Anti-viral agent

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.

 

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

Development and the search for effective drugs for the prevention and treatment of viral infections is one of the topical problems of modern pharmaceutical industry. If the problem of combating diseases of bacterial etiology is successfully solved by the creation of new antibiotics and other effective means, having a protective activity, anti-viral diseases is limited.

Biogenic antiviral agent interferon is used for the prevention of influenza. Given the analogues suggest that the creation of antiviral drugs is not solved task. Therefore, the search for new means of protection against viral infections is very important. Known antiviral substances isolated from marine animals. This polityczne sulfates, effective against HIV [EP 0495116 A1, 22.07.92)], and triculation triterpene glycosides from the Antarctic sea cucumbers Staurocucumis liouvillei, effective against herpes virus [J. Nat. Prod. 2001 Jun; 64 (6): 732-6].

Long and intensive search for anti-viral compounds led to the creation of a limited set of pharmacologically active drugs. M the OIG of them have a narrow spectrum of activity and in most cases, toxic, that significantly limits their use as medicines. Small synthetic antiviral agents - Medinan, methisazone - find limited prophylactic use, idoxuridine, oksolina are applied topically in viral diseases of the skin, and the toxicity of the first very high [Pharmacology. M, Medicine, 1981, s-401].

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

The technical result is to create a dosage form of pulsewire for rectal administration. Rectal administration of pulsewire has no effect on hematological parameters of peripheral blood, as well as damaging effects on the organs and systems of the body.

To solve the problem proposed antiviral agent, executed in the form of a suppository, and contains the active substance forever (sodium salt fulleropyrrolidines acid) and an excipient is propylene glycol and witepsol W-35 and/or N-15 in the following ratio of ingredients in 1 candle by weight of 2 g:

forever20 mg
propylene glycol200 mg
witepsolthe rest is th

Forever® is a water-soluble derivative of fullerene (75 mg/ml) sodium salt filerenameoperations acid with the number of connected groups of at least 2, the General formula

C60Hn[NH(CH2)5C(O)O-]n,

where C60- fullerene core,

NH(CH2)5C(O)O-- aminocapronic anion,

n is an integer not less than 2.

The molecular weight depends on the number of attached amino acids:

for the sodium salt fullerene-tetrahydro-tetraaminobiphenyl acid60H4[NH(CH2)5C(O)O-]44Na+equal 1333,2 g/mol.

Most chemical reactions with the fullerenes are not selective, which complicates the synthesis of individual compounds. Therefore, in the product are present as position isomers, and compounds that differ in the number of attached amino acids.

Method of delivery:

10 suppository - pound 200 mg of the substance in 2 g poliatilenglikola at 40°until dissolved. Witepsol (17.8 g) melted and cooled to 35°C. the basis with constant stirring bring in forever. Candles form a mass of 2 g ± 5%.

Experimental studies were performed on the antiviral activity of pulsewire.

The most adequate way about Enki antiviral effect of drugs against HIV is a model of human cells, infected with a virus. The criterion of effectiveness of the drug is the absence or reduction in the number of infectious virus in cells infected by the virus.

Used human lymphoblastoid cells of a human MT-4. Cells were cultured at a concentration of 3.0 to 5.0·105cells in 1 ml of medium RPMI 1640 (manufactured by research Institute of poliomyelitis and viral encephalitides RAMS) with 10% serum of cow embryos (produced by the research Institute of poliomyelitis and viral encephalitides RAMS), 100 µg/ml gentamicin (Pharmachim, Bulgaria). Cell viability was determined by the color of 0.4% solution Trypanosoma blue (Sigma, USA).

As a source of virus used strains of HIV-1BRUHIV-1899A collection of strains of human immunodeficiency Institute of Virology. Dijanoveckog RAMS.

Researched drug Forever® in powder form.

As antiretroviral reference drug used drug Retrovir (azidothymidine) manufactured by GlaxoSmithKline (Belgium).

The research structure

1) study of the cytotoxic drug action

To the cells was added to the study drug in different concentrations. Incubated cells with S° in an atmosphere with 5% CO2and 98% humidity 3-6 days. Next, we determined the viability and number of cells using a dye.

