Gel of hyaluronic acid for intracutaneous injection

FIELD: medicine.

SUBSTANCE: invention relates to medicine. Described is implant, which can be injected in subcutaneous or intracutaneous way in form of monophase hydrogel, which contains gel, obtained from cross-linked hyaluronic acid and one of its physiologically acceptable salts.

EFFECT: obtaining subcutaneous implant used for filling wrinkles and stimulation of epidermal cells and/or supporting mechanical properties of skin density and elasticity.

15 cl, 2 ex

 

The present invention relates to implants based on hyaluronic acid, which can be injected subcutaneously or intradermally.

Hyaluronic acid (ha) in its acid form or in salt form (hyaluronate) is a major component of the extracellular matrix. She, in particular, presents in the connective tissues, called "soft", in contrast to other glycosaminoglycans, such as chondroitinase acid present in the tissues, called "hard", such as cartilage. Thus, it is found in large quantities in the skin.

HK is a linear desulfation of glycosaminoglycan consisting of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine (Tammi R., Agren U.M., Tuhkanen A.L, Tammi M. Hyaluronan metabolism in skin. Progress in Histochemistry &Cytochemistry. 29(2): 1-81, 1994).

In normal skin, ha is synthesized mainly dermal fibroblasts and epidermal keratinocytes (Tammi R., CIT. above). Due to the negatively charged residues Ledger plays the role of a water pump that allows the maintenance of skin elasticity. Ledger plays a major role in the regulation of diffusion of nutrients, hormones, vitamins and inorganic salts of connective tissue and excretion of waste products of metabolism, capable of induction of inflammatory reactions. With age, the number of GC and the degree of polymeri the emission decreases, resulting in reduction of the amount of water held in the connective tissue. Then, the skin undergoes the aging process, which leads to increased fibrosis and drop the content of elastic fibers. In normal human skin Ledger exists in the form of high molecular weight polymer (600000-1000000 Yes). Physiological dissolution of ha in the skin occurs through (i) internalization by keratinocytes and (ii) intracellular fragmentation of hyaluronidase fragments of intermediate size (600000-300000 Yes). Fragmented Ledger is released by keratinocytes, crosses the basal membrane and is released directly into the lymphatic vessels (Tammi R. et al., CIT. above).

The role of hyaluronic acid (ha) in dermatology is well known in various fields, including wound healing and hydration. Hyaluronic acid is often operates through its interactions with binding proteins and, in particular, with the transmembrane receptor CD44 (Tool V.R. 2001, Sem. Cell. Devel. Biol. 12: 79-87, Liao Y-H., A.J. Stuart, Drug Delivery, 12: 327-342, 2005). Activation of this receptor is expressed through its role in morphogenesis, reproduction and cell proliferation, angiogenesis and cell migration (G.Weindl, M.Schaller, Skin Pharm. Physiol. 2004; 17; 207-213). Data in the literature suggest that these different actions are a function of a cellular environment, molecular mA the son of hyaluronic acid and its concentration. For example, higher molecular weight inhibit angiogenesis, whereas oligosaccharides stimulate it.

In Japanese patent application JP 11279042 demonstrated appropriate action sulfated fragments of hyaluronic acid (PGA) (molecular weight of between 1 and 50 kDa), which could give the opportunity to maintain skin elasticity and prevent keratinization.

In a recent study conducted on fragments of hyaluronic acid (molecular weight between 50 and 750 kDa), respectively, demonstrated increased synthesis of hyaluronic acid by keratinocytes (patent application FR 2865651, WO 2005 082327). This proliferative activity induced PGA exact molecular masses, so this activity is not apparent at higher or lower molecular masses.

In the field of cosmetology, there are various injectable preparations based on hyaluronic acid. In mesotherapy apply solutions of vitamins, antioxidants, inorganic salts or hyaluronic acid. Vitamins are present to stimulate and maintain cellular metabolism and, thus, increase the production of collagen, antioxidants fight against aging, and inorganic salts essential for cellular enzymatic activities. The role of hyaluronic acid is a software prob is gnosti maintain volume and hydration of the skin and create a protective screen against the destructive action of free radicals (H.Trommer, S.Wartewig, Int. Journ. Of Pharm. 254 (2003) 223-234). When sufficient concentrations of hyaluronic acid creates an optimal environment for cell proliferation and new collagen synthesis.

