WO2010005344A1 - Forme médicamenteuse nanosomale d'une préparation à action prolongée servant au traitement de l'hépatite c (variantes) - Google Patents

Forme médicamenteuse nanosomale d'une préparation à action prolongée servant au traitement de l'hépatite c (variantes) Download PDF

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WO2010005344A1
WO2010005344A1 PCT/RU2009/000345 RU2009000345W WO2010005344A1 WO 2010005344 A1 WO2010005344 A1 WO 2010005344A1 RU 2009000345 W RU2009000345 W RU 2009000345W WO 2010005344 A1 WO2010005344 A1 WO 2010005344A1
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samples
hepatitis
drug
sample
preparation
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Геворкбек Гайкович БАРСЕГЯН
Евгений Алексеевич ВОРОНЦОВ
Виктор Константинович МАРКОВ
Кирилл Юльевич ШУБСКИЙ
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Общество С Ограниченной Ответственностью "Akba-Aльяhc"
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A61K38/063—Glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • the invention relates to medicine, and in particular to a nanosomal dosage form of a sustained release preparation for the treatment of hepatitis C.
  • Hepatitis C is a liver disease caused by the hepatitis C virus (HCV).
  • HCV hepatitis C virus
  • hepatitis can also be caused by other viruses, such as cytomegavirus (CMV) or the herpes virus.
  • CMV cytomegavirus
  • WHO World Health Organization
  • Hepatitis C can be acute and chronic. Acute hepatitis C is diagnosed very rarely and more often by accident. There are three scenarios - a variant of events that occur after acute hepatitis C.
  • liver damage The dramatic and often hidden transition of acute hepatitis C into chronic occurs gradually and does not depend on the degree of manifestations of the acute phase. Over several years, damage to liver cells increases, fibrosis develops. The main functions of the liver can be preserved for a long time. The disease progresses slowly, pathological manifestations increase gradually, therefore, as hepatologists warn, the problem of adverse outcomes of hepatitis C will be faced by civilization in one or several decades, when the experience of observing a large population of people who are currently ill will be analyzed.
  • hepatitis C The pronounced pathological effects of hepatitis C are manifested in 20% of patients with a chronic form of HCV and the active course of hepatitis, while there is a high risk of transformation of hepatitis into cirrhosis of the liver. And 5% of patients with cirrhosis may develop primary liver cancer. The likelihood of developing liver cancer is higher with the simultaneous course of two infections - hepatitis B and hepatitis C. Long-term alcohol consumption is also associated with a higher risk of developing liver cancer, which imposes a restriction on the use of alcohol-containing drugs. for the treatment of hepatitis.
  • interferon alpha IFN
  • ribavirin ribavirin
  • H. Blokhina New patients interferon therapy strategies chronic hepatitis C 5 Viral hepatitis - achievements and prospects, Information Bulletin, 1999, NaI (2), pp. 9-12
  • Petrov BA Zabolotnaya G.A., Interferon Inductors in the Treatment and Prevention of Viral Infections, New Drugs and Pharmacoeurs News FDI, 2000, Na 8, pp. 7-12
  • Rosen HR Gretch DR
  • Neratitis C virus current understanding add-ons for future futures terariies, MoI., Med., Todau, 1999, v.5, N ° 9, p .393-399).
  • Interferon alfa inhibits the replication and transcription of viruses and chlamydia. It has an antiviral effect, inducing a state of resistance to viral infections and modulating the response of the immune system aimed at neutralizing viruses or killing infected cells.
  • the antiviral mechanism of action is to create protective mechanisms in cells not infected with the virus.
  • interferon alpha alters the properties of the cell membrane, prevents the adhesion and penetration of the virus into the cell, stimulates specific enzymes, acts on RNA and inhibits the synthesis of virus proteins.
  • Interferon alfa Due to the immunomodulating activity of Interferon alfa, normalization of the immune status occurs. Its immunomodulatory effect is due to the stimulation of macrophage activity (phagocytic activity) and natural killer cells (NK cells). Interferon alpha stimulates the process of antigen presentation by macrophages to immunocompetent cells. However, interferon alfa preparations can lead to the appearance of antibodies to interferon, which, as a result, reduces their therapeutic effect. Interferon alfa is ineffective for the treatment of hepatitis C. So, in case of chronic hepatitis C, 3-6 million ME are prescribed s / c or i / m 3 times a week, the duration of treatment is 12 weeks. Most patients respond to therapy with a decrease in transaminase levels 12 weeks after the start of treatment. However, if within 16 weeks from the start of therapy there is no decrease in the content of transaminases (ALAT), then it is recommended to stop treatment.
