COMBINED MEDICINAL PREPARATION FOR TREATING VIRAL INFECTIONS

Abstract

The present invention relates to a novel combination drug in a solid oral dosage form comprising, as one of the three active ingredients, elsulfavirine sodium that may be suitable for medical use when treating viral infections including HIV and HBV.

An antiviral combination drug in a solid oral dosage form comprising, as one of the three active ingredients, a therapeutically effective amount of elsulfavirine sodium of formula 1a in a crystalline or polycrystalline form optionally in combination with auxiliary agents:

##STR00001##

Claims

1. A combination drug in a solid dosage form for the treatment of viral infections comprising, as one of the three active ingredients, a therapeutically effective amount of elsulfavirine sodium of formula 1a in a crystalline or polycrystalline form optionally in combination with auxiliary agents: ##STR00008##

2. The combination drug according to claim 1 for the treatment of human immunodeficiency virus.

3. The combination drug according to claim 1 for the treatment of Hepatitis B Virus (HBV).

4. The combination drug according to claim 1 comprising in a crystalline or polycrystalline form, as the other two active ingredients, a therapeutically effective amount of a Nucleoside Reverse Transcriptase Inhibitor (NRTI) precursor of formulas 2a-2j or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of a Nucleotide Reverse Transcriptase Inhibitor (NtRTI) precursor of formula 3 or 4a-4m or a pharmaceutically acceptable salt thereof: ##STR00009##

5. The combination drug according to any of claims 1 to 4 in the form of tablets, gelatin capsules, pills, powders, or chewing gums.

6. The combination drug according to any of claims 1 to 5 comprising, as active ingredients, elsulfavirine sodium of formula 1a, an NRTI precursor of formulas 2a-2j or a salt thereof, and tenofovir disoproxil fumarate of formula 3 in a mass ratio of 1a: 2a-2j or a salt thereof: 31:10:15.

7. The combination drug according to any of claims 1 to 5 comprising, as active ingredients, 15-25 mg of elsulfavirine sodium of formula 1a, 150-300 mg of an NRTI precursor of formulas 2a-2j or a salt thereof, and 250-350 mg of tenofovir disoproxil fumarate of formula 3.

8. The combination drug according to claims 6, 7 in tablets comprising, as excipients, lactose monohydrate 200, microcrystalline cellulose 102, croscarmellose sodium, pre-gelled starch, Povidone K30, magnesium stearate, and, as a film coating, Vivacoat PC-8T-181, with the mass ratio depending on their nature and mode of production.

9. The combination drug according to claim 8, wherein each tablet comprises 20.7 mg of elsulfavirine sodium of formula 1a, 200-300 mg of an NRTI precursor of formulas 2a-2j, 300 mg of tenofovir disoproxil fumarate of formula 3, 386.9 mg of lactose monohydrate 200, 134.2 mg of monocrystalline cellulose 102, 67.1 mg of croscarmellose sodium, 33.4 mg of pre-gelled starch, 15.0 mg of Povidone K30, 10.7 mg of magnesium stearate, and, as a film coating, 50.0 mg of Vivacoat PC-8T-181.

10. The combination drug according to any of claims 1 to 5 comprising, as active ingredients, elsulfavirine sodium of formula 1a, an NRTI precursor of formulas 2a-2j or a salt thereof, and tenofovir of formulas 4a-4m in a mass ratio of 1a:2a-2j or a salt thereof: 4a-4m1:10:1,25.

11. The combination drug according to any of claims 1 to 5 comprising, as active ingredients, 15-25 mg of elsulfavirine sodium of formula 1a, 150-300 mg of an NRTI precursor of formulas 2a-2j or a salt thereof, and 10-35 mg of tenofovir of formulas 4a-4m.

12. The combination drug according to any of claims 1 to 5 comprising, as active ingredients, 15-25 mg of elsulfavirine sodium of formula 1a, 150-300 mg of an NRTI precursor of formulas 2a-2j or a salt thereof, and 10-35 mg of tenofovir of formula 4h or 4m.

