 1 suppository rectally 1 time in three days.

Contraindication for treatment is severe renal failure.

The dose of the drug fullevir with creatinine clearance of 50% of the norm should be reduced by half. The drug fullevir is contraindicated in individuals if the clearance is 30% of the norm.

Contraindication may be benign prostatic hyperplasia, causing urination disorders.

Hypoalbuminemia can lead to an increase in the pharmacological effect.

The drug is not prescribed for patients with hypersensitivity to the components of the drug.

Claims (1)

An antiviral agent, characterized in that it is in the form of a suppository and contains the active substance fullevir (sodium salt of fullerenpolyaminocaproic acid) and excipients - propylene glycol and Witepsol W-35 and / or H-15 in the following ratio of ingredients per 1 candle weighing 2 g: fullvir 20 mg propylene glycol 200 mg vitepsol rest