  • AAT trans
  • ribavrin activity against hepatitis C virus has been shown. Although the mechanism of action has not been fully identified, it is assumed that ribavirin triphosphate accumulating as phosphorylation competitively inhibits the formation of guanosine triphosphate, thereby reducing the synthesis of viral RNA. It is also believed that the mechanism of synergistic action of ribavirin and interferon alpha against hepatitis C virus is due to increased phosphorylation of ribavirin by interferon.
  • interferons The mechanism of action of interferons is associated with a simultaneous antiviral intracellular effect by activation of cellular genes, resulting in the synthesis of proteins that inhibit the synthesis of viral DNA (RNA) and systemic immunomodulatory effect, which is realized by enhancing the expression of HLA antigens on cell membranes and increasing the activity of cytotoxic T-cells and natural killer cells (Ahmad A., Alvarez F., RoIe schreibf NK and NKT cells immodepocephase Hclupeduce .VioL, 2004, v. 76, p.
  • Interferons on the one hand stimulate phagocytosis, the activity of natural killer cells, and the expression of antigens. On the other hand, they can inhibit the formation of antibodies, provoke the development of anaphylactic shock, inflammation, delayed-type hypersensitivity. Therefore, most authors today share the opinion that the treatment of viral hepatitis can no longer and should not be carried out using IFN therapy alone (Clinical experience with the use of IFN alfa-2b in hepatitis, http://medi.ru/doc/081015. htm; Deryabin P.G., Isaeva E.I., Grenkova E.P.
  • One of the first drugs to solve this problem is a drug consisting of disodium salt of oxidized glutathione with cis-diaminodichloroplatin (GSSG Pt).
  • GSSG Pt disodium salt of oxidized glutathione with cis-diaminodichloroplatin
  • GSSG Pt and its pharmaceutically acceptable derivatives, in particular salts, in the treatment of oncological, infectious (viral) diseases, including Hepatitis C was explained both by stimulation of the production of a wide palette of endogenous cytokines and by its unique ability to activate apoptotic death of exclusively transformed cells .
  • Most of the therapeutic effects of GSSG Pt and its pharmaceutically acceptable derivatives, both under experimental and clinical conditions, are associated with the properties of GSSG Pt and its dosage forms stimulate / modulate the endogenous production of cytokines or reproduce their effects with respect to the regulation (restoration of the normal ratio) of the proliferation and differentiation of normal cells and, at the same time, activate apoptotic death of exclusively transformed cells.
  • the technical problem to which the present invention is directed is to develop the drug and its specific forms, with which it is possible to provide effective treatment for viral hepatitis C (HCV) by ensuring the delivery and localization of drugs in the main target organ (liver) affected by the HCV and inside liver cells (in hepatocytes), in doses that are effective for therapeutic action.
  • HCV viral hepatitis C
  • the nanosomal dosage form of a long-acting drug for the treatment of hepatitis C containing a complex of active components including glutathione ( ⁇ -glutaminyl-cystsinyl-glycine) and inosine (1,9-dihydro-9-beta-D-ribofyranosyl-6H-pyrene -6-one), taken in the ratio 1: 1, as well as the platinum salt (in the particular case it can be cisplatin or carboplatin) in the amount of 0.05%, while the complex of active components is immobilized due to adsorption on a biodegradable polymer carrier in the form polylactide glycolide nanoparticles.
  • active components including glutathione ( ⁇ -glutaminyl-cystsinyl-glycine) and inosine (1,9-dihydro-9-beta-D-ribofyranosyl-6H-pyrene -6-one
  • the solution in the form of a solution contains, wt.%: A complex of active components - 0.35-0.37 polylactide glycolide - 2.45-2.72 solvent - the rest.
  • the preparation in the form of a solution additionally contains a surfactant in an amount of 0.80 wt.%, And a solution of dimethyl sulfoxide in water is used as a solvent.
  • the preparation in the form of a powder contains, wt.%: A complex of active components - 4 polylactide glycolide - 16 surfactant - 60 means of cryoprotection and resuspension - 20.
  • the nanosomal dosage form of a sustained-release preparation for the treatment of hepatitis C also contains a complex of active components, which in this case includes only glutathione and inosine taken in a 1: 1 ratio.
  • the complex of active components in this embodiment is immobilized due to adsorption on a biodegradable polymer carrier in the form of polylactide glycolide nanoparticles.
  • the preparation in the form of a powder contains, wt.%: A complex of active components - 4 polylactide glycolide - 16 surfactant - 60 means of cryoprotection and resuspension - 20.
  • polyvinyl alcohol or polysorbate 80 can be used as a surfactant.
  • D-Mannitol is used as a means of cryoprotection (prevention of destruction and adhesion of nanoparticles during freeze drying) and resuspension (prevention of aggregation or adhesion).
