WO2012143502A1

WO2012143502A1 - Composition imitating intraarticular liquid

Info

Publication number: WO2012143502A1
Application number: PCT/EP2012/057257
Authority: WO
Inventors: Ara Arshavirovich Abramyan; Viacheslav Ivanovich Beklemyshev; Konstantin Vitalievich Filippov; Igor Ivanovich Makhonin; Umberto Orazio Giuseppe Maugeri; Vladimir Aleksandrovich Solodovnikov
Original assignee: Closed Stock Company "Institute Of Applied Nanotechnology"; Fondazione Salvatore Maugeri Clinica Del Lavoro E Della Riabilitazione; Sib Laboratories Limited
Priority date: 2011-04-21
Filing date: 2012-04-20
Publication date: 2012-10-26
Also published as: RU2473352C2; RU2011115535A

Abstract

The invention concerns new compositions which imitates intraarticular liquid and thus can be introduced into an articulate cavity for replacement or correction of an intraarticular liquid lacking or pathologically modified. The new compositions consist essentially of water solutions of polyvinylpirrolidone and of an additive having antimicrobic activity, constituted by silicon nanoparticles modified with silver. The rheological properties of the new compositions can be improved by adding a water solution of sodium salt of hyaluronic acid.

Description

COMPOSITION IMITATING INTRAARTICULAR LIQUID

Field of the invention

The invention concerns pharmacology and medicine and concerns a composition imitating the intraarticular liquid (synovia) as well as a method for obtaining an additive suitable to improve the properties of this liquid, which is useful for preventive maintenance and treatment of degenerative-dystrophic lesions of large joints (knee and elbow), and articulate cartilages in case of osteoarthritis and rheumatoid arthritis. The new composition is integrated into an articulate cavity for replacing or correcting the intraarticular liquid in case of its pathological changes.

With osteoarthritis and rheumatoid arthritis, a pathological change takes place of parameters of intraarticular liquid, i.e. amount, biochemical and cytological composition, rheological and greasing properties so as protective and recycling properties.

State of the art

For preventive maintenance, correction and treatment of degenerative-dystrophic lesions, various compositions have been described, imitating the properties of the intraarticular liquid (synovia) and used by integration into joints for improvement of rheological, greasing and other properties affected by pathological changes of intraarticular liquid.

A composition containing 15% water solution of middle molecular weight polyvinylpirrolidone is known in applied medicine. This composition is suitable for injection and is a temporary substitute of intraarticular liquid in case of diseases of joints (arthrosis) (see R.Hasan, V.Shibalkin "Experience of treatment of patients with deforming osteoarthrosis of the knee, with the help of polyvinylpirrolidone." Rheumatology - 1985, N. 3, p. 40-43; A.Matulis, I.Dadonene, R.Grigene. "New chondroprotector- polyvinylpirrolidone for treatment of patients with osteoarthritis." Therapeutic archive; 1990, v.62.5, p. 95-98).

However, since a 15% solution of polyvinylpirrolidone possesses determined greasing, immunoregulatory and exchange properties in an articulate cavity, it does not display the needed rheological and thixotropic properties at dynamic loadings, in a required extent (see "Artificial synovia for intraarticular treatment of rheumatoid arthritis and osteoarthrosis" (Development, a clinical-experimental and biochemical substantiation). Therapeutic archive - 1984, N. 11, p. 73-77). Moreover the composition does not possess antimicrobic properties against endogenous infections.

Compositions containing water solutions of biopolymeric components as hyaluronan (hyaluronic acid) or derivatives thereof are described in the specification of patents (see patents: RU No. 2268052, published in 2006; RU No. 2394552, pub. 2010; RU No. 2197238, published in 2003; US No. 7485629, published in 2009; application RU No. 2007141864, published in 2009). These compositions are suitable for integration into an articulate cavity, for treatment of inflammatory and degenerative diseases of joints (arthritis, arthrosis).

Biopolymeric components as hyaluronan (nonsulphated glycosaminoglycans), derivatives of hyaluronan (for example, solutions of sodium salt of hyaluronic acid) concern the components-analogues present in the biological environment of natural intraarticular liquid (synovia). Their presence promotes improvement of greasing, rheological, chondroprotective, and anti-inflammatory properties of intraarticular liquid.