Oxidative stress by generating free radicals in the dermis and epidermis that are responsible for skin aging and appearance of wrinkles, fine wrinkles and sagging tissue.

In addition, it also demonstrated that free radicals are also responsible for the depolymerization of hyaluronic acid in situ, which further contributes to the sagging of tissues and premature aging (Mendoza G., et al. Carbohydrate Research 342, 2007, 96-102).

Extracellular matrix (ACM) is a dynamic structure that plays a structural and regulatory role in tissue. VKM consists of collagen fibers and elastin, as well as from the primary environment (mainly water, mineral salts and proteoglycans). This matrix gives the skin its elasticity and its mechanical properties density, elasticity and tone.

Macromolecule collagen are fibrillar proteins formed by three polypeptide chains crosslinked by covalent and hydrogen bonds. Known nineteen types of collagen, where half of them are located in the skin. The majority of skin collagen belongs to the fibrillar collagens I, III and V.

Young adults with the Tav derma 80% collagen type I and 20% collagen type III; however, this ratio changes with age as a consequence of aging.

The elastin are proteins, organized into fibers inside the skin, and they give the skin properties of elasticity and flexibility. These elastin-enriched amino acid hydrophobic character.

The collapse of the ACM is involved in several physiological processes such as wound healing, embryonic development or angiogenesis, as well as in pathological situations, such as arthritis, Atheros or atherosclerosis, and, of course, during the phases of progression of the tumor with metastasis (Fisher et al., 1997. N England J. Med., 337, 1419-28; Shapiro, 1998. Current Opinions in Cell Biology, 10, 602-608).

Components VKM, mainly broken down by enzymes endopeptidase called the matrix metalloproteinase or MMD. These MMD actively contribute to the process of wound healing, as well as contribute to the relaxation of the skin and wrinkles, which are the first signs of skin aging. The MMP family consists of approximately 22 enzymes, which are distinguished by their specificity towards the substrate, which they split.

MMP-1 or interstitial collagenase, predominantly cleaves the triple helix fibrillar collagens type III, but also the collagens I, II, VII, VIII and X.

MMP-3 cleaves glycoproteins, such as fibronectin and laminin, some Proteas ikani, elastin, gelatin and collagen IV and V. These two MMP expressed by keratinocytes and fibroblasts.

To fill wrinkles chemically cross-linked gels of ha injected intradermally to fill depressions caused by the wrinkle. The stitching gives the possibility to increase the stability of the drug within the dermis. Thus, if the drug is properly injected in accordance with the genetic profile of each individual, this drug allows filling in for 4 to 6 or even 8 months. Then he undergoes a complete resorption in the dermis.

Such gels based on GC or sewn Ledger give the possibility of reducing wrinkles due to the effect of the mechanical filling of the depression in the skin, which is the result of the wrinkles. These drugs are endowed with only this mechanical effect and does not contribute either in the prevention or treatment of skin ageing and destruction of ACM that are essential for maintaining the mechanical properties of the skin, such as its elasticity and density. Such implants, if they give the ability to remove wrinkles or fine and fine lines, creating an effect that is limited in time, and which only partially masks the effects inherent in the skin-ageing concerns of the supporting structures represented by ACM.

To date, several drugs used in this field. the spruce or silicone oils are easy to apply, but they have a disadvantage due to migration in the tissue located directly below the point of injection. Thus, the result of chronic inflammation or allergic reactions. In addition, the silicone is not biodegradable and is in some organs such as the liver. Also proposed various suspension polymer particles, but most caused rejection, infection or inflammation. Finally, in the last few years using the collagen suspension. However, collagen undergoes resorption relatively quickly (between 1 and 3 months) and causes some allergic reactions thanks to its origin, because it usually has a bovine or porcine origin.

There remains a need for an injectable implant that is able to function as fill wrinkles and restore the vital functions of the dermis and epidermis and contribute to the limitation of the process of cellular aging associated with aging skin who do not have the abovementioned disadvantages, or they are much less pronounced, and it all comes easy and enhanced convenience in use.