  • biodegradable polymer nanoparticles ensures their optimal distribution and bioavailability for the therapeutic effect, as well as the prolongation of release, which occurs as the particles in the organs, tissues and cells break down, where they penetrate due to the mechanism of endocytosis.
  • the amount of drugs delivered by nanoparticles is sufficient both for direct extracellular virucidal action and for complex effects at the cellular level. That is, the inclusion of drugs in biodegradable polymer nanoparticles provides optimal bioavailability and an adequate distribution of drug substances in therapeutic quantities. In addition, a beneficial effect on the immune system is achieved. Along with this, thanks to the invention, prolongation of the circulation of drugs in the bloodstream (including the capillary system), accumulation and prolonged release (release) of drugs is achieved, which contributes to the direct antiviral effect of the drug providing highly effective treatment for acute and chronic forms of hepatitis.
  • a method of treatment is possible by introducing into the body of mammals infected with hepatitis C virus, a prolonged-acting pharmaceutical nanosomal preparation containing an effective amount of GSSG-inosine-PT, with the aim of directly inhibiting the ATP-azo / helicase activity of hepatitis C virus NS3 to stop replicative activity and elimination of hepatitis C virus, therefore, achieving a therapeutic effect, along with ensuring the cytoprotective effects of healthy cells.
  • the FAS / APO-1 antigen CD95 + on the membranes of virus-infected cells, which helps to eliminate virus-infected cells, reducing the pool of affected cells and thereby contributing to healing.
  • the therapeutic effect is also achieved by stimulating T-cell and humoral anti-infection immunity.
  • the ratio of components - GSSG rinosine: Pt (1: 1: 0.001) was selected experimentally by a set of indicators, namely, efficacy, toxicity, therapeutic breadth, side effects.
  • the individual components that make up the composite are registered as active substances of known drugs.
  • Inosine is the active substance of the drug riboxin (FS 42-2069-97), and the disodium salt of glutathione is bis- ( ⁇ -L-glutamyl) -L-cysteinyl-bis-glycinate sodium - is the active substance of the drug Glutoxim® (VFS 42-3194-98), platinum compounds - cisplatin and carboplatin are registered drugs.
  • the implementation of the invention is illustrated by the example of technologies for two nanosomal dosage forms of the drug: liquid - samples MO-1/1, MO-1/2 (containing a complex of active components of 0.35 and 0.37 wt.%, Respectively); powdered - sample MO-7, MO-9.
  • the first embodiment of the invention includes samples MO-1/1, MO-1/2 and MO-7.
  • a second embodiment of the invention relates to sample MO-9.
  • a LASTEL ® Poly (DZ, -lactide-co-glusolide) copolymer of lactic and glycolic acid (PLGA aeg (50: 50) (0.37) was used as the polymer matrix - carrier of active substances or the polymer base of the resulting nanoparticles ) (firms of ⁇ borbable ⁇ olumers ⁇ ptematiopal, USA).
  • particles were obtained either by emulsification of a PLGA polymer solution with auxiliary components, or by condensation in a reactive medium.
  • the solvents used were dimethyl sulfoxide (DMSO,> 99.5%) (Ridel-de Naep, Germany), acetone (osch) (Khimmed, RF), and also distilled water (DE-4 aqua distiller model 737, RF).
  • Polysorbate 80 (Twep ® 80) (Serva, USA) and polyvinyl alcohol (PVA) mol were used as surfactants. wt. 30000 ⁇ 70,000 (Sigma, USA).
  • PVA polyvinyl alcohol
  • the three component substance of RGCS consists of glutathione (GSSG), inosine (Iposip) taken in the ratio 1: 1 (1: 1 mol / mol) and platinum (Pt 2+ ) in an amount of 0.05%. 0.70 ml of an acetate buffer solution (pH 6.05) was dissolved with stirring on a magnetic stirrer (IKA ® RH-CT / S, Germany) (glutathione (GSSG), inosine (Iposipe) taken in the ratio 1: 1 (1: 1 mol / mol ) and added platinum (Pt 2+ ) with a total weight of 54.9 mg (solution “A”).
  • Particle size and size distribution were determined by autocorrelation spectroscopy.
  • a Soulter N4MD submicron laser spectrometer Soulter Electropics, France-USA
  • the measurement algorithm is based on the CONTIN program.
  • the three component substance of the liquid preparation MO-1/2 consists of glutathione (GSSG), inosine (Iposip) taken in a ratio of 1: 1 (1: 1 mol / mol) and platinum (Pt 2+ ) in an amount of 0.003 mol.
  • the ratio of the components of GSSG: Iposip: Pt 2+ is (1: 1: 0.003).
  • the specific amount of cisplatin was increased to experimentally test the effect of platinum ions (Pt 2+ ) on the effectiveness and toxicity of the drug.