However hyaluronan and derivatives of hyaluronan may cause allergic reactions in case of exacerbation of osteoarthrosis and arthritis of inflammatory genesis. Compositions based on them need injection of additional additives such as preservatives for their stabilization and compounds for preventing development of microorganisms (see: patent RU N. 2268052, and the book of S.Ermakov, V.Rodnenkov, et al. "Liquid crystals in technics and medicine." Minsk. "Asar", 2002, p. 331-334).

Compositions simulating intraarticular liquid (synovia), containing sodium carboxymethylcellulose (NaCMC), esters of cholesterol and gemodez are described in technical specifications (see patents: RU No. 2002461, pub. 1993; RU No. 2007173, published in 1994; RU No. 20331621, published in 1995).

The presence of such components as gemodez and esters of cholesterol allows imitation of the structure of the intraarticular liquid which contains the compounds of cholesterol, able to promote decrease in friction in a joint, due to their orientation to be adsorbed on a microrelief of cartilage in direction of sliding of the articulate surfaces (see: I.Kupchinov, S.Ermakov et al. Orthopedics and traumatology - 1989, No. 10, p. 7-11).

The presence in known compositions of NaCMC (as thickener and softener) promotes improvement of pheological properties of compositions. However all the given compositions do not possess antimicrobic activity in relation to endogenous infections and also expressed anti-inflammatory and reparative properties.

Besides, the considered available art testifies, as a whole, that when reproducing the composition of intraarticular liquid, the experts imitated its properties using components without the necessary consideration of their biocompatibility. However development and expansion of means containing biocompatible as well as functionally and biologically active components of intraarticular liquid, is clearly indicated as important in modern medicine.

Summary of the invention

One of the objects of the present invention was the provision of improved technical results in the development of a biologically compatible composition, imitating intraarticular liquid, effective on rheological properties and antimicrobic activity and in promoting regeneration of cartilage.

The composition containing 15% water solution of middle molecular weight polyvinylpirrolidone, used as a substitute of intraarticular liquid in case of diseases of joints (arthrosis), is chosen as the closest prior art for the solution of the put technical problem, regarding development of a composition imitating intraarticular liquid (see: R.Hasan, V.Shibalkin. "Experience of treatment of patients with deforming osteoarthrosis of the knee with the help of polyvinylpirrolidone." Rheumatology - 1985, No. 3, p. 40-43; A.Matulis, I.Dadonene, R.Grigene. "New chondroprotector-polyvinylpirrolidone for treatment of patients with osteoarthritis." Therapeutic archive - 1990, v. 62.5, p. 95-98).

As it was marked above, said known composition is inefficient on the rheological and thixotropic properties and does not possess antimicrobic activity.

In view of these ascertained facts, one aspect of the present invention was the creation of a biologically compatible additive, effectively influencing the rheological and thixotropic properties of the composition, promoting improvement of reparative (regenerating) properties of cartilage and giving antimicrobic activity to the composition imitating the intraarticular liquid.

Biological compatibility and applicability in pharmacological and medical practice of the following materials, were taken into account in the search of a suitable additive for the given composition: 1) nanoparticles of metals of antimicrobial action (Ag, Zn, Cu, Au, Pt, Sn, Sb) in various solutions, including in solutions of polymers for medical purposes (for example, polyvinylpirrolidone); (see for example: patent RU No. 2131269 (WO 94046003/14), published on 10 June 1999; medical preparations «ARGONICA», «ARGOVIT»);

2) coatings with nanostructured porous polysilicon on the surface of implants, promoting regeneration of bonds and cartilage (see for example: US patent No. 7186267, published on 3 June 2007; US application No. 2004/000313, published on 15 January 2004);

Biocompatibility of porous polysilicon, presence of adhesion to cellular structures, bones and formation of calcium-phosphatic bonds on its surface, absence of cytotoxicity, is confirmed by research works, including the following: S.C.Bayliss, P.J.Harris and others "Phosphate and cell growth on nanostructured semiconductors." Journal of Materials Science Letters - 1997: 16:737-740; Yangyang Lu, Fan Yang, Linto Cai. "Osteoblast adhesion on porous silicon." Bulleten of Advanced Technology Research - 2009, Jan., Vol.3, No.l, 25-28; S.D.Alvarez, A.M.Defrus and others. "The compatibility of hepatocytes with chemically modified porous silicon with reference to in vitro biosensors." Biomaterials - 2009:30:26-34;

3) silicon nanoparticles (see for example: patent WO 2010/096733 A2, pub. 20.02.2010. "Luminescent porous silicon nanoparticles, method of making and using same"; application RU No. 2003137823, published 27.03.2005. "Skeletons for engineering bones of a human being, methods of their manufacturing and their application".