The present invention thus relates to a subcutaneously or intradermally injectable implant in the form of a monophasic Hydra the gel, characterized in that it contains from 0.5% to 5% wt./wt., preferably from 0.5% to 4%, more preferably 2% hyaluronic acid, and in which:

- from 50% to 95%, preferably from 60% to 90%, even more preferably 85% wt./wt. hyaluronic acid is in the form of a cross-linked gel;

- from 5% to 50%, preferably from 10% to 30%, even more preferably 15% wt./wt. hyaluronic acid is in the free form or in the form of one of the physiologically acceptable salts Ledger molecular weight between 500 and 2800 kDa, preferably between 750 and 2600 kDa, more preferably between 800 and 2500 kDa, even more preferably between 1000 and CD,

in a physiologically acceptable liquid carrier, where the ratio between the mass of the crosslinked gel of hyaluronic acid and lots of free hyaluronic acid is between 1:1 and 1:0,05.

In a particular embodiment of the invention, the implant may contain about 80% of the crosslinked hyaluronic acid and about 20% of free hyaluronic acid, which is fundamentally important in the context of the use of the implant according to the invention for the treatment of thin and wrinkles. In the case of treatment of deep wrinkles, the number of crosslinked hyaluronic acid should preferably be approximately 85%, and the amount of free hyaluronic acid for approximately 15%.

In the context of the present invention hyaluronic is iSlate or Ledger is defined as desulfuromonas linear glycosaminoglycan, consisting of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine.

Monophase hydrogel means hydrogel in a single homogeneous phase.

Physiologically acceptable salt of hyaluronic acid means, in particular, the sodium and potassium salts, and mixtures thereof. Mostly salt is a sodium salt.

Mostly gel, made from cross-linked hyaluronic acid according to the invention has a viscosity between 200 and 2000 PA·s, more specifically between 500 and 1800 PA·s, even more particularly between 1000 and 1800 PA·S.

In the case of the implant for the treatment of deep wrinkles, containing 85% crosslinked hyaluronic acid, the viscosity of the latter set about 1000-1500 PA·s, in particular, about 1200 PA·C. In the case of the implant, mainly for shallow wrinkles, therefore, with a lower content of crosslinked hyaluronic acid, i.e. about 80% of crosslinked hyaluronic acid, the viscosity of the implant is installed around 200-500 PA·s, more specifically about 350 PA·S.

In this specification, these viscosities correspond to the values measured for a shear rate of 0.01-1.

Mainly crosslinked hyaluronic acid, which is a gel according to the invention has a molecular weight between 1000 and 6000 kDa, more preferably between 1000 and 4000 kDa.

According to a preferred and is the aspect of the invention injectable implant also contains chondroitin sulfate.

Mainly the amount of chondroitin sulfate is between 0.05% and 5% wt./wt. from the total mass.

Mainly chondroitin sulfate has a molecular mass of between 2 and 80 kDa, more preferably between 20 and 50 kDa.

The implant according to the invention can contain various conventional additives. For example, one can mention dyes, pigments, oils, thickeners, pH modifiers and controllers osmotolerance.

Free hyaluronic acid or one of its physiologically acceptable salts mainly distributed homogeneous inside the gel crosslinked hyaluronic acid.

The carrier fluid is primarily sterile pyrogen-free isotonic buffer.

Injectable implant according to the invention also mainly contains at least one other active substance used in dermocosmetic.

Mainly dermocosmetics active substance selected from vitamins, antioxidants, inorganic salts, antiseptics and chondroitin sulphate.

In a particular embodiment of the invention injectable implant contains mannitol, which acts as an antioxidant at the level of the dermis and contributes to the protection Ledger from depolymerization induced by free radicals generated inside the dermis as a result, the oxidant is on stress. Indeed, it is described that the combination of mannitol with SC enhances the protection against damage induced by free radicals (Belda et al., 2005, J. Cataract Refract. Surg., 31: 1213-1218). Thus, the next aspect of the present invention is to develop an injectable implant, as defined above, and containing mannitol as an antioxidant.

Another aspect of the present invention is the use of mannitol in injectable implant according to the present invention in order to protect the skin from free radicals and/or limitations of depolymerization of hyaluronic acid contained therein.

The invention thus also relates to the use of hyaluronic acid in free form or in the form of one of its physiologically acceptable salts of molecular weight between 500 and 2800 kDa, preferably between 750 and 2600 kDa, more preferably between 800 and 2500 kDa, even more preferably between 1000 and 1500 kDa in the presence of an antioxidant, such as mannitol, for the manufacture of the implant, designed to protect the skin from free radicals and/or to limit the depolymerization of hyaluronic acid in the dermis and/or for the prevention of skin aging.