  • composition of the drug is MO 1/2, in May. %:
  • composition of the drug MO-7 (calculated on a completely dry mixture), May. %:
  • composition of the drug MO-9 (calculated on a completely dry mixture), May. %:
  • the sizes of the obtained particles were also determined by photon correlation spectroscopy (FCC) using a nanosizer (sub-micron spectrometer ⁇ ult Foodr N4MD from ⁇ ult Canalr ⁇ lestgopis, France-USA).
  • FCC photon correlation spectroscopy
  • nanosizer sub-micron spectrometer ⁇ ult Camillr N4MD from ⁇ ult Canalr ⁇ lestgopis, France-USA.
  • the measurement algorithm is based on the CONTIN program.
  • the contents of the vial are made up to a volume of 2 ml with bidistilled water and gently shaken for 2-4 minutes.
  • the effect of resuspension was determined visually by the absence of agglomerates or sediment.
  • a normally restored sample is an opalizing colloidal solution without visible agglomerates or inclusions.
  • a portion of the recovered sample (50 ⁇ l) was placed in a nanosizer flask containing 3 ml of double-distilled water. The flask was continuously shaken and v was placed in a nanosizer. Measurements are taken immediately.
  • the obtained characteristics of the produced samples show that according to the registered values of the particle sizes and the existing criteria for assessing the particle size, the created samples are classified as nanoparticles, and the average particle sizes are close to 100 nm (101-121 nm).
  • the accumulated samples are classified as polydisperse.
  • Analysis of the distribution of particle sizes by fractions shows a bimodal picture of the distribution by fractions of all samples.
  • the minimum recorded particle size was 2.3 nm for sample MO-1/2, and the maximum 146 nm for sample MO-7. From the analysis of the particle size distribution by fractions, it follows that for the sample MO-1/1, the nanoparticles with a size of 119 nm account for 90%, and the mode — the average value over the entire array of nanoparticles — was 108 nm.
  • nanoparticles with a size of 146 nm were 83%, and the mode was 121 nm.
  • the nanoparticles with a size of 131 nm are 84%, the mode is 111 nm.
  • the obtained samples MO-1/1, MO-1/2, MO-7, MO-9 in accordance with the accepted classification are nanosomal substances (hereinafter referred to as the nanosomal form of the preparation RGKS - NRGKS).
  • the particle sizes of the aged samples and their size distribution were determined on a nanosizer using autocorrelation spectroscopy.
  • a Soulter N4MD submicron laser spectrometer Soulter Electropics, France-USA was used.
  • the sample of MO- 1/2 before aging was a white or slightly creamy powder (powder dosage form for oral administration). After exposure, the sample slightly changed color, remaining transparent.
  • Sample MO-7 before aging was a powder of white or light cream color (powdered dosage form for oral administration). After exposure, the sample practically did not change color, remaining light cream.
  • the established particle sizes of sample MO-7 (s) in an aqueous medium are presented in table 8.
  • Sample MO-9 before aging, was a white powder (powder dosage form for oral administration). After exposure, the sample practically did not change color.
  • the studies analyzed the substance of RGCS, glutathione, inosine (Sigma), and the obtained samples of H-RGCS.
  • the dialysis bags used were the Dialysis tubi ⁇ g, high retiop, Size: 23 mm x l5 mm (Sigma- ⁇ ldrivich) tape.
  • the exclusion limit is 12 kDa.
  • sections of the dialysis tape (10 cm) were boiled in a ⁇ 0.001 M solution of ethylenediaminetetraacetic acid for 1 h. Distilled water obtained using a DE-4 model 737 akvadistillyator (RF) was used.
  • RF akvadistillyator
  • Dialysis vessels were 250 ml measuring cylinders with caps. Mixing of the dialysis fluid in laminar mode was carried out using magnetic stirrers in a Teflon shell and an RH-CT / S magnetic stirrer (IKA R , Germany). The optical densities of the solutions were measured on spectrophotometers Hitachi 557 (Japan) and Ultrosres 2000 (Pharmacia, Sweden). For weighing, a VIBRA analytical balance (max / d 220 / 0.0001 g) (Japan) was used. Pipettes and plastic tips (BIOHIT) (Finland) were used for sampling.
  • the tubes were heated in a boiling water bath for 30 minutes. Samples acquired a characteristic blue-violet coloration (Rueman purple). The concentration of oxidized glutathione (GSSG) was determined spectrophotometrically at a wavelength of 570 nm using a calibration curve.
  • GSSG oxidized glutathione
  • Fig L presents a chromatogram of a sample of the preparation of RGKS. There are 2 peaks of the main components of the drug: oxidized glutathione (GSSG) and inosine (Iposip).