13. The combination drug according to any of claims 1 to 5, 11 and 12 comprising, as active ingredients, 15-25 mg of elsulfavirine sodium of formula 1a, 150-300 mg of an NRTI precursor of formulas 2a-2j or a salt thereof, 10-35 mg of tenofovir of formulas 4a-4m; as excipients, lactose monohydrate 200, microcrystalline cellulose 102, croscarmellose sodium, pre-gelled starch, Povidone K30, magnesium stearate; and, as a film coating, Vivacoat PC-8T-181.

14. The combination drug according to claim 13 comprising 20.7 mg of elsulfavirine sodium of formula 1a, 200-300 mg of an NRTI precursor of formulas 2a-2j, 25 mg of tenofovir of formula 4f, 4h or 4m, 386.9 mg of lactose monohydrate 200, 134.2 of mg monocrystalline cellulose 102, 67.1 mg of croscarmellose sodium, 33.4 mg of pre-gelled starch, 15.0 mg of Povidone K30, 10.7 mg of magnesium stearate, and, as a film coating, 50.0 mg of Vivacoat PC-8T-181.

15. The combination drug according to claim 12 comprising, as active ingredients, 10-25 mg of elsulfavirine sodium of formula 1a, 150-350 mg of an NRTI precursor of formulas 2a-2j, 5-35 mg of tenofovir of formula 4f, 4h or 4m, 20-35 mg of croscarmellose sodium, 70-120 mg of microcystalline cellulose, and 1-7 mg of magnesium stearate.

16. The combination drug according to claim 12 comprising, as active ingredients, 20.7 mg of elsulfavirine sodium of formula 1a, 200-300 mg of an NRTI precursor of formulas 2a-2j, 25 mg of tenofovir of formula 4f, 4h or 4m, 28 mg of croscarmellose sodium, 105.56 mg of cellulose microcrystals, 5.25 mg of magnesium stearate and a film coating consisting of Vivacoat PC-8T-181.

17. A method for producing a combination drug formulated in tablets by mixing therapeutically effective amounts of elsulfavirine sodium of formula 1a, an NRTI precursor of formulas 2a-2j or a pharmaceutically acceptable salt thereof, and an NtRTI precursor of formula 3 or formulas 4a-4m or a pharmaceutically acceptable salt thereof with auxiliary agents followed by compression.

18. A method of treatment or prevention of viral diseases by oral administration to the patient of the novel combination drug according to claim 1.

19. The method according to claim 18 for treating or preventing HIV.

20. The method according to claim 18 for treating or preventing HBV.

Description

PREFERRED EMBODIMENT

[0102] The present invention will now be described in terms of certain embodiments, which are not intended to limit its scope. On the contrary, the present invention covers all alternatives, modifications, and equivalents that can be included within the scope of the claims. Thus, the following examples, which include specific embodiments, will illustrate this invention without limiting it.

EXAMPLE 1

General Procedure for Preparing Solid Oral Dosage Forms (SODFs).

[0103] Elsulfavirine sodium of formula 1a (2.0-2.5 g), an NRTI precursor of formulas 2a-2j (20.0-30.0 g), and tenofovir disoproxil fumarate of formula 3 (30.0 g) or tenofovir of formulas 4a-4m (15-35 mg) are carefully ground and mixed. The resulting SODF (Table 3) is used to produce a combination drug in tablets, gelatin capsules, pills, powders, granules, or medicated chewing gums by known process.

TABLE-US-00003 TABLE 3 The content of active ingredients in SODF Solid Oral Dosage Forms (SODFs) SODF SODF SODF SODF SODF SODF SODF 1 2 3 4 5 6 7 Ingredient Content of active ingredients (g) in SODF 1a 2.07 2.07 2.07 2.07 2.07 2.07 2.07 2b 20.0 20.0 20.0 20.0 2j 25.0 25.0 25.0 3 30.0 30.0 4f 2.5 4h 2.5 2.5 4m 2.5 2.5

EXAMPLE 2

Evaluation of Anti-HIV Activity, Cytotoxicity, and Selectivity Index for Tenofovir of Formulas 4f, 4h, 4k, and 4m.

