RU2331420C2 - Antiviral agent (versions) and treatment method based on it - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and viral infection treatment, particularly combined antiviral agent with anti-HIV action, namely the combined antiviral agent, comprising the composition of (2R, cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolane-5-yl)-1H-pyrimidine-2-on (lamivudine) and 5'-H-phosphonate-3'-azido-3'-desoxythymidine sodium salt (phosphazide) in 250:1 to 1:250 weight ratio, and the treatment for HIV-infection method.

EFFECT: producing of combined antiviral agent with anti-HIV action, providing the increase in treatment effectiveness along with significant reduction of toxic manifestations.

12 cl, 2 tbl, 7 ex

Description

The invention is intended for the treatment of viral infections and relates to combinations of antiviral agents having anti-HIV activity, namely, a combination of lamivudine (3TC) with phosphazide (5'-H-phosphonate-3'-azido-3'-deoxythymidine, sodium salt; Nikavir ^® ).

HIV infection is a long-term infectious disease that develops as a result of infection with the human immunodeficiency virus (HIV). With HIV infection, damage to the immune system progresses, leading to a condition known as Acquired Immune Deficiency Syndrome (AIDS), in which the patient develops “opportunistic diseases”: severe infections caused by opportunistic pathogens, and some oncological diseases .

An infected person remains a source of infection for life. Untreated HIV infection progresses within 3-20 years and ends with the death of the infected person.

A variety of clinical manifestations of HIV infection and opportunistic diseases arising against it, a long course of the disease made the development of drugs for the treatment of HIV-infected and AIDS patients relevant. Currently, the following groups of drugs are known: nucleoside, nucleotide and non-nucleoside inhibitors of viral reverse transcriptase; protease inhibitors; fusion inhibitors. A number of compounds are being developed that are active against other HIV sites.

The most widely used are nucleoside inhibitors of viral reverse transcriptase. Among them - zidovudine (AZT), lamivudine (3TC), didanosine (DDI), etc. Moreover, the first of the developed drugs is still the most widely used - zidovudine.

The need for a long, almost life-long use of drugs leads to toxic manifestations and the development of drug resistance of the virus (resistance). According to some reports, resistance to AZT develops after 6 infectious cycles, to 3TC - through 4 infectious cycles. Known drug phosphazide (5'-H-phosphonate-3'-azido-3'-deoxythymidine, sodium salt, Nikavir ^® ) [Kravchenko A.V. The use of Nikavir as part of antiretroviral therapy // Medical Department, 2004. - No. 2. - S.90-92, 166-172], the use of which is characterized by better tolerance and a lower rate of occurrence of resistance to it compared to zidovudine [Machardo J., Salomon H., Oliveiro M., et al. Antiviral activity and resistance profile of phosphazide, a novel prodrug of AZT. Nucleosides &Nucleotides;1999; 18: 901-906]. The noted benefits can be explained by phosphazide metabolism data [Y. Skoblov et al. / Antiviral Research 63 (2004) 107-113].

The use of combinations of antiviral drugs can more effectively influence HIV due to the emerging synergistic effect. So the authors of the patent [Patent RU No. 2139059] showed that in combination with the second component, lamivudine (3TC) either enhances the action of the second component (synergistic effect), or reduces the cytotoxic effect of the second component, or has both of these properties.

The disadvantages of the known combination of lamivudine with zidovudine include:

- toxicity, manifested mainly in the development of anemia, neutropenia and leukopenia;

- mutagenic effect, manifested in the ability to induce gene mutations in indicator microorganisms in the metabolic activation system in vitro;

- insufficient treatment effectiveness.

The objective of the invention is to develop a combination agent with activity against HIV infection, devoid of these disadvantages.

The authors of this application found that the replacement of zidovudine with phosphazide in a known combination with lamivudine leads to a significant reduction in toxic effects characteristic of patients receiving zidovudine, the absence of a mutagenic effect when taking into account gene mutations in microorganisms and a significant increase in the effectiveness of treatment. Due to the fact that phosphazide in the body of a mammal partially generates zidovudine [Y. Skoblov et al. / Antiviral Research 63 (2004) 107-113], its use in a known combination instead of zidovudine should not lead to these advantages. Therefore, the results can be classified as unexpected, which was impossible to predict in advance.