  • GSSG oxidized glutathione
  • Iposip inosine
  • Figure 2 illustrates the dynamics of the release of glutathione and inosine from the RGCS preparation at 252 nm (inosine) and at 570 nm (oxidized glutathione) obtained by equilibrium dialysis in a model experiment.
  • Fig. 3 illustrates the dynamics of the release of inosine from the RGCS preparation and samples MO-1/1, MO-7, MO-9 at 252 nm (inosine); obtained by equilibrium dialysis.
  • the experiments were performed on white sexually mature Wistar male rats of standard weight 200-220 g. Substances were injected into the tail vein. In decapitation animals collected blood and received blood plasma. Then 1 ml of the obtained blood plasma was placed in a test tube with a ground stopper with a capacity of 5 ml and 2 ml of acetonitrile containing 15 ⁇ l of trifluoroacetic acid was added. The tube was fixed in a shaker so that the carriage moved along the tube’s main axis. It was extracted for 10 min, then the contents of the tube were centrifuged and the supernatant was decanted into another tube with a ground stopper with a capacity of 10 ml. 6 ml of methylene chloride was added, extracted again for 10 minutes and centrifuged. 50 ⁇ l of the obtained supernatant was introduced into the chromatograph.
  • a chromatography mode was used, which is a combination of isocratic and linear gradient elution. Spectrophotometric detection was performed at a working wavelength of 205 nm. Computer processing of the experimental data was carried out using a calibration graph previously checked against standard samples at three concentration levels.
  • RGKS sample - 1 ml of the drug contains 10 mg of oxidized glutathione.
  • the dose is 10 mg / kg in terms of oxidized glutathione.
  • Blood samples were taken after 1, 2, 5, 10, 20, 40 and 60 minutes, during the study of the drug RGKS, in addition after 1.5, 3, 24, 48 and 72 hours, when examining the samples of the drug N-RGKS-liquid form MO-1/1 and powdered form MO-7; Plasma was separated in the collected blood samples and the concentration of oxidized glutathione was determined. At least 6 animals were taken for each point.
  • Table 10 shows data on the concentration of oxidized glutathione in the blood plasma of Wistar rats after iv administration of the drug MO-1/1.
  • Figure 4 illustrates the pharmacokinetics of the drug RGKS. The dependence of the concentration of oxidized glutathione in the blood plasma of rats on time.
  • Table 11 shows data on the concentration of glutathione in the blood plasma of Wistar rats after iv administration of a sample of MO-1/1.
  • FIG. 5 illustrates the pharmacokinetics of sample MO-7.
  • Figure 6 illustrates the pharmacokinetics of the preparation of RGCS and prolong form MO-7.
  • Table 12 presents data on the concentration of glutathione in the blood plasma of Wistar rats after iv administration of sample MO-7.
  • sample MO- 1/1 liquid form sample MO- 1/2 liquid form
  • MO-7 powder sample MO-9 powder against viral infection caused by the cytopathogenic variant of HCV in cell cultures.
  • a cytopathogenic strain of hepatitis C virus was used in the experiments, isolated from the blood of a chronically infected patient, in the blood serum of which antibodies to HCV and HCV RNA were detected.
  • the virus holding material was. " culture fluid collected from virus-infected SPEV cell cultures at the height of cytopathic manifestations.
  • Samples of the preparations MO-7 and MO-9 arrived in the form of a dry substance, soluble in the medium, which was used after dissolution in the nutrient medium immediately for processing cells and 7 days after storage of the solution at room temperature. Samples of the preparations MO-1/1 and MO-1/2 were provided as a liquid solution. All preparations were used to treat HCV-infected cell cultures in dilutions from 1: 2 to 1: 20.
  • the virucidal activity of samples of the preparations MO-1/1 / MO-1/2, MO-7, MO-9 was determined by exposure of non-toxic doses of the drug with virus-containing material in a ratio of 1: 1 for 10 and 30 minutes, after which the samples were determined residual infectious activity of HCV by titration of residual infectious activity in SPEV cell cultures.
  • the research results indicate that all samples of the preparations, except MO-1/2, in the dilutions used do not possess cytotoxic properties for SPEV.
  • the preparation MO-1/2 at a 1: 2 dilution caused 100% death of SPEV cells, treatment of cells with this drug at a dilution of 1: 4 led to 35% cell death. Therefore, in further studies, the preparation MO-1/2 was diluted 8 times or more before use.
  • the drugs do not have the ability to protect HCV-infected SPEV cells from the lytic effect of the virus for 6 days (observation period).
  • Samples of the preparations MO-7 and MO-9 retained the ability to suppress the infectious activity of HCV in the case of treatment of infected cells after infection with the virus, to a greater extent than with the preventive use of the drug.
  • the experimental animals were divided into 10 equal groups of 10 animals each; 6 groups with a model of toxic hepatitis, 2 groups - control.