[0104] The anti-HIV activity of active ingredients (test compounds) was evaluated using SupT1 cells. The cells were infected with the NL4.3 HIV strain carrying a gene of green fluorescence protein (NL4.3-GFP). The virus preparation was obtained by transfection of 293T cells of proviral DNA. Forty-eight hours after the transfection, the preparation was frozen and stored until use. To enhance the infection efficiency, the suspension of SupT1 cells was precipitated from the infection mixture by centrifugation. Test compounds were added to the cells immediately before virus addition. After a 2-hour incubation, the infection mixture was replaced by fresh culture medium with test compounds. The infection efficiency was evaluated following 45 hours by counting the percent of fluorescing cells against noninfected cell cultures. The cytotoxicity of test compounds was evaluated simultaneously at the same, but not infected, SupT1 cellular line using the XTT reagent. To this end, serial ten-fold dilutions of test compounds were prepared (starting with 10 M to evaluate viral activity or with 100 M to evaluate cytotoxicity). For negative control, 0.1% DMSO was used. As a result, the values of activity EC.sub.50, cytotoxicity CC.sub.50, and selectivity index SI were calculated. The test quality was evaluated using the following controls: signal to background ratio, integrase inhibitor raltegravir (1 M), and reproducibility of the test. The drug emetine was used to control the validity of cytotoxicity obtained (0.03, 0.09, and 0.2 M). The results are summarized in Table 1.

EXAMPLE 3

Evaluation of Anti-HBV Activity, Cytotoxicity, and Selectivity Index for Tenofovirs of Formulas 4f, 4h, 4k, 4m.

[0105] Anti-HBV activity of tenofovir of formulas 4f, 4h, 4k, 4m (test compounds) was evaluated in the cell line of human hepatoma AD38 carrying integrated HBV DNA with terminal repeats [Lander S, et. al, Antimicrobial Agents and Chemotherapy, 1997, pp. 1715-1720]. The cell line was made available by Dr. C. Seeger, Fox Chase Cancer Center, Philadelphia, Pa.). Simultaneously, cytotoxicity was evaluated.

[0106] The cells were cultivated in complete DMEM/F12 culture medium containing 2 mM of L-glutamine (Thermo Scientific, Cat #11320033), 10% fetal bovine serum (ThermoFisher Scientific, Cat#), 1% antibiotic-antimycotic solution (ThermoFisher Scientific, Cat#15240096), and 0.3 g/ml of tetracycline (Sigma, Cat # T7660-5G). The cells were seeded into 96-well Corning Biocoat plates (Corning, Cat # 356407) in 225 l of complete medium without tetracycline, 20 000 cells per well. The test compounds were dissolved first in DMSO (Sigma cat. D2650), then in DMEM/F12 medium, and 9 dilutions in log 3 steps (225 l) were added to the cells. The final concentrations of test compounds varied from 10 M to 1 nM. Each dilution was tested in three identical wells. For inhibition control, cells cultivated in the presence of tetracycline were used, because tetracycline completely stops HBV replication in this cell line. The cells were then incubated for 4 days under a humidified 5% CO.sub.2 atmosphere at 37 C.

[0107] Isolation of secreted HBV DNA. Following a 4-day incubation, the viral DNA was isolated from cultural supernatants using the PureLink Pro 96 Genomic DNA Purification Kit (ThermoFisher Scientific, Cat # K183104A) according to the manufacturer's instructions. After elution, the purified DNA was stored at 20 C.

[0108] The technique of real-time quantitative polymerase chain reaction (RT-qPCR) was applied using the CFX96TM Real-Time System instrument (Bio-Rad, Hercules, Calif.) and polymerase AmpliTaq Gold DNA Polymerase (Applied Biosystems).