Silicon nanoparticles, as well as films of nanostructured porous polysilicon, show effect of biocompatibility with cellular structures; (see: L.A.Osminkina, E.N.Lukjanova, M.B.Gongalsky et al. "Influence of nanostructured silicon on processes of proliferation of stem and cancer cells." Bulletin of experimental biology and medicine - 2011. Volume 151, N. 1, p. 91-95; WO 2010/096733 A2, published on 20 February 2010. "Luminescent porous silicon nanoparticles method of making and using same".

The process of obtaining silicon nanoparticles by laser pyrolysis of silicomethane (SiH₄), with dissociation of silicomethane molecules at heating its jet with radiation of the CW C0₂-laser (laser-induced chemical vapour deposition - LICVD) is the most preferable. Thus the jet is surrounded with a stream of buffer gas (see: N.N.Kononov, G.P.Kuzmin, A.H. Orlov et al. "Optical and electric properties of the thin plates made from nanocrystal powders of silicon." Physics and technics of semiconductors - 2005. Volume 39, 7, p. 868- 873; A.G.Vladimir, S.B.Korovin, V.I.Pustovoj. "A luminescence of silicon nanoparticles General Physics Institute named A.M.Prokhorov of the Russian Academy of Science. The collection of theses of reports of the Third International Forum on Nanotechnologies RUSNANOTECH. Moscow. 2008).

The main feature of the given process is to obtain silicon nanoparticles with distribution of the sizes of nanoparticles in the range of 10-40 nm. That is optimal according to the hygienic specifications establishing allowable ranges of the used sizes of nanoparticles.

Taking into account the known available art, this technical disclosure is chosen as the closest precedent to the choice and creation of a biocompatible additive improving properties of intraarticular liquid (see: N.N.Kononov, G.P.Kuzmin, A.H. Orlov et al. "Optical and electric properties of the thin plates made from nanocrystal powders of silicon." Physics and technics of semiconductors. 2005. Volume 39, 7, p. 868-873; A.G.Vladimir, S.B.Korovin, V.I.Pustovoj. "A luminescence of silicon nanoparticles." General Physics Institute named A.M.Prokhorov of the Russian Academy of Science. The collection of theses of reports of the Third International Forum on Nanotechnologies RUSNANOTECH. Moscow. 2008). The considered technical disclosure consists in obtaining silicon nanoparticles in the size of 10-40 nm by laser pyrolysis of silicomethane (SiH₄).

The composition imitating intraarticular liquid, containing 15% water solution of middle molecular weight polyvinylpirrolidone is described as a solution of the put technical problem regarding development of a composition. Thus the solution contains (0,5-1,5)· 10^" % by weight (b.w.) of the additive with antimicrobic properties on the basis of silicon nanoparticles, modified with silver, at a size of the nanoparticles of 10-40 nm and with an amount of silver in the additive of 0,8-1,1 % b.w..

According to the present invention, the composition imitating intraarticular liquid contains in addition a 0,05-2,5% water solution of sodium salt of hyaluronic acid in a ratio by weight of water solution of polyvinylpirrolidone : water solution of sodium salt of hyaluronic acid, as 1 : (0,2-0,3).

Sodium salt of hyaluronic acid with molecular weight of 0,5-1,8 MDa, obtained by biological fermentation, is used in the composition according to the present invention. According to the present invention the composition contains 15% water solution of polyvinylpirrolidone with molecular weight 35000 + 5000 Da.

The method of obtaining the additive for the composition imitating intraarticular liquid, consisting in obtaining silicon nanoparticles in the size of 10-40 nm by laser pyrolysis of silicomethane (SiH₄), was described as a solution of the put technical problem. Thus a surface of silicon nanoparticles was modified by its processing in a 2-5% solution of silver nitrate in deionized water at the weight ratio, solution: nanoparticles, as 1:0,02, with a subsequent centrifugation of the solution and repeated washings of the deposit in deionized water with centrifugation at each step of washing.

According to the present invention the repeated washings of the deposit with deionized water was carried out at initial specific resistance of water not below 10 MOm-cm and up to obtaining a specific resistance not below 1 MOm-cm.