  • Group 2 an experimental-formed model of hepatitis, the treatment was carried out with a comparison drug “Essential-H” iv at a dose of 15 mg / kg;
  • Organs of the chest cavity Localization preserved. Pathological changes in the structure were not detected. Light light pink, clean, without seals and neoplasms. Heart without pathologies, moderate blood supply.
  • Organs of the abdominal and pelvic cavities Localization preserved, no redness. Scanty or moderate deposition of fatty tissue.
  • the surface is smooth. There are no pathological changes at the macroscopic level. Spleen of dense uniform consistency, red-brown saturated color. Size saved.
  • the surface is smooth, the edges are even. There are no pathological changes at the macroscopic level.
  • the liver is slightly enlarged, brownish-gray. The edges are even, the surface is smooth.
  • the consistency is uniform, elastic or slightly flabby, easily torn.
  • the section has a slight bulging. A greasy residue remains on the scalpel blade. Capsule without damage.
  • Organs of the chest cavity Localization preserved. Pathological changes in the structure were not detected. Light light pink, clean, without seals and neoplasms. Heart without pathologies, moderate blood supply.
  • Organs of the abdominal and pelvic cavities Localization preserved, swelling, no redness. Scanty or moderate deposition of fatty tissue.
  • the surface is smooth. There are no pathological changes at the macroscopic level. Spleen of dense uniform consistency, red-brown saturated color. Size saved.
  • the surface is smooth, the edges are even. There are no pathological changes at the macroscopic level.
  • the liver is slightly enlarged, red-brown in purple, the texture is dense, uniform.
  • the surface is smooth, the edges are even. On a section without bulging.
  • Organs of the chest cavity Localization of organs preserved. Pathological changes in the structure were not detected. Light light pink, clean, without seals and neoplasms. Heart without pathologies, moderate blood supply.
  • Organs of the abdominal and pelvic cavities Localization preserved, swelling, no redness. Scanty or moderate deposition of fatty tissue.
  • the surface is smooth. There are no pathological changes at the macroscopic level. Spleen of dense uniform consistency, red-brown saturated color. Size saved.
  • the surface is smooth, the edges are even. There are no pathological changes at the macroscopic level.
  • the liver is slightly enlarged, red-brown to purple, the texture is dense, uniform.
  • the surface is smooth, the edges are even. On a section without bulging.
  • Organs of the chest cavity Localization preserved. Pathological changes in the structure were not detected. Light light pink, clean, without seals and neoplasms. Heart without pathologies, moderate blood supply.
  • Organs of the abdominal and pelvic cavities Localization preserved, swelling, no redness. Scanty or moderate deposition of fatty tissue.
  • Buds of dense uniform consistency red-brown without foci of necrosis on the surface and incision.
  • the surface is smooth. There are no pathological changes at the macroscopic level.
  • Spleen of dense uniform consistency red-brown saturated color.
  • the surface is smooth, the edges are even.
  • the liver is reddish brown. Size saved.
  • the consistency is dense, uniform.
  • the edges are even, smooth.
  • the surface is smooth. On a section without bulging.
  • Organs of the chest cavity Localization preserved. Pathological changes in the structure were not detected. Light light pink, clean, without seals and neoplasms. Heart without pathologies, moderate blood supply.
  • Organs of the abdominal and pelvic cavities Localization preserved, swelling, no redness. Scanty or moderate deposition of fatty tissue.
  • Buds of dense uniform consistency red-brown without foci of necrosis on the surface and incision.
  • the surface is smooth. There are no pathological changes at the macroscopic level.
  • Spleen of dense uniform consistency red-brown saturated color.
  • the surface is smooth, the edges are even.
  • the liver is reddish brown. Size saved.
  • the consistency is dense, uniform.
  • the edges are even, smooth.
  • the surface is smooth. On a section without bulging.
  • Group I (toxic hepatitis, Fig. 7 is a photograph of a section of a rat liver). Microscopy of the slices reveals an unclear, smeared lobular structure. Hepatic beams are visible only in 1/3 segments near the venule. Vessels of moderate blood supply. Tissue is infiltrated by macrophages and lymphocytes. Hepatocytes without clear boundaries, the cytoplasm is pale, low-grain, glycogen content is reduced. Fatty and vacuole right up to balloon dystrophy. The periportal areas are characterized by areas of necrosis and apoptosis.
  • Fig. 8 is a photograph of a section of a rat liver.
  • the lobed structure is moderately lubricated.
  • the vessels are blood-filled, the tissue is not infiltrated.
  • Hepatic beams are clearly visible.
  • the size of the hepatocytes is preserved, the boundaries are not clear.
  • the nuclei with nucleoli are clearly visible.
  • the cytoplasm is granular, moderately hyperchromic.