The implant according to the invention is injected into a superficial, medium or deep dermis.

The advantage is the ability to arouse in her the injection activation of fibroblasts by stimulating cell proliferation and new collagen synthesis. Then activate fibroblasts creates a modulation of the mechanisms involved in remodeling of the extracellular matrix, which is expressed in the restoration of vital functions of the dermis.

Double advantage of the implant according to the present invention is to obtain a direct and immediate effect of reducing wrinkles in the mechanical fill skin depressions and indirect effect in the long term cellular regeneration through stimulation of collagen synthesis and the regulation of MMP.

Crosslinked hyaluronic acid contributes directly to the effect of mechanical filling and allows, through its cross-linked nature, to obtain such an effect lasting for longer periods than unstitched Ledger.

In addition, free Ledger contained in the implant according to the invention, inhibits the overexpression of MMP-1 and overexpression of collagen III. In addition to its regulatory action on MMP-1, it contributes to the limitation of the breaker and the destruction of the CMC, is indispensable to the structure to maintain the mechanical properties of the density and elasticity of the skin.

In addition, demonstrated that the ratio of collagen III/I collagen increases during aging (Weber et al, 1984, J. Invest. Dermatol., 82, 156-60). Knowing that in the context of this invention watched h what about the fraction of free Ledger used in the implant according to the invention, inhibits the expression of collagen type III, not affecting the expression of collagen type I, it is reasonable to assume that the implant according to the invention is able to restore the ratio of collagen III/I collagen to the ratio measured in young tissues.

Thus, the purpose of this invention is the use of the implant according to the invention for the manufacture of a medicinal product intended to maintain and/or restore the ratio of collagen III/I collagen to the ratio measured in young tissues.

The consequence of the combination of free GC molecular weight between 500 and 2800 kDa by gel crosslinked ha is immediate hydration and the maintenance of the volume of the skin. In addition, it induces the activation of fibroblasts of the dermis, mainly due to the presence of free Ledger, and the activation of fibroblasts prolonged in time as far as progression in vivo decomposition of the crosslinked gel Ledger.

The implant according to the present invention can be presented in the form of a three-dimensional mesh, made of stitched Ledger, containing within it the free molecules Ledger, capable of inducing stimulation of fibroblasts, inhibition of dissolution of VKM by inhibiting MMP and regulation of collagen synthesis by orientation of the latter in the direction status is I, the corresponding observed in young tissue.

These molecules are free Ledger progressive released from this three-dimensional matrix stitched Ledger as passive diffusion through decay with time matrix stitched CC in the weeks and months after injection. This progressive release gives the possibility of such a rejuvenating effect stimulation of cells in situ these molecules free GK, which are protected from biological degradation within the specified matrix for several weeks, and can therefore operate over a longer period than when injecting them alone.

Thus, the present invention also relates to the use of hyaluronic acid in its free form or in the form of one of its physiologically acceptable salts, of molecular weight between 500 and 2800 kDa, preferably between 750 and 2600 kDa, more preferably between 800 and 2500 kDa, even more preferably between 1000 and 1500 kDa, distributed within the gel crosslinked hyaluronic acid, for the manufacture of a subcutaneous implant for filling wrinkles and stimulate epidermal cells and/or maintaining mechanical properties the density and elasticity of the skin.

Thus, the implant according to the invention combines the mechanical effect of cross-linked gel, which swells and Vosstania the es form of wrinkles, biological action of the free group.

The invention also relates to a set in the form of a syringe containing injectable implant, such as described above.

The invention also relates to an implant, as described above, as a medicine.

The invention also relates to the use of injectable implant, as described above, to fill wrinkles, thin and wrinkles, skin depressions and/or scarring, including subcutaneous injection of the implant.

Injectable implant according to the invention can be used for the manufacture of drugs intended for the stimulation of epidermal and dermal metabolism and/or to stimulate epidermal cell activity.

Injectable implant according to the invention can be used for the manufacture of drugs intended for the stimulation of antioxidant activity of the dermis and/or prevention of skin aging.

The invention also relates to a cosmetic method for the filling of wrinkles and/or fine lines, which is injected at least one injectable implant according to the invention.