[0109] Reaction mixture composition:

TABLE-US-00004 10 reaction buffer 2 l 25 mM MgCl.sub.2 2 l dNTP mix (ATP, GTP, CTP - 2 mM, 2 l UTP - 4 mM) ROX, 50 M (Fisher Scientific117545000) 0.04 l 20 primers/probe mix (primers 6 M, 1 l probe 5 M) Uracil-DNA Glycosylase (1 U/l) 0.15 l AmpliTaq Gold DNA Polymerase 0.1 l (5 U/l) Water 12.11 (up to 19.4 l) DNA HBV 0.6 l

[0110] Cycle program:

TABLE-US-00005 50 C. 2 minutes 95 C. 10 minutes 40 cycles: 95 C. 15 seconds 60 C. 30 seconds 72 C. 30 seconds + 1 second per each subsequent cycle.

[0111] The fluorescent signal was read at the end of each cycle.

[0112] Primers and fluorescent samples were obtained from IDT (San Diego, Calif.):

TABLE-US-00006 HBV_rcDNA-S_FAM 56FAM/ATCCTCAAC/ZEN/CACCAGCACGGGACCA/3IABkFQ; HBV_rcDNA-S_RGAGGGATACATAGAGGTTCCTTGA; HBV_rcDNA-S_FGTTGCCCGTTTGTCCTCTAATTC.

[0113] The values of Ct (threshold cycle, in which HBV DNA amplification becomes appreciable) normalized to cell cultures without test compounds were found by using the formula E=(1/(1+100%))(Ct[test compound]-Ct[K-]), where E is a normalized level of HBV DNA, Ct[K-] and Ct[test compound] are Ct values for samples without and with test compounds, respectively. EC.sub.50 values (Table 2) were computed using the Graph Prizm software.

[0114] The cytotoxicity of test compounds was determined simultaneously on the same AD38 cell line. The cells were cultivated in a 96-well black plate with a transparent bottom (10.sup.4 cells/well) in complete DMEM/F12 medium containing 2 mM of L-glutamine (Thermo Scientific, Cat #11320033), 10% fetal bovine serum (ThermoFisher Scientific, Cat#), and 1% antibiotic-antimycotic solution (ThermoFisher Scientific, Cat#15240096). The AD38 cells were seeded in 96-well plates (7.510.sup.3 cells/well in 100 l of medium). The solutions of test compounds in DMEM were prepared immediately before use. All in all, there were 9 serial 3-fold dilutions. Four hours following cell seeding, the serial dilutions of preparations were added to the cells (100 l per well). The final concentration of test compounds varied from 30 M to 10 nM and that of DMSO, 0.5%. Whenever necessary, higher concentrations of test compounds were studied. The cells were then incubated for 3 days under a humidified 5% CO.sub.2 atmosphere at 37 C. The number of living cells was counted using the ATPLite kit (Perkin Elmer, Boston, USA) in compliance with manufacturer's instructions. Three independent repeats were provided for each compound. Each cell was washed three times with PBS (0.2 ml/well) and then lysed by adding cell buffer (50 l/well). All reagents mentioned hereinabove were included in the ATPLite kit. The microplate was incubated for 5 minutes on a rocking platform at 600 rev/min, following which a substrate solution (part of the ATPLite kit) was added (50 l/well). After additional 5-minute incubation on a rocking platform at 600 rev/min, the plates were kept in dark for 10 minutes, and the luminescence was read using TopCount NXT (Packard, Perkin Elmer). For quantitative evaluation of cytotoxicity, the CC.sub.50 parameter (a concentration that kills 50% of cells) was used. Calculation of CC.sub.50: inhibition efficacy (% Inh) was calculated by using the formula: % Inh=[(L.sup.posL.sup.ex)/(L.sup.posL.sup.neg)]*100%, where L.sup.pos is positive control, luminescence in wells containing cells without compound medium; L.sup.neg is negative control, luminescence in wells containing medium without cells; L.sup.ex is luminescence in wells containing medium in a particular concentration. CC.sub.50 values (Table 2) were then determined using XLfit 4 software.