The agent according to the invention may be a pharmaceutical composition containing, as an active principle, a combination of nucleoside inhibitors of the viral reverse transcriptase of lamivudine (a) and phosphazide (b) or a kit where components (a) and (b) are each provided in a form (which can be the same or different) suitable for administration in combination with each other.

Made according to any of the options, the tool contains a combination of lamivudine and phosphazide in the ratio of active components from 1: 250 to 250: 1, preferably 1: 5-50: 1, and more preferably about 1: 10-10: 1.

The antiviral combination of the present invention can be used in a method of treating HIV infection. In this case, both compounds can be introduced either simultaneously, in a mixture, either separately or sequentially. As in the prototype, with sequential administration, the time interval between the administration of inhibitors should not be too large. It can be from a few minutes to 3 hours. Simultaneous administration is preferred. The amount of lamivudine and phosphazide required for treatment may vary depending on the route of administration, the course of HIV infection, the age and condition of the patient, and can be determined in each case by the attending physician. The doses used are in the range of 2 to 750 mg per kg of body weight per day, preferably in the range of 6-90 mg / kg / day, and most preferably 15-60 mg / kg / day of each inhibitor.

Lamivudine and phosphazide can be used for therapeutic purposes in the form of substances: (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxacisolan-5-yl) - (1H) -pyrimidin-2-one and 5'-H-phosphonate-3'-azido-3'-deoxythymidine, sodium salt. However, it is preferable to administer said inhibitors in the form of a composition with pharmaceutically acceptable carriers.

As acceptable carriers, various additives can be used that are used in the manufacture of finished dosage forms (tablets, capsules, suppositories, infusion solutions, etc.) that are compatible with other ingredients of the drug and do not adversely affect the patient.

Due to the properties of the molecules of lamivudine and phosphazide, the peculiarities of the mechanism of their effect on HIV inside the cells, preparations for oral, parenteral and rectal administration are of the greatest interest. Also, the preparations can be made by any of the methods known in the pharmaceutical industry, which conceptually include a number of steps: mixing and granulating the inhibitors with solid or liquid carriers; product formation in the form of discrete unit dosage forms.

For oral administration, tablets, capsules, oral solutions and other standard dosage forms known in the pharmaceutical industry can be used. Tablets and capsules may contain standard excipients, necessary both for the technology of their manufacture, and to achieve the necessary qualitative and quantitative indicators that meet the requirements of the corresponding pharmacopeia. Examples of fillers may be lactose, mannitol, starches, modified starches, cellulose and its derivatives, gelatin, stearic acid or its salts, moisturizers, perfumes, dyes.

Oral liquid preparations can be made either in the form of solutions (aqueous or oily suspensions, solutions, emulsions, syrups, etc.), or in the form of dry products that must be diluted with water or other solvents before direct use.

Preparations for parenteral administration (for example, for infusion) can be made in the form of standard vials, ampoules, filled syringes, etc. Also, drugs must be manufactured under sterile conditions and may contain various preservatives, buffering agents, stabilizers, isotonic agents. Lyophilized forms must be mixed with a pyrogen-free sterile solvent before use.

Formulations for rectal administration may be formulated as suppositories in unitary forms. Solid forms are also made, usually using a suitable carrier, by mixing the inhibitor with a solid or molten carrier, followed by molding from the melt upon cooling.

In addition to the aforementioned, pharmaceutical compositions can be prepared using modified compounds leading to a prolonged release of inhibitors within the body.

The present invention is illustrated, but not limited to, the following examples.

Example 1

Capsules of the following composition:

Phosphazide 300 mg

Lamivudine 150 mg

Lactose 240 mg

Magnesium Stearate 20 mg

Example 2

A set in the form of a blister pack containing two rows of capsules of 10 pieces in different colors.

