RU2316321C1 - Anti-viral agent for systemic application - 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 solution for intravenous administering contains per 1 ampoule 3 % concentrate of Fullevir (fullerenopolyaminocaproic acid sodium salt)1 g and water for injection up to 1 ml. Additionally in contains 0.9 % solution of sodium chloride or 10 % solution of human albumen in amount of 100 ml per 1 ampoule.

EFFECT: decreased viral load in lymphocytes and blood serum; increased amount of CD-4 cells.

2 cl

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).

Over the past decade, dozens of antiviral drugs have appeared that have markedly increased the effectiveness of HIV treatment. However, the activity of most drugs shortly after the start of therapy begins to decline due to the emergence of HIV-resistant strains of drugs. This phenomenon depends on many factors, including the duration of treatment.

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, in the form of a concentrate for the preparation of a solution for infusion.

To solve this problem, an antiviral agent for systemic use is proposed, made in the form of a solution for intravenous administration and containing 3% fullevir concentrate (fullerenpolyaminocaproic acid sodium salt) and water for injection in one ampoule with the following ingredients per 1 ampoule of the product:

fullvir 30 mg water for injections up to 1 ml

however, when administered intravenously to a patient, the agent additionally contains 0.9% sodium chloride solution or 10% human albumin solution at the rate of 100 ml per 1 ampoule.

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, of 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.

The method of obtaining the claimed dosage form is carried out according to the generally accepted method for preparing ampouled preparations.

The resulting solution is reddish brown and opaque.

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 _899A from the collection of human immunodeficiency virus strains of the 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 (the design of the experiment may be different) - human cells infect HIV.

b) The introduction of the drug - the drug was added to the infected virus cells. 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 from 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 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.

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 52-54% of the administered dose is excreted in the urine in three days.

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, and the prevention of opportunistic infections associated with 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.

Fullevir 3% concentrate for the preparation of infusions is intended only for intravenous infusions (directly into a vein) and can be used only in diluted form.

For intravenous administration, a drug solution prepared ex tempore from the concentrate is used by adding 100 ml of 0.9% sodium chloride solution or 100 ml of 10% human albumin solution to each ampoule containing 1 ml of 3% fullevir concentrate.

The drug is administered intravenously, drip at a rate of 3 ml (50-60 drops) per minute.

Fullevir in an acidic environment (pH below 5.6) goes into an insoluble form, so it is necessary to control the pH of the solutions used to dilute the drug.

Fullevir is capable of complexation with iron and its salts. Ready-to-use solution is incubated for 20 minutes at room temperature. Use only clear solutions.

The infusion solution is prepared anew every time before use

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.

Albumin should not be used for thrombosis, internal bleeding, hypersensitivity to blood protein preparations.

Claims (2)

1. An antiviral agent for systemic use, characterized in that it is in the form of a solution for intravenous administration and contains in one ampoule a 3% concentrate of fullevir (sodium salt of fullerenpolyaminocaproic acid) and water for injection with the following ingredients per 1 ampoule of the agent: fullvir 30 mg water for injections up to 1 ml 2. The antiviral agent according to claim 1, which, when administered intravenously to the patient, further comprises a 0.9% solution of sodium chloride or a 10% solution of human albumin at the rate of 100 ml per 1 ampoule.