  • Group IV control, olive oil, figure 10 is a photograph of a slice of a rat liver. Microscopy revealed the preserved structure of the hepatic lobules and acini. The structure of the beams is not broken. Vessels of moderate blood supply, no infiltration. Hepatocytes are preserved, size is within normal limits, cell outlines are somewhat blurred. A round nucleus with nucleoli in the center of the cell is clearly visible. The cytoplasm is granular, rich in glycogen. The diameter of the sinusoids is 19-21 microns.
  • Group V (intact, 11 is a photograph of a section of a rat liver). The structure of the hepatic lobules and acini is preserved. Hepatic beams saved.
  • the toxicity test of the developed drug was carried out in accordance with the requirements for experimental (preclinical) study of new pharmacological substances.
  • the test was performed on white mice of the Balb / s line and white Wistar rats of both sexes.
  • the drugs were administered to animals in two ways: intragastrically (iv), intravenously (iv) in increasing doses according to Litchfield-Wilcoxon. To achieve large doses of the drug when administered orally, the animal was administered repeatedly to the animals at intervals of 20-30 minutes for 2-3 hours.
  • Table 15 shows the results of evaluating the survival of white Balb / s mice after a single intravenous administration of H-RGCS preparations.
  • Table 16 shows the survival data for white mice of the Balb / s line after a single intragastric administration of the preparations.
  • MO-1/1 MO-1/1 (s) in animals observed inhibition, stunning, decreased breathing and heart rate.
  • a dose of 800 mg / kg on the day after administration of sample MO-1/1, 1 male fell on day 4 after administration of sample MO-1/1, 1 male fell on day 3 after administration of sample MO-1/1 (c).
  • a dose of 800 mg / kg after the administration of the sample MO-1/1 and after the administration of the sample MO-l / l (c)
  • 2 mice died - one animal for 3-4 days.
  • samples of the N-RGKS preparation (samples MO-1/1, MO-l / l (c)) at a dose of 1000 mg / kg, 3 mice out of 10 subjects fell among males, 5 mice out of 10 subjects fell among females.
  • samples of the N-RGKS preparation (samples MO-1/1, MO-1/1 (s)) at a dose of 1200 mg / kg in animals, inhibition, lethargy, increasing phenomena of respiratory depression, lateral position, stiffness and death during first days after the introduction of samples MO-I and MO-l (c): among males, 5 out of 10, and among females, 6 out of 10. No seizures were observed.
  • the number of dead animals among females with the on / in the introduction of the drug N-RGKS exceeded the number of dead animals for doses of 800 mg / kg, 1000 mg / kg and 1200 mg / kg, respectively, per animal for each dose.
  • the toxicity of the powdered samples MO-7 and MO-7 (c) turned out to be less than the toxicity of the liquid samples MO-1/2 and MO-l / 2 (c) and. MO-1/1 and MO-l / l (c).
  • a toxicity test with iv administration of a liquid sample MO-1/2 showed that this sample has significantly greater ( ⁇ 2 times) toxicity than samples MO-1/1, MO-l / l (s).
  • Mortality after a single intravenous injection of a MO-1/2 liquid sample was observed at a dose of 300 mg / kg - 2 males out of 10 and 2 females respectively, and at a dose of 500 mg / kg, 3 males out of 10 and 4 females respectively out of 10, at a dose of 700 mg / kg - 7 out of 1 OB fell to the group of males and females.
  • mice of the control groups in / in the introduction of isotonic saline mortality was not observed for the entire observation period.
  • a comparative characteristic of the average toxic dose for iv administration of drugs to Balb / c mice shows that the LDso value is significantly higher than that of the comparison drug. Therefore, the acute toxicity of the developed samples is less than that of the "H-escent".
  • Table 17 shows the survival data of Wistar rats after a single intravenous administration of the N-RGCS sample MO-1/1.
  • Table 18 shows the survival data of Wistar rats after a single intravenous administration of the N-RHCS preparation, a powdered sample of MO-7.
  • a dose of 550 mg / kg on the 5th day after administration of the drug 1 male and 1 female fell, on the 7th day another 1 female. Subsequently, the general condition and behavior of the surviving experimental animals did not differ from the control groups. There were no gender differences during intoxication, but the mortality rate in females was slightly higher than in males.
  • lethargy, decreased feed intake, and increased water intake were noted.
  • At a dose of 1550 mg / kg on the 5th day after the administration of the drug 1 male and 1 female fell. Subsequently, the general condition and behavior of the surviving rats did not differ from the indices of the control group.
  • Intragastric administration After a single intragastric (i / f) administration to rats of solutions of the N-RGKS preparation, a sample of MO- 1/1 in increasing doses of 7.0 g / kg, 8.0 g / kg, 9.0 g / kg, 12 , 0 g / kg, 14 g / kg, 16.0 g / kg fatalities were observed only at doses greater than 8.0 g / kg.