EXAMPLE 4

Evaluation of Acute Toxicity and Tolerated Toxic and Lethal Doses of SODFs.

[0115] Evaluation of SODF acute toxicity and tolerated toxic and lethal doses for single intragastric administration to male and female mice and rats. Evaluation was carried out for 24 male rats weighing 235-260 g and 28 female rats weighing 225-250 g as well as for 24 male mice weighing 21-25 g and 24 female mice weighing 20-24 g. All in all, there were 8 groups in each of the four categories. SODF was administered in the largest possible volume 10 ml/kg three times a day at a 40-minute interval. Prior to administration, SODFs were dispersed, then ground in a mortar and mixed with a 0.5% Tween 80 solution to obtain a suspension suitable for intragastric administration to animals at a dose of 10 ml/kg. Solutions for administration were always prepared on the day of administration. Prepared SODF suspensions were administered at the same time every day (within a deviation of maximum 4 hours). Prior to SODF administration, animal weights were recorded; after administration, experimental animals' condition was observed for 1 hour. On each of the 14 days following SODF administration, the weights of rats and mice were recorded and the animals were examined to detect any cases of death or abnormal condition. In this way, parameters of acute SODF toxicity were established. It was technically impossible to estimate LD50 for SODF, as the administration of maximum permissible SODF doses did not lead to animal death. Thus, it was found that intragastric SODF administration at doses like 600/6000/9000 mg/kg did not affect body weight gain just like in groups receiving reference drugs (compound of formula 1a) at a dose of 3000 mg/kg and Truvada (compound of formula 2b+compound of formula 3) at a dose of 6000/9000 mg/kg. The latter did not differ from control or experimental groups of mice or rats (both males and females). In addition, no reduced feed or water intake was observed in all experimental and control groups. The intragastric administration of SODF and reference drugs did not affect the relative weight of animals' organs in experimental groups, and no statistically significant difference with control groups was detected. The gross examination of animals treated with drugs did not reveal any differences from control groups.

EXAMPLE 5

A SODF Combination Drug in Tablets

[0116] Tableted SODFs were prepared using a dry granulation technique followed by compressing and film coating. Dry granulation by roller compaction was performed to minimize SODF moisture exposure during granulation process. The general production process involved SODF lubrication using intragranular fillers followed by roller compaction and milling. Resulting SODF composition granules were mixed and lubricated with extragranular excipients to obtain final powder SODF mixtures, which were then compressed into tablets and coated with the Vivacoat PC-8T-181 or Opadry II White 85F18422 films.

[0117] API content in one tablet is given in Table 4.

TABLE-US-00007 TABLE 4 Active ingredient and excipient contents in each tablet SODF tablet SODF SODF SODF SODF SODF SODF SODF Ingredient 1 2 3 4 5 6 7 Active ingredient content (mg) in SODF 1a 20.7 20.7 20.7 20.7 20.7 20.7 20.7 2b 200 200 200 200 2j 250 250 250 3 300 300 4f 25 4h 25 25 4m 25 25 Total.sup.a (mg) 520.7 570.7 245.7 295.7 295.7 295.7 295.7 Excipient content in a tablet (mg) LM.sup.b 386.94 MCC.sup.c 134.20 CS.sup.d 67.08 GS.sup.e 33.4 mg Povidone K30 15.0 mg MS.sup.f 10.68 EW.sup.g (mg) 697.3 TCW.sup.h 1168 1218 893 943 943 943 943 FC.sup.i 50,00 TW.sup.j (mg) 1218 1268 943 993 993 993 993 .sup.aTotal weight (mg) of active ingredients in a tablet, .sup.bLactose monohydrate (200 mesh). .sup.cMicrocrystalline cellulose 102. .sup.dCroscarmellose sodium. .sup.eGelled starch. .sup.fMagnesium stearate. .sup.gExcipient weight. .sup.hTablet core weight. .sup.iFilm coat Vivacoat PC-8T-181. .sup.jCoated tablet weight.

INDUSTRIAL APPLICABILITY

[0118] The invention could be used in medicine and veterinary.