The invention also relates to a method of manufacturing an injectable implant, as described above. It can be made using any methods known to experts in the art, for example, using diepoxy in particular, potentialapplications ether (BDDE) or 1,2,7,8-diepoxyoctane. When stitching in a basic environment, the concentration of diepoxy can vary, for example between 5 and 15% compared to hyaluronic acid to bind in a water bath at 45-55°C for 1.5-6 hours Then cross-linked gel purified using the classical techniques of professionals in the art: various baths with deionized water, alcohol precipitation, dialysis, etc. to remove traces of residual cross-linking agent. To this purified gel add Ledger appropriate molecular weight, pre-gidratirovannuyu in a suitable buffer. Finally, the finished product Tegaserod, placed in a syringe or any other suitable container and sterilized by autoclaving.

The preferred technique according to the invention comprises the following stages:

1) preparation of cross-linked gel in accordance with the following stages:

- adding hyaluronic acid to the base fluid,

- swelling, homogenization with slow stirring and the crosslinking when heated,

- neutralization and swelling cross-linked gel in a buffer solution at a pH of about 7 by adding the agent, giving isoosmolar,

- removal of cross-linking agent,

2) preparation of gel-free hyaluronic acid by:

- adding hyaluronic acid to the buffer Rast the oru with a pH of approximately 7, isoosmolar:

- swelling,

3) mixing the cross-linked gel obtained in stage 1), with gel-free hyaluronic acid, obtained in stage 2),

4) possible degassing and possible packaging in bottles or syringes, and then sterilization.

Mainly the pH of the gel after sterilization is approximately 7, and osmotolerant is about 250-350 mOsm, preferably between 300 and 320 mOsm.

Further, the invention is illustrated, without limiting it, the following examples.

Example 1:

Feature activity Ledger of different molecular weight on:

healthy fibroblasts;

- senescent fibroblasts (through oxidative stress H2O2).

MMP-1 (collagenase 1) is an interstitial collagenase, which breaks down triple helix fibrillar collagens, such as collagen I and III. In the skin it is expressed and secreted by fibroblasts and keratinocytes. MMP-1 is involved in aging. Indeed, her overproduce in the aging process may be involved in the loss of density and elasticity and wrinkles. When the aging induced by N2O2in fibroblasts see a significant increase in MMP-1. The main active substances capable of reducing overproduction, therefore, may have the characteristics of "anti-aging".

the Fibroblasts were obtained from skin, originating from the remnants of operating from young subjects. The samples were washed in FSB and ethanol. Cut small pieces of skin were placed in culture plates and immersed in an environment that is conducive for the proliferation of fibroblasts (DMEM + 10% SVF). In this environment was added primocon. The latter is a antibiotic, antifungal agent and an anti-Mycoplasma. Cultural cups were placed in an incubator.

For the induction of aging fibroblasts were sown in the culture Cup, After 24 hours, add the tested active substance at an appropriate concentration to DMEM. After 24 incubation, the cells subjected to oxidative stress. Cells were incubated for 2 hours at 37°C in 75 μm solution of H2O2the FSB. Involved acute stress, which causes aging of cells. Then fibroblasts were again placed in complete DMEM with 10% SVF. Fibroblasts return to aging 72 hours after the end of stress.

1.1. The incubation Protocol

For senescent fibroblasts: GK added to a final concentration of 1 μg/ml, leave them in the course of stress H2O2, i.e. 24 h incubation.

For healthy fibroblasts: GK also incubated for 24 h with 1 μg/ml

1.2. The allocation of total RNA using a kit for isolation Rneasy (Qiagen) at the end of the experiment (day 5, i.e. 72 h after stress).

1.3. EN is Liz total RNA

Qualitative and quantitative analysis of total RNA is carried out using an analytical kit RNA 6000 Nano LabChip and apparatus 2100 Bioanalyzer (Agilent). It is based on the principle of electrophoretic migration of the samples in nanochips. Calculate the ratio of the ribosomal RNA 28S/18S; it is informative in assessing the integrity of the RNA. In addition, assess the purity of RNA the ratio of genomic DNA.

1.4. Analysis of the rate of transcription using real-time PCR allows quantification of the level of transcriptional expression of the gene of interest by amplification of cDNA obtained by reverse transcription of mRNA of the gene present in the cell lysate. The reaction mixture is prepared from the cDNA mixture and groups interested primers with a solution IQ SybrGreen Supermix (Biorad)containing, among other things, Taq polymerase and intercalating agent minor grooves of the DNA double helix: Sybr Green (fluorescent intercalating agent). The reaction is carried out in thermocycler Icycler IQ (Biorad); involved a sequence of cycles denaturation/hybridization/elongation.