1 row - yellow capsules of the following composition:

Phosphazide 300 mg

Calcium Carbonate 170 mg

Microcrystalline cellulose 55 mg

Aerosil 20 mg

Magnesium Stearate 15 mg

2 row - white capsules of the following composition:

Lamivudine 150 mg

Lactose 85 mg

Magnesium Stearate 5 mg

Example 3

A set in the form of a blister pack containing two rows of tablets of 10 pieces in different colors.

1 row - yellow tablets of the following composition:

phosphazide 100 mg

calcium carbonate 55 mg

microcrystalline cellulose 18 mg

aerosil 6 mg

magnesium stearate 6 mg

quinoline yellow E104

2 row - white tablets of the following composition:

lamivudine 200 mg

lactose 21 mg

hydroxypropyl methylcellulose 45 mg

povidone 12 mg

magnesium stearate 2 mg

Example 4

Injection

Lamivudine 200 mg

Phosphazide 20 mg

Benzyl alcohol 100 mg

Glucofurol 75 1000 mg

Water for injection up to 3 ml

Example 5

Candles of the following composition:

Lamivudine 2 mg

Phosphazide 500 mg

Vitepsol 1498 mg

Example 6

In vitro trials of a combination of lamivudine with zidovudine and lamivudine with phosphazide to evaluate mutagenic activity.

A method was used to take into account the ability of a compound to induce gene mutations in indicator microorganisms in an in vitro metabolic activation system.

Histidine auxotrophic strains of Salmonella typhimurium TA 98, TA 100 and TA1537 were used as indicator microorganisms.

Aqueous solutions of substances (lamivudine + zidovudine; lamivudine + phosphazide) in concentrations of each from 0.2 to 2000 μg / cup were sterilized using membrane filters with a pore diameter of 0.2 microns from GELMAN. Selective semi-enriched agar in test tubes in a volume of 2 ml was melted in a water bath at a temperature of 100 ° C, cooled in a thermostatic water bath to a temperature of 44-45 ° C. 100 μl of a solution of substances, 100 μl of a suspension of bacteria overnight culture, 100 μl of rat liver fraction 59 and cofactors were added to agar tubes. The microsomal fraction of the rat liver induced by Aroclor was obtained as a lyophilisate from Labsystems (Finland). We conducted an experiment with both a complete microsomal activating mixture of PAMS and an incomplete activating microsomal mixture of NAMS. These mixtures were added after extraction of the tubes from a thermostatic bath and semi-liquid agar was immediately applied to a layer of minimal agar on Petri dishes. After complete solidification of the agar, the plates were transferred to a thermostat at 37 ° C. For each dose of the drug, 3 repetitions with PAMS and 3 repetitions with NAMS were done. The account of prototrophic revertants was performed after 48 hours of incubation at 37 ° C. For each option, the geometric mean number of revertants was calculated

Xi = ³ N Xi ₁ Xxi ₂ Xxi ₃

where Xi is the number of revertants in Petri dishes 1, 2, 3 at a dose i.

For each experimental variant, the excess of the geometric mean number of revertants in the experiment over the control was found and compared with the critical value (1.9 for strain TA 1537 and 1.5 for strain TA 98 and TA 100).

The mutagenic effect of solutions with zidovudine was detected for strains TA 98 and TA 1537 both in the presence of a microsomal mixture and without it at doses of 2 μg per cup or higher.

Solutions of lamivudine with phosphazide do not have a mutagenic effect in the Ames test.

Example 7

The FNMC AIDS analyzed the effectiveness and safety of antiretroviral therapy, including including a combination of phosphazide - 0.6 g per day and lamivudine - 0.3 g (in the ratio of examples 1, 2) per day in 23 women with HIV infection . In any case, there were no adverse events associated with the use of phosphazide or lamivudine and requiring correction of therapy.

An analysis of the dynamics of laboratory markers of the effectiveness of antiretroviral therapy (the amount of HIV RNA and CB4 lymphocytes in the blood) yielded the following results:

- The median HIV RNA level (viral load) before treatment was 15,500 copies per ml (4.19 log10), the maximum value in the groups was 136,000 copies per ml (5.13 log10), the minimum was 1510 copies per ml (3.10 log10). After 12 weeks of treatment, the median is below the detection level (<400 copies per ml - 2.6 log10), maximum - 28200 copies / ml (4.45 log10).