  • Table 19 shows the survival data of Wistar rats after a single IV injection of the MO-1/1 sample.
  • the autopsy of the dead with iv administration of rats revealed, in addition to venous congestion of the internal organs, signs of dilatation of the stomach and paralytic expansion of the intestine, which is probably due to the large volume of the drug administered.
  • An autopsy of the rats who died after IV injection of the MO-7 sample revealed venous congestion of the internal organs, signs of gastric dilatation and paralytic expansion of the intestine, which could be associated with a large volume of the administered drug.
  • the value of the average effective hepatoprotective dose of the drug taking into account the prolonged action, may be significantly less than 5 g / kg, which indicates the possibility of a greater therapeutic breadth than the estimated value.

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Abstract

La présente invention vise à élaborer une préparation et des formes concrètes de celle-ci afin de traiter efficacement le virus de l'hépatite C par administration et localisation de doses thérapeutiquement efficaces de substances médicamenteuses dans l'organe cible principal (foie) et à l'intérieur de cellules hépatiques (hépatocytes). La préparation de cette invention comprend un complexe de composants actifs contenant du glutathione et de l'inosine dans un rapport de 1:1, ainsi que de la cisplatine en une quantité égale à 0,05 %, ledit complexe de composants actifs étant immobilisé par adsorption sur un support polymère se présentant sous la forme de nanoparticules de polylactide-glycolide. Dans une deuxième variante, le complexe de composants actifs contient uniquement du glutathione et de l'inosine dans un rapport de 1:1.
PCT/RU2009/000345 2008-07-09 2009-07-08 Forme médicamenteuse nanosomale d'une préparation à action prolongée servant au traitement de l'hépatite c (variantes) WO2010005344A1 (fr)

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EA200801819A EA014044B1 (ru) 2008-07-09 2008-07-09 Наносомальная лекарственная форма препарата пролонгированного действия для лечения гепатита с (варианты)
EA200801819 2008-07-09

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RU2506074C1 (ru) * 2013-01-25 2014-02-10 Николай Борисович Леонидов Антигипоксант и способ его получения
RU2508098C1 (ru) * 2013-01-25 2014-02-27 Николай Борисович Леонидов Противосудорожное средство и способ его получения

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RU2153351C2 (ru) * 1995-12-14 2000-07-27 Закрытое акционерное общество "ВАМ" Средство для регулирования эндогенной продукции цитокинов и гемопоэтических факторов (варианты) и способ его использования
WO2007110152A2 (fr) * 2006-03-24 2007-10-04 Lts Lohmann Therapie-Systeme Ag Nanoparticule de polylactide
RU2318513C1 (ru) * 2007-04-10 2008-03-10 Автономная некоммерческая организация "Институт молекулярной диагностики" (АНО "ИнМоДи") Лекарственное средство на основе d-циклосерина, препарат пролонгированного действия, содержащий наночастицы, способ его получения
WO2008041246A2 (fr) * 2006-10-05 2008-04-10 Panacea Biotec Ltd. Nouvelles compositions en dépôt injectables et leur procédé de fabrication
RU2327459C1 (ru) * 2007-06-26 2008-06-27 Евгений Сергеевич Северин Лекарственное средство противомикробного действия, способ получения лекарственного препарата направленного действия, содержащего наночастицы

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JP2004508420A (ja) * 2000-09-18 2004-03-18 アイデック　ファーマスーティカルズ　コーポレイション Ｂ細胞枯渇抗体／免疫調節性抗体の組合せを用いて自己免疫疾患を治療するための併用療法

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RU2153351C2 (ru) * 1995-12-14 2000-07-27 Закрытое акционерное общество "ВАМ" Средство для регулирования эндогенной продукции цитокинов и гемопоэтических факторов (варианты) и способ его использования
WO2007110152A2 (fr) * 2006-03-24 2007-10-04 Lts Lohmann Therapie-Systeme Ag Nanoparticule de polylactide
WO2008041246A2 (fr) * 2006-10-05 2008-04-10 Panacea Biotec Ltd. Nouvelles compositions en dépôt injectables et leur procédé de fabrication
RU2318513C1 (ru) * 2007-04-10 2008-03-10 Автономная некоммерческая организация "Институт молекулярной диагностики" (АНО "ИнМоДи") Лекарственное средство на основе d-циклосерина, препарат пролонгированного действия, содержащий наночастицы, способ его получения
RU2327459C1 (ru) * 2007-06-26 2008-06-27 Евгений Сергеевич Северин Лекарственное средство противомикробного действия, способ получения лекарственного препарата направленного действия, содержащего наночастицы

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