1.4.1. Reverse transcription: total RNA "back Transcriber" on cDNA using Reverse reduced System (Promega) and a Gene Amp PCR System 2400 (Perkin Elmer).

1.4.2. The measurement of the rate of transcription of the gene of interest: the level of expression of the gene of interest normalize the expression level of reference genes was kotarumalos varies with aging. The level of expression of the gene of interest calculated in accordance with the formula: 2 (CTmin-CT), where CT denotes the "threshold cycle".

The expression level normalize with respect to the expression of three reference genes according to the calculation: 2 (STT-ST)/normalization factor.

1.5. Results expression of MMP-1

Fibroblasts subjected to aging by incubation with H1O2, expressed in 2.14 times more MMP-1 than normal fibroblasts (thus, aging properly induced). In the presence of vitamin E (positive control) overexpression of MMP-1 in senescent fibroblasts was reduced by 40%.

Overexpression of MMP-1 was reduced by 40% in the presence of HK 450 kDa (10 μg/ml), 75% in the presence of GK 800 kDa (10 μg/ml), 71% in the presence of GL 1500 kDa (10 μg/ml) and 83% in the presence of SC 2600 kDa (10 µg/ml).

1.6. The results on the expression of collagen type I and type III. In fibroblasts subjected to aging by incubation with H2O2was measured variable inhibition of transcription of collagen type I; this inhibition seemed, however, dependent on donors. One of the donors, for which the transcription of the type I was ingibirovany 24% stress H2O2various Ledger of various sizes were not restored the synthesis of collagen.

In fibroblasts undergoing aging, was the measured temperature is +163% - +304% transcription of collagen type III. On the donor, for which the transcription of collagen type III stimulated by +304% stress H2O2free GK various sizes inhibit the synthesis of collagen. HK 450 kDa inhibited by approximately 25%, whereas GC mass greater than 800 kDa, inhibits transcription by about 100%, returning to levels close to the levels of young fibroblasts.

1.7. Conclusion

The conclusion of this experiment is that it turns out that GK has anti-MMP-1 activity (hence, antioxidant, thus having potential activity against aging) in this model induced aging. Ledger higher molecular weight (800, 1500 and 2600 kDa) are more effective than HK 450 kDa.

In these experimental conditions the GC molecular weight of 450 kDa active against MMP-1. Ledger molecular weight higher than 800 kDa, more active, because they inhibit the overexpression of from -75 to -83%.

On the other hand, these results show that various free Ledger no effect on the synthesis of collagen type I.

Finally, the available GC inhibit the overexpression of collagen type III, and this is particularly noticeable for the Ledger molecular mass of 800 kDa and above, which inhibits the overexpression from -118 to -93%.

Thus, in addition to their regulatory effect on MMP-1, free the e Ledger contained in the implant according to this invention, contribute to the limitation of destruction and the breaker VKM. In addition, knowing that it was demonstrated that the ratio of collagen III/I collagen increases in the aging process, the Ledger contained in the implant according to the invention, provide an opportunity to restore balance, measured in young tissues.

Example 2: Preparation of the implant according to the invention.

2.1. Preparation of crosslinked Ledger

5 g of sodium hyaluronate molecular weight of 1.6 MDA add 1% sodium hydroxide (35,6 g). The mixture is left for periodic homogenization for 1 h and 30 min Then to the mixture GK/NaOH, which was homogenized add 315 mg BDDE, close, and then placed in a water bath at 50°C for 2 hours, the Mixture is neutralized by adding 5 g of 1 N. HCl.

The gel thus obtained, was added at the desired concentration by adding EDI and salts, providing isoosmolar, as well as a stable neutral pH, to obtain the gel at 20 mg/g in the Ledger.

2.2. Preparation of implant

To this purified gel add CC to 1.2 MDA, pre gidratirovannuyu in phosphate buffer. Then 60 g of gel CC of 1.2 MDA concentration 20 mg/g add to 226 g of the obtained cross-linked gel. These 2 gel homogenized in a standard paddle mixer for 1-2 hours.