According to the present invention processing silicon nanoparticles in a water solution of silver nitrate was carried out within 12-36 hours.

At realization of the present invention a biologically compatible composition imitating intraarticular liquid with effective rheological properties, possessing antimicrobic activity and promoting reparative action on cartilage was obtained. This result is explained as follows:

- application of a biologically compatible polymeric agent widely used in medical practice, including for intraarticular liquids, i.e. polyvinylpirrolidone;

- application of a biocompatible additive on the basis of silicon nanoparticles, whose surface is modified by silver and which confer to the composition effective antimicrobic properties of prolonged action. Moreover the rheological properties and the reparative activity on cartilage are improved;

- presence of polyvinylpirrolidone (PVP) and sodium salt of hyaluronic acid

(NaHA), forming tridimensional mesh structure of a polymeric complex, owing to formation of bonds between carbonyl groups (C=0) in monomer units of PVP and cations of sodium in monomer units of NaHA. This additive promotes improvement of rheological properties of the composition;

- presence of synergically compatible polymeric agents on the basis of polyvinylpirrolidone and sodium salt of hyaluronic acid, forming polymeric electrolytic complexes, which are adsorbed on the surface of the silicon nanoparticles. That leads to structuring a polymeric matrix and to improve the rheological and thixotropic properties of the composition. In result efficiency of interaction of intraarticular liquid with the cartilage of an articulate cavity increases.

Analysis of the available art did not reveal technical solutions with a set of the characteristics corresponding to the declared technical solution and providing results as described above.

The analysis of available art testifies the conformity of the declared technical solution to criteria of "novelty" and "degree of inventiveness".

The declared technical solution can be industrially realized by using known technological processes as well as equipments and materials used in pharmacology and medicine.

Brief description of the figure

The invention is explained by in detail as follows:

- Figure 1 is the photo of silicon nanoparticles, obtained with use of a scanning electronic microscope (SEM);

Detailed description of the invention

The following equipment and materials, known in the industry, are used for realization of the invention:

- 15% water solution of polyvinylpirrolidone of medium MW and preferably with molecular weight 35000+5000 Da; the given solution is used for injection as an artificial substitute of intraarticular liquid (synovia) in case of diseases of joints accompanied by its shortage in an articular cavity;

- sodium salt of hyaluronic acid ([(C₁₄H₂oNOn)Na]_n) with molecular weight 0,5-1,8 MDa. It is obtained by the method of bacterial (cellular) fermentation which is optimal for therapeutic effect, due to the absence in the preparation of allogenic proteins, in particular animal proteins. The specified molecular weight is optimal for medical action as it is proved by testing compositions of intraarticular liquids: "Ostenil" (TRB Chemedica AG, Germany, <http://www.trbchemedica.com>; <http://www.ostenil.ru/>) and "Synocrom" (Croma Pharma GmbH, Austria, <http://www.croma.at>; <http://www.synocrom.com.ua/croma-pharma.htm>) containing sodium salts of hyaluronic acid. Sodium hyaluronate with molecular weight 1,6 MDa is preferably used for realization of the invention. A 0,05-2,5% solution of sodium salt of hyaluronic acid in deionized water is used for obtaining the composition imitating the intraarticular liquid. A solution of sodium salt of hyaluronic acid in deionized water is obtained by mixing, preferably at temperatures of 40-50°C.

Materials used in the composition according to the invention:

- water solutions of polyvinylpirrolidone and sodium salts of hyaluronic acid in a weight ratio as 1 : (0,2-0,3). This ratio is the optimum for obtaining a physiologically active composition imitating intraarticular liquid, as it is proved by the results of the researches reported hereinafter.

- the additive with antimicrobic properties on the basis of silicon nanoparticles modified with silver (Ag) with a size of nanoparticles of 10-40 nm.

The following steps of process are used for obtaining the given additive:

1st step - obtaining silicon nanoparticles.

For realization of the given step it was used the technology of synthesis described in the following works: N.N.Kononov, G.P.Kuzmin, A.H. Orlov. "Optical and electric properties of the thin plates made from nanocrystal powders of silicon." Physics and technics of semiconductors - 2005. Volume 39, 7, p. 868-873; A.G.Vladimir, S.B.Korovin, V.I.Pustovoj. "Luminescence of silicon nanoparticles." General Physics Institute named A.M.Prokhorov of the Russian Academy of Science. The collection of theses of reports of the Third International Forum on Nanotechnologies RUSNANOTECH. Moscow. 2008).