- A significant decrease in the level of “viral load” (by more than 0.7 log10) by the 12th week of treatment was noted in 13 of 13 cases (100%) in the first group and in 9 cases out of 10 in the second (90%).

- The number of patients with a relatively high level of “viral load” (> 30,000 copies per ml) before treatment was 8 out of 23 (35%), and in 2 (9%) patients the viral load was very high (more than 100,000 copies per ml). After 12 weeks of treatment, the viral load in no case exceeded 30,000 copies per ml.

- Viral load above 1000 copies per ml (indication for delivery of HIV-infected pregnant by planned cesarean section) before treatment was observed in all patients, after 12 weeks of treatment - in 4 (17%).

- Viral load below the level of determination (400 copies per ml), not encountered before treatment in any case, after 12 weeks of treatment was observed in 16 (70%) patients.

- The median level of CD4 lymphocytes before treatment was 0.35 billion / L, the maximum value in the group was 0.8 billion / L, the minimum was 0.19 billion / L. After 12 weeks of treatment, the median is 0.4, the maximum is 0.87, and the minimum is 0.15.

- An increase in the level of CD4 lymphocytes by the 12th week of treatment took place in 16 (70%) patients, a decrease in 7 (30%).

- The number of patients with a relative level of CD4 lymphocytes of less than 0.35 billion / l before treatment is 11 people (48%), after 12 weeks of treatment - 7 people (30%).

Thus, in both groups of patients, by the 12th week of treatment, a statistically significant decrease in the “viral load” was observed. In 22 of 23 patients, therapy according to the criterion of reducing the "viral load" was effective.

The anti-HIV-1 activity of the combination was studied on a MT-4 cell culture highly sensitive to human immunodeficiency virus. MT-4 cells were infected with a virus stock (strain HIV-1 _EVK ) with a multiplicity of infection of 0.2-0.5 infectious units per cell and incubated at 37 ° for 1 hour (virus adsorption). The suspension of infected cells was diluted with RPMI-1640 growth medium containing 10% ICN cow serum (USA), 0.06% L-glutamine, 100 μg / ml gentamicin and 60 μg / ml lincomycin, to a seed concentration of 0, 5 × 10 ⁶ cells / ml and introduced into the wells of a 96-well culture plate. Experimental (with the drug) and control (without the drug) infected cells and control uninfected cells (without the drug) were cultured at 37 ° C and 5% CO ₂ for 4 days. At the end of the incubation, the fraction of viable cells in the Goryaev’s chamber is calculated after staining with a 0.4% trypan blue solution and the accumulation level of the virus-specific p24 protein is checked by the enzyme immunoassay, as described in [Fedyuk N.V., Konovalov E.E., Loktev V. B., Uryvaev L.V., Kulyandin S.A., Pokrovsky A.G. Identification and quantification of human immunodeficiency virus antigens of types 1 and 2. Vopr. virusol. (1992). No. 3: 135-138].

The experimental data are given in the tables.