The final product can is about then degassing, packaged in syringes and sterilized by steam autoclaving. by execution of the loop, for example, 125°C for 7 min, 127°C for 4 min or 130°C for 3 minutes

After sterilization the pH of the preparation is 7.1, osmotolerant 320 mOsm, and rheological properties are the elastic modulus G' 45 PA·s at a frequency of 1 Hz. The final concentration of the gel was evaluated as to 19.8 mg/g (analysis carbazole in accordance with the method of the European Pharmacopoeia) for the expected concentration of 20 mg/g

1. The implant, which is injectable subcutaneously or intradermally in the form of a monophase hydrogel, characterized in that it contains from 0.5% to 5% wt./wt., preferably from 0.5% to 4%, more preferably 2% hyaluronic acid, where:
- from 50% to 95%, more preferably from 60% to 90%, most preferably 85% wt./wt. hyaluronic acid is in the form of a cross-linked gel,
- from 5% to 50%, preferably from 10% to 30%, most preferably 15% wt./wt. hyaluronic acid is in the free form or in the form of one of its physiologically acceptable salts of molecular weight between 500 and 2800 kDa, preferably between 750 and 2600 kDa, more preferably between 800 and 2500 kDa, most preferably between 1000 and 1500 kDa,
in a physiologically acceptable liquid carrier, where the ratio between the mass of the crosslinked gel of hyaluronic acid and Holy mass is free of hyaluronic acid is between 1:1 and 1:0,05.

2. Injectable implant according to claim 1, characterized in that the gel obtained from cross-linked hyaluronic acid has a viscosity between 200 and 2000 PA·s, preferably between 1000 and 1800 PA·S.

3. Injectable implant according to claim 1, characterized in that the hyaluronic acid component is cross-linked gel has a molecular weight between 1000 and 6000 kDa, preferably between 1000 and 4000 kDa.

4. Injectable implant according to claim 1, characterized in that it further comprises a chondroitin sulfate having a molecular weight of between 2 and 80 kDa, preferably between 20 and 50 kDa.

5. Injectable implant according to claim 1, characterized in that the free hyaluronic acid or one of its physiologically acceptable salts uniformly distributed inside the gel crosslinked hyaluronic acid.

6. Injectable implant according to claim 1, characterized in that the carrier fluid is a sterile pyrogen-free isotonic buffer.

7. Injectable implant according to claim 1, characterized in that it additionally contains at least one other active substance used in dermocosmetic.

8. Injectable implant according to claim 7, characterized in that dermocosmetics active substance selected from vitamins, antioxidants, inorganic salts, antiseptics and chondroitin sulphate.

9. Injectable implant according to claim 8 characterized in that what an antioxidant is a mannitol.

10. Injectable implant according to any one of claims 1 to 9 as a medicine, in particular, to stimulate antioxidant activity of the dermis, and/or for the prevention of skin aging, and/or to maintain and/or restore the ratio of collagen III/I collagen to the ratio measured in young tissues.

11. The set is presented in the form of a syringe containing injectable implant according to any one of claims 1 to 9.

12. The use of hyaluronic acid in free form or in the form of any of its physiologically acceptable salts of molecular weight between 500 and 2800 kDa, preferably between 750 and 2600 kDa, more preferably between 800 and 2500 kDa, most preferably between 1000 and 1500 kDa, in the presence of an antioxidant, such as mannitol, for the manufacture of an implant in the form of a monophase hydrogel designed to protect the skin from free radicals and/or to limit the depolymerization of hyaluronic acid in the dermis.

13. The use of injectable implant according to any one of claims 1 to 9, or set by clause 11 to fill wrinkles, thin and wrinkles, skin depressions and/or scarring, including subcutaneous injection of the implant.

14. The use of hyaluronic acid in free form or in the form of any of its physiologically acceptable salts of molecular weight between 500 and 2800 kDa, preferably between 750 and 2600 kDa, more preferably between 800 and 2500 kDa, most preferably between 1000 and 1500 kDa, distributed in the gel crosslinked hyaluronic acid, for the manufacture of a subcutaneous implant in the form of a monophase hydrogel designed to fill wrinkles and to stimulate epidermal cell activity, and/or to maintain the mechanical properties of the density and elasticity of the skin and/or stimulating epidermal and dermal metabolism, and/or to stimulate antioxidant activity of the dermis, and/or prevention of skin aging.