Synthesis of silicon nanoparticles is carried out through laser pyrolysis of silicomethane (SiH₄). The process is carried out in a flowing reactor by heating a jet of silicomethane with radiation of the WC C0₂-laser (laser-induced chemical vapour deposition - LICVD). A jet of silicomethane is surrounded with a stream of inert buffer gas (for example Ar). Parameters of power of C0₂-laser, speeds of streams of buffer gas and silicomethane (SiH₄) are adjusted for obtaining nanoparticles with the specified parameters of the size.

Specifically the process for obtaining silicon nanoparticles is carried out at the following parameters of synthesis (pyrolysis) of silicomethane: speed of the stream of silicomethane - 0,2 1/min; speed of the stream of buffer gas - 12,5 1/min; temperature - 650°C; pressure in the chamber 200 Torr; power of the C0₂-laser - 600 W. Main feature of the given process is to obtain silicon nanoparticles with distribution of the sizes in a range of 10-40 nm. This result was proved with a test using a scanning electronic microscope (see Fig. 1).

2nd step - modification of the surface of silicon nanoparticles.

The given step is carried out with the following sequence of operations::

1. A solution of silver nitrate (AgN0₃) in deionized water is used. Ion-exchange properties of the given salt in solution are widely used in medical practice for obtaining preparations with antimicrobic properties.

The silicon nanoparticles obtained in the first step are processed in a water solution of silver nitrate (AgN0 ).

Silver recovery, at silicon thermodynamic balance between two redox-couples, is determined by the difference of their standard potentials. Thus the following procedure is carried out: the conjugate pair with rather high (more positive) potential connects electrons from the conjugate pair with rather low (more negative) potential. The conjugate pair for Ag, at recovery of silicon nanoparticles on a surface, will be : pair : SiH_x - (4-x)e = Si + (4- x)H⁺. The potential of the given electrode reaction makes 0,148 V. Value of potential is determined starting from value of energy of activation of thermal desorption SiH_x-groups from the silicon surface. Such value of potential allows carrying out processes of deposition of other substances, whose potential of recovery is higher than 0,148 V. Silver is recovered in water solutions: Ag⁺ + e^~ = Agcp°. Standard electrode potential of Ag is 0,799 V (see V.A.Rabinovich et al. «The brief chemical reference*. Edition 3. Leningrad. "Chemistry". Leningrad branch, 1991 p. 353, 363).

According to the invention a 2-5% water solution of silver nitrate is used in a weight ratio of a 1 : 0,02 with respect to a solution of modified nanoparticles, by processing said nanoparticles within 12-36 hours so that the deposition of zero-valent silver on the surface of silicon nanoparticles is realized.

The parameters of water solution of silver nitrate, the quantitative ratio between said solution and nanoparticles and the modalities of processing silicon nanoparticles in said solution of AgN0₃ as specified, are the optimum to realize the present invention.. Change of the specified technological modalities and parameters will lead to reduction of the quantitative content of silver on the modified surface of silicon nanoparticles (what decreases antimicrobic activity of intraarticular liquid), or to increase of the quantitative content of silver on the surface of silicon nanoparticles (what worsens biocompatibility of nanoparticles).

2. Centrifugation of the suspension of the silicon nanoparticles in the water solution is carried out. The suspension is separated in components with the help of multipurpose centrifuge Thermo Scientific SL40, with a maximal speed of 15200 rev/min. A solution of silver nitrate is merged and a deposit of silicon nanoparticles is obtained.

3. The deposit of silicon nanoparticles was repeatedly washed out with deionized water, with centrifugation at each step of washing.

Deionized water at initial specific resistance not below lOMOm-cm, according to OST 11 029.003-80 "Products of electronic technics." is used. Marks, technical requirements, methods of cleaning and control, was used for washing.

The process of washing and centrifugation comes to the end at a specific resistance of water not below 1 MOm-cm. The block of control BKVR-24-003 is used for the control of the specific resistance of water.

In result silicon nanoparticles, which surface is modified by silver deposited on it, are obtained.

The process of centrifugation as realizated according to step 2 is optimal in the technology of processing and obtaining nanoparticles from water solutions.