Table 1
Anti-HIV-1 activity of a combination of nikavir and lamivudine against HIV-1 _EVK strain in MT-4 cell culture A drug Concentration Viability Content Inhibition drug cells,% p24, ng / ml p24,% mcg / ml nikavir 5 92.26 ± 0.96 138.52 ± 9.2518 91.39 ± 0.58 - one 91.21 ± 0.27 136.30 ± 3.39 91.52 ± 0.21 - 0.5 94.76 ± 2.00 133.33 ± 2.22 91.71 ± 0.14 - 0.1 94.36 ± 1.54 204.14 ± 18.66 87.31 ± 1.16 - 0.05 87.35 ± 2.28 508.24 ± 103.38 68.40 ± 6.43 - 0.01 42.9 ± 6.33 1428.80 ± 439.63 11.16 ± 27.34 - 0.005 41.56 ± 7.21 1614.28 ± 97.47 -0.38 ± 6.06 nikavir 1 + 2 70.89 ± 9.83 79.26 ± 14.80 95.07 ± 0.92 lamivudine - 0.5 + 1 87.60 ± 7.64 101.48 ± 5.13 93.69 ± 0.32 - 0.05 + 0.1 95.87 ± 4.10 312.01 ± 26.71 80.60 ± 1.66 - 0.01 + 0.02 57.58 ± 8.84 906.17 ± 191.68 43.65 ± 11.92 - 0.005 + 0.01 33.66 ± 5.97 981.93 ± 110.95 38.94 ± 6.90 lamivudine 2 86.16 ± 4.28 163.07 ± 1.06 89.86 ± 0.07 - one 79.92 ± 12.45 171.52 ± 13.99 89.33 ± 0.87 - 0.2 89.35 ± 1.72 538.91 ± 56.60 66.49 ± 3.52 - 0.1 32.82 ± 4.44 845.36 ± 38.92 47.43 ± 2.42 - 0.02 31.00 ± 6.28 992.90 ± 193.84 38.26 ± 2.42 - 0.01 37.28 ± 1.07 911.15 ± 53.86 43.34 ± 3.35 - 0.002 46.47 ± 3.96 936.07 ± 0.00 41.79 ± 0.00 - 0.001 19.36 ± 6.38 1161.37 ± 68.87 27.78 ± 4.28 MT-4 - 95.62 ± 1.96 - - MT-4 (HIV1) - 24.45 ± 5.03 1608.21 ± 221.91 -

table 2
Quantitative Characteristics of HIV-1 _EVK Inhibition by Nicavir and Lamivudine Combination A drug ID ₅₀ μm Fic individual in a mixture 0.715 nikavir 0.11 0.056 lamivudine 0,097 0,021

Thus, as a result of studying the anti-HIV activity of nucleoside inhibitors of phosphazide (nikavir), lamivudine (3TC) and their combinations in relation to the HIV-1 strain of HCV 4005 on MT-4 cell culture, it was found that combinations of nikavir and lamivudine exhibit synergism with respect to inhibition reproductions of HIV-1.

When the virus was passaged under the described conditions, it was found that the drop in the effective concentration of phosphazide in the presence of lamivudine occurs 60-70 days later compared to using phosphazide alone. Concentrations of phosphazide in comparative experiments were identical.

Claims (12)

1. Combined antiviral agent with activity against HIV infection, characterized in that it is a composition containing (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl ) -1H-pyrimidin-2-one (lamivudine), and 5'-H-phosphonate-3'-azido-3'-deoxythymidine, sodium salt (phosphazide) in a ratio of from 250: 1 to 1: 250 by weight. 2. The tool according to claim 1, characterized in that the ratio of lamivudine to phosphazide is from 1:10 to 10: 1. 3. The tool according to claim 1 or 2, characterized in that the composition further comprises a pharmaceutically acceptable carrier. 4. The tool according to claim 3, characterized in that it is intended for oral administration. 5. The tool according to claim 4, characterized in that it is made in the form of a tablet. 6. The tool according to claim 4, characterized in that it is made in the form of a capsule. 7. Combined antiviral agent with activity against HIV infection, characterized in that it is a kit consisting of (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5- il) -1H-pyrimidin-2-one (lamivudine), and 5'-H-phosphonate-3'-azido-3'-deoxythymidine, sodium salt (phosphazide) in a ratio of from 250: 1 to 1: 250 by weight. 8. The tool according to claim 7, characterized in that at least one active component is introduced into a pharmaceutically acceptable carrier. 9. A method of treating a mammal, including a person infected or susceptible to HIV infection, characterized in that he is administered an antiviral agent according to any one of claims 1 to 8 in an effective amount. 10. The method according to claim 9, in which lamivudine and phosphazide are administered simultaneously. 11. The method of claim 10, in which lamivudine and phosphazide are administered sequentially. 12. The method according to claim 11, in which lamivudine and phosphazide are administered in the form of a single pharmaceutical composition.