15. The method of obtaining injectable implant according to any one of claims 1 to 9, characterized in that it comprises the following stages:
1) preparation of cross-linked gel in accordance with the following stages:
- adding hyaluronic acid to the base fluid,
- swelling, homogenization with slow stirring and the crosslinking when heated,
- neutralization and swelling cross-linked gel in a buffer solution at a pH of about 7 by adding the agent, giving isoosmolar,
- removal of cross-linking agent,
2) preparation of gel-free hyaluronic acid by:
- adding hyaluronic acid to the buffer solution with a pH of about 7, isoosmolar:
- swelling,
3) mixing the cross-linked gel obtained in stage 1), with gel-free hyaluronic acid derived n is stage 2),
4) possible degassing and possible packaging in bottles or syringes and subsequent sterilization.



 

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3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to cosmetology and discloses an product for temporary keratin fibre deformation and hair spray containing it. Said agent contains in a cosmetically acceptable carrier: a) at least one copolymer A which is formed of at least one monomer A1 specified in acrylic acid, methacrylic acid, alkyl esters of acrylic acid and alkyl esters of methacrylic acid, at least one monomer A2 specified in hydroxyalkyl esters of acrylic acid and hydroxyalkyl esters of methacrylic acid and at least one monomer A3 specified in methylsuccinate and ethylsuccinate, and b) copolymer of N-octylacrylamide/acrylic acid/tert-butylaminoethylmetacrylate.

EFFECT: invention provides high degree of resistance with preserved flexibility, elasticity and plasticity.

7 cl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present group of inventions refers to aqueous oral care compositions. The compositions contain: a) 0.2% to 3% of bivalent metal ions including 0.1% to 1.5% zinc ions and 0.1% to 2% tin ions (II); and b) fluoride ion source; c) silicone dioxide dental abrasive; d) one or more chelating agents having MW less than 1000 and able to form water-soluble zinc ion complexes wherein the chelating agents contain at least 0.2% of linear polyphosohates of the chain length of four or more; e) orally acceptable carrier containing at least 20% of total water amount. The pH value of the composition varies within the range 5 to 6.5. The molar ratio of the chelating agents to bivalent metal ions makes at least 0.70:1, and at least 80 wt % of total zinc ion amount solubilised in the composition.

EFFECT: compositions provide better antimicrobial activity as compared with the zinc/tin combination with no considerable flavouring, staining or stability problems as compared with the compositions having the low content of chelating agents.

18 cl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to cosmetology and represents a method of light-sensitive make-up on a human keratin material wherein: i. keratin material is coated with a layer of a heat-resistant photochromic composition containing a heat-resistant photochromic agent visualised under UV radiation, and optical agent which shields UV radiation; and ii the layer of the composition is exposed to non-uniform UV radiation to excite the heat-resistant photochromic agent and to create a light-sensitive make-up wherein the screening capacity F of the composition in relation to solar UV radiation (within 280 nm to 400 nm) makes 2 or more.

EFFECT: invention provides delayed deterioration of the light-sensitive make-up under natural light and improved contrast range and colour retention of the light-sensitive make-up.

14 cl, 4 ex, 16 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to cosmetology and represents a silicone-free skin protector, particularly a cold protector containing ingredients - each per total composition of the skin protectors: a) 10 to 50 wt % of at least one oil having a pour point according to DIN ISO 30 16≤10°C, specified in hydrocarbons and carbonates, b) 10 to 40 wt % of at least one polyols specified in physiologically tolerable polyalcohols or polyhydroxycompounds with 2-15 carbon atoms and at least 2 hydroxyl groups, c) 1 to 10 wt % of at least one emulsifier specified in polyhydroxystearic acid and/or polyricin esters and d) target additives to 100% with viscosity variations within the temperature range +4°C to +50°C between the highest and lowest viscosity of the skin protector having the value within the range 0 to ≤20000 mPa·s.

EFFECT: invention provides skin protection against cold; it is applied at min -18°C from a tube; herewith it remains paste-like or flowing and/or spread; it is stable and carries a load of freezing-thawing cycle for 3 times.

9 cl, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to cosmetology and represents a silicone-free skin protector, particularly a cold protector containing ingredients - each per total composition of the skin protectors: a) 10 to 50 wt % of at least one oil having a pour point according to DIN ISO 30 16≤10°C, specified in hydrocarbons and carbonates, b) 10 to 40 wt % of at least one polyols specified in physiologically tolerable polyalcohols or polyhydroxycompounds with 2-15 carbon atoms and at least 2 hydroxyl groups, c) 1 to 10 wt % of at least one emulsifier specified in polyhydroxystearic acid and/or