2) Study protivovirusnogo drug action

a) Infection of cells* (* the scheme of the experiment may be different) - human cells infect HIV.

b) adding the agent to the infected cells were added to the drug. Incubation of infected cells with S° in an atmosphere with 5% CO2and 98% humidity 5-7 days. Results: light microscopy study of the cytopathic effect of the virus (JRC) and virusinduced syncytium formation (syncytium - a conglomerate of several cells from the total cell membrane, formed by the merger of their membranes).

The degree of cytodestructive was evaluated under a microscope by the common chetyrehrazovoe the system by the signs + or - respectively the number of dead cells in each of the four holes corresponding to one of the investigated parameter.

++++ - 100%cell death in four holes used in the experience of one breeding

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

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

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

+- - beginning degeneration

- - no cytodestructive.

Enzyme-linked immunosorbent assay using commercial immunoassay kits firms BioRad, Organon Technika (the Netherlands), were used to define the virus antigen in the culture fluid.

Determination of optimal conditions is royalene antiviral drug action:

- introduction of the drug to the infection;

- introduction of the drug simultaneously with the virus;

- introduction of the drug after infection of cells.

The data obtained showed that at a concentration of 1-100 μg/ml Pullover® had no significant cytotoxic activity in lymphoblastoid cells. IR50drug (50%inhibitory concentration) was 400 µg/ml

In the study of antiviral activity of the drug against human immunodeficiency virus detected antiviral effect.

The greatest activity of the drug is observed at a concentration of 10 μg/ml - 95%protection of the cells. Dose of Pulsewire® 1 µg/ml accounted for 80%of the resistance of cells to the action of the virus. At these doses indicated the disappearance in the cells of virus-induced entities.

Data immunoassay analysis confirmed the presence of the detected dose dependence: at concentrations of 0.5 µg/ml reduction of virus antigen in the culture fluid of 25.4%at 1 μg/ml of 63.7%, with 10 µg/ml 95.3 per cent. Thus, EC95(95%effective concentration) is 10 µg/ml.

Study the antiviral effect of Pulsewire® under various schemes of its introduction (preventive and curative) showed its presence in all the studied schemes.

Thus, the study of drug Forever® on models cultures of human cells detected its pronounced antiviral activity against human immunodeficiency virus in doses of 1-10 µg/ml for prophylactic and therapeutic scheme introduction.

A study was conducted General toxic effect of the substance and dosage form for rectal drug Forever®.

Acute toxicity of the substance of Forever® was studied in mice and rats. Indicators LD50when administered intravenously to mice was within 198-234 mg/kg; intravenously to rats - 312-383 mg/kg; oral introduction exceeded for mice, 8000 mg/kg, for rats - 6250 mg/kg by rectal way of introducing exceeded 3125 mg/kg Difference in index LD50depending on the sex of animals is insignificant. The death of animals occurred in the first day of the clinical manifestations of a General nature: oppression, denial of food and water, visible cyanosis of the mucous membranes. At postmortem examination showed a substantial volume of parenchymatous organs, focal hemorrhage in the lungs and kidneys.

In the chronic experiment, the substance of Forever® experienced on white outbred rats at doses of 0.3 mg/kg of body weight (equitisations), 1.5 and 3.0 mg/kg rectally administered. Just used 192 animals (96 females and 96 males). the via 1, 3 and 6 months, and 2 weeks recovery period equal to the number of groups of animals were selected for the study, samples of blood and urine, and then cut and conducted post-mortem examination. Throughout the period of introduction of the substance of Forever® rats and during the recovery period was not marked by signs of changes in the clinical condition of the animals. The drug had no effect on the behavior, condition of coat, visible mucous membranes in experimental animals.

Long, within 6 months, rectal administration of the substance Forever® rats had no effect on the peripheral blood. Changes of biochemical parameters of blood serum, resulting in some periods of rats substance of Forever®not have dose, time or other dependence. The level of these changes is not significant, values not beyond the norm for this species. In the postmortem studies have not established a morphological signs of toxic effects of the substance Forever® on the body of Wistar rats in a rectal for 6 months at doses of 0.3-3.0 mg/kg

Acute toxicity dosage forms of Forever® studied at single anal vodenitsata and rats. The administered dose of the dosage form, Forever® was as follows: rabbit 10 mg/kg of body weight (in substance that the dosage form is 1 suppository mass of 2 g for rabbit weighing 2 kg); for rats - 7.5 mg/kg of body weight (substance). When observing animals was not observed any signs of intoxication or local irritants after drug administration.