Specifically the following procedure was used for realization of the given step: A 3% solution of silver nitrate (AgN0₃) in deionized water, in amount of 50 ml per

1 g of silicon nanoparticles is processed in a suspension over 24 hours; the suspension of the silicon nanoparticles after their processing in a water solution of silver nitrate is centrifuged and subjected to repeated washings and centrifugations.

In result a deposit of silicon nanoparticles in amount of 0,98 is obtained. The silicon nanoparticles obtained were tested for determinating the amount of silver in them.

The method of the quantitative analysis based on measurement of volume or weight of a reagent required for reaction with researched substance, i.e. the titrimetric analysis, was used for researches. The titrimetric analysis for determination of the amount of silver was carried out with use of the indicators fixing a point of equivalence of titration.

The following reagents were used during the titrimetric analysis for definition of % b.w. content of silver: a mix of sulfuric and nitric acids; titrant on the basis of a solution of ammonium thiocyanate (or potassium); as the indicator - a solution of ferrum-ammonium alums.

It was established as a result of the carried out researches that there was 0,9 % b.w. of silver (9,0- 10^" g) in tested amount of silicon nanoparticles.

The silicon nanoparticles obtained at the second step whose surface was modified with silver, were used for obtaining the composition imitating intraarticular liquid.

15% water solution of polyvinylpirrolidone of middle molecular weight, and preferably with a molecular weight of 35000+5000 Da and silicon nanoparticles obtained in the step 2, were used for preparation of the compositions according to the invention.

Slurryfication of components was carried out with use of ultrasonic processing.

(0,5- 1,5) -10^" % b.w. of the additive with antimicrobic properties, on the basis of silicon nanoparticles modified by silver with a size of nanoparticles of 10-40 nm and a content of silver in silicon nanoparticles of 0,8-1,1 % b.w., were added in 15% water solution of middle molecular weight polyvinylpirrolidone for obtaining the compositions according to the invention.

The contents of the additive with antimicrobic properties on the basis of silicon nanoparticles modified by silver with the specified size of nanoparticles and an amount of silver in silicon nanoparticles as specified in the invention are optimum and also correspond to hygienic specifications on concentration of nanoparticles in water solutions (GN 1.2.2633-10. Hygienic specifications of the contents of priority nanomaterials in objects of an environment. It is authorized 25.05.2010 by the Main state health Officer of the Russian Federation).

Increase or decrease in the specified parameters will lead to deterioration of antimicrobic activity of the composition or to decrease in a level of its biocompatibility with tissues in joints, or to increase in costs for obtaining the composition.

The quantitative contents of the additive on the basis of silicon nanoparticles modified by silver, with the specified size of nanoparticles and the amount of silver in silicon nanoparticles as specified in the invention are the optimum for creation of compositions possessing effective rheological and thixotropic properties.

The following compositions imitating intraarticular liquid were prepared:

Example 1 (according to the invention) The composition contains 10 ml of 15% water solution of polyvinylpirrolidone with molecular weight 35000 MDa and 1,5-10^" % b.w. of silicon nanoparticles modified with silver, with the size of nanoparticles of 10-40 nm and an amount of silver in silicon nanoparticles of 0,9 % b.w..

The suspension was mixed with use of ultrasonic processing.

Example 2 (according to the invention)

The composition contains 8 ml of 15% water solution of polyvinylpirrolidone with molecular weight 35000 MDa, 2 ml of 0,1% water solution of sodium salt of hyaluronic acid obtained by biological fermentation and with molecular weight 1,6 MDa and 1,0- 10^" weight % of silicon nanoparticles modified with silver with the size of nanoparticles of 10- 40 nm and an amount of silver in silicon nanoparticles of 0,9 % b.w..

Water solutions of polyvinylpirrolidone and sodium salt of hyaluronic acid were preliminary mixed with use of ultrasonic processing, before injection of silicon nanoparticles in them.

Example 3 (control)

The composition contains 10 ml of 15% water solution of polyvinylpirrolidone with molecular weight 35000 MDa.

Example 4 (control)

The composition contains 8 ml of 15% water solution of polyvinylpirrolidone with molecular weight 35000 MDa, 2 ml of 0,1% water solution of sodium salt of hyaluronic acid obtained by biological fermentation and with molecular weight 1,6 MDa.

Compositions according to the Examples 1-4 were tested for estimation of their rheological and antimicrobial properties.

Estimation of antimicrobic properties of the compositions according to the Examples 1-4 was carried out on the basis of the disk-diffusion method, with use of Methodical instructions (MUK 4.2.1890-04) «Determination of sensitivity of microorganisms to antibacterial preparations*, authorized on 4 March 2004 by the Main state health officer of the Russian Federation (Ministry of Health of the Russian Federation).

The disk-diffusion method (DDM) for determination of sensitivity of microorganisms (bacteria and funguses) is based on ability of the antimicrobic agents included in the composition, to diffuse in nutrient mediums from the cardboard (paper) disks impregnated with them, preventing growth of the microorganisms sown on a surface of nutrient medium.

Inflammatory affection of joints is characterized by the specific changes revealed at physical, chemical, biochemical and cytological tests of synovial effusion (synovia), obtained through a puncture of a cavity of a joint. Synovial effusion contains microflora and it can contain various endogenous infections. Color and transparency of synovial effusion depend on the contents of pathological impurities and their nature. Muddy-yellow effusion named serous is characteristic for the majority of arthritises. Muddy- white effusion, sometimes with a sage-green stint, flakes and a bloody impurity, indicates usually its purulent character and is a typical attribute of an acute bacterial arthritis.

In view of what above said, the following strains of kinds of bacteria were used as test-microorganisms for estimation of antimicrobic activity of the compositions according to the Examples 1-4: Staphylococcus aureus ATCC 25923; Staphylococcus aureus ATCC 43300 MRSA; Pseudomonas aeruginosa.

Bacteria of kind Staphylococcus aureus are one of the most resistant representatives of gram-positive microflora of a human being and are activators of pustular infections.

Bacteria of kind Pseudomonas aeruginosa are one of the most resistant representatives of gram-negative microflora; they possess high stability to physical and chemical factors. They frequently show stability to many medicinal and to disinfectants.

A trypcase-soy agar was used as a dense nutrient medium for carrying out tests.

Preparation of Petri dishes with a dense nutrient medium was carried out according to the instructions for the manufacturer of a nutrient medium, from methodical instructions MUK 4.2.1890-04.

Standard disks with a diameter of 6 mm, from a special filtering cardboard (paper), were used as diagnostic disks.

Disks were exposed to unitary processing (impregnation) with the tested compositions (Examples 1-4).

Suspensions (inoculums) of each test-culture of bacteria in sterile physiological solutions were prepared, with density (turbidity) 0,5-0,6 according to the standard of McFarland. Those suspensions contained nearly (1,5-2,0)· 10 CFU/ml (colony forming units/ml). Inoculums (within 15 minutes after preparation) were applied for inoculation of dense nutrient mediums in Petri dishes as mentioned above.

Inoculums were rendered on a surface of nutrient medium, in volume of 1-3 ml, by a pipette and were allocated on the surface at regular intervals. Slightly opened Petri dishes were dried at room temperature within 15-20 minutes.

After inoculation the disks processed with the tested compositions (Examples 1-4) were placed on the surface of a nutrient medium (with the help of a sterile tweezers). Uniform and dense contact of disks to a surface of environment was provided.

Petri dishes were placed in a thermostat directly after application of disks and then were incubated at a temperature of 25-35°C, during 18-72 hours. Measurements of diameter of zones of growth inhibition of test- microorganisms (around of disks), accurate within 1 mm, were carried out after incubation. Zones of full suppression of seen growth were chosen as landmarks during measurements.

Testing the disks with tested compositions (Examples 1-4) was repeated three times for each test-microorganism, to increase reliability of tests; average value of diameter of zone of growth inhibition was estimated.

As a result of the tests what follows was established:

- Diameters of zones of growth inhibition of test-microorganisms around of the disks processed with the compositions according to the Examples 1 and 2 were respectively 17 mm and 15 mm;

- Diameters of a zone of growth inhibition of test-microorganisms around of the disks processed with the compositions according to the Examples 3 and 4 were respectively 2 mm and 3 mm.

Measurements of rheological characteristics of the samples of the compositions imitating intraarticular liquid, were carried out with use of rotational viscometer Rheotest RN4.1 (manufacturer - PKG "GRANAT") intended for quality assurance in manufacture and laboratory researches of products: oils, pastes, solutions, suspensions, emulsions.

Measurements of dynamic viscosity of samples of the compositions were carried out according to the Examples 1-3.