Medicinal form of Follower® experienced in chronic experiment on rabbits by rectal injection in two doses (in substance): 0.3 mg/kg (equitisations) and 3.0 mg/kg For each dose were groups of rabbits for 24 animals each. The same number of rabbits formed the control group. The total number of rabbits in the experiment 72 of the head.

Throughout the 6-month rate of introduction of the dosage form, Forever® and during the recovery period was not marked by signs of changes in the clinical condition of the animals. The body weight of experimental and control animals during all periods of observation were not statistically significant differences.

Rectal administration of the dosage form, Forever® rabbits have no effect on hematological parameters of peripheral blood. Conducted postmortem examination is not installed morphological traits toxic the impact of substance, Forever® on the body of Guinea pigs with rectal for 6 months in equitisations dose of 0.3 mg/kg (on substance). When exposed to a dose of 3.0 mg/kg for 3 months in one case were installed signs of previous small damage to the kidneys, presumably postinfarction nature. Any other pathology was not found.

Thus, conducted on laboratory animal studies the substance of a new drug, Forever® and its dosage forms for rectal installed the absence of significant damaging effect on the organs and systems of the body in the range of doses from equitisations - 0.3 mg/kg of body weight up to ten times its excess. The drug can be recommended for clinical trials.

Forever® in laboratory studies on animals has no allergenic activity. Not detected occurrence of hypersensitivity immediate or delayed-type to the drug with the different methods of ingestion doses up to 300 mg/kg In these doses is not installed immunotoxic effect of the drug. Forever® does not possess mutagenic activity against mammalian cells in doses up to 15 mg/kg

Pharmacokinetics and distribution of the drug, Forever® the body is m were studied after a single injection at doses of 0.25 and 2.5 mg/kg on two kinds of animals: mice and rabbits.

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

Installed the linearity of the change in concentration (C) of drug Forever® plasma from doses in the studied range of doses (0.25 and 2.5 mg/kg).

Analysis of data on tissue availability (fT), characterizing the penetration of the drug in peripheral tissues, indicates that Forever® penetrates all bodies. The most intense of Forever® enters the liver, spleen, kidneys, lungs, thymus and heart. Inaccessible to drug the brain and seal.

The mean retention time (MRTtfor all organs exceeds the average retention time in plasma.

Forever® in therapeutic doses well associated with plasma proteins. Forever® eliminated in unchanged form. The study of the excretion of the drug Forever® showed that for three days with urine output 52-54% of the administered dose.

Thus experimental studies of drug follower showed that it is effective against HIV infection.

The use of the drug, forever in the treatment of patients with HIV infection leads to a decrease in viral load in lymphocytes and serum, the increase in the number of CD-4 cells, the increase in the duration of remission, to prevent opportunist who ical infections, arising on the background of immunodeficiency, and can be shown to apply when AIDS and HIV-related diseases.

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

You must also regularly monitor kidney function, to define the content of the formed elements of the blood to carry out a neurological examination.

It is not recommended during treatment of the stated means be combined with neuro - and neurotoxic drugs.

Treatment steals up individually and depends on the severity of the disease, as well as patient response to treatment. Effective combination of one-time infusion of drug forever.

The recommended course of treatment:

intensive therapy: 1 suppository rectally every day for 3 months. There are repeated treatments after doctor's consultation;

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

Contraindication to treatment is severe renal failure.

Dose of forever when creatinine clearance is 50% from the norm must be the mind is ishana twice. Preparation of forever contraindicated in patients if the clearance is 30% of normal.

Contraindication may be benign hyperplasia of the prostate gland that causes violations of mocheispuskanija.

Hypoalbuminemia may cause increased pharmacological effect.

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

Antiviral agent, characterized in that it is made in the form of a suppository and contains the active substance forever (sodium salt fulleropyrrolidines acid) and an excipient is propylene glycol and witepsol W-35 and/or N-15 in the following ratio of ingredients in 1 candle by weight of 2 g:

forever20 mg
propylene glycol200 mg
witepsolrest



 

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