Measurements of dynamic viscosity (mPas) were carried out at temperature of 25°C of the samples of the composition, at speeds (gradients) of shift: 1,0 c^"1; 10 c^"1; 100 c^"1 . The results of measurements of dynamic viscosity of the samples of the composition are submitted in Table 1.

Table 1

Rheological properties of the samples of the compositions imitating intraarticular liquid

The results of measurements testify that addition of silicon nanoparticles (according to Example 1) and silicon nanoparticles and a water solution of sodium salt of hyaluronic acid (according to Example 2) in the composition imitating intraarticular liquid, increases (improves) dynamic viscosity and thixotropic properties of the composition, with respect to native intraarticular liquid (synovia).

The values determined for dynamic viscosity of samples of the compositions, correspond to the range of acceptable values of dynamic viscosity of native intraarticular liquid (synovia).

According to biological and rheological tests performed, dynamic viscosity of native intraarticular liquid (synovia) can be in a wide range of values - from 10 mPas up to 1000 mPas- depending on the condition of the joint of the patient, loadings, the gradient of shift and chemical and biological factors (see: for example: Dan Mazzucco, Gareth McKinley et al.. "Rheology of joint fluid in total knee arthroplasty patients." Journal of Orthopedic Research - 2002:20: 1157-1163; Schurz J., Ribitsch V. "Rheology of synovial fluid". Biorheology - 1987: 24:385-399; Fama H., Bryant JT., Kontopoulou M. "Rheological properties of synovial fluids." Biorheology - 2007:44:59-74; Bryan Preston Conrad, "The effects of glucosamine and chondroitin on the viscosity of synovia".

Thus, the researches carried out confirm, as a whole, that the compositions imitating intraarticular liquid described in the invention posses biocompatible properties; moreover they have effective rheological properties and posses antimicrobic activity.

The utility of using the additive on the basis of silicon nanoparticles, in the compositions according to the invention, is proved by the results of the researches specified in the description.

Claims

1. Compositions imitating intraarticular liquid, based on a 15% water solution of middle molecular weight polyvinylpirrolidone, characterized in that it contains (0,5-1,5) · 10- 3 % b.w. of an additive with antimicrobic properties essentially consisting of silicon nanoparticles having a size of 10-40 nm, modified with silver.

2. Compositions according to claim 1, wherein the polyvinylpirrolidone has a molecular weight of 35.000+5.000 Da.

3. Compositions according to claim 1, wherein the amount of silver which modifies the silicon nanoparticles is of 0,8 - 1,1% b.w. with respect to the nanoparticles.

4. Compositions according to claim 1, further containing a solution at 0,05-2,5% b.w. of sodium salt of hyaluronic acid in deionized water, the ratio between the solution of polyvinylpirrolidone and the solution of sodium salt of hyaluronic acid being equal to 1 : (0,2 - 0,3) in parts b.w..

5. Compositions according to claim 4, wherein the sodium salt of hyaluronic acid is obtained by biological fermentation with a molecular weight of 0,5 - 1,8 MDa.

6. Method for the preparation of the additive with antimicrobic properties to be added to a solution of polyvinylpirrolidone according to claim 1, characterized in that silicon nanoparticles in the size of 10-40 nm are obtained by laser pyrolysis of silicomethane (SiH₄), then the surface of the nanoparticles is modified by treatment with a 2-5% solution of silver nitrate in deiniozed water, with a ratio silver solution: nanoparticles equal to 1 : 0,002, followed by centrifugation and washing with deionized water of the decanted solid.

7. Method according to claim 6, wherein the treatment of the silicon nanoparticles with the solution of silver nitrate is performed over 12-36 hours.

8. Method according to claim 6, wherein the washing of the decanted solid with deionized water is performed initially with deionized water having a specific resistance not below 10 MOm-cm, and is repeated until a specific resistance of washing water not below 1 MOm-cm is obtained.

9. Additive with antimicrobic properties to be added to a composition imitating intraarticular liquid based on a 15% water solution of middle molecular weight polyvinylpirrolidone, consisting of silicon nanoparticles having a size of 10-40nm, modified with silver. Additive to be added to compositions imitating intraarticular liquid, consisting of a solution of polyvinylpirrolidone of middle molecular weight and of silicon nanoparticles modified with silver, in order to confer to the compositions improved rheological. properties, which additive consists of a water solution of sodium salt of hyaluronic acid obtained by biological fermentation with molecular weight of 0,5 - 1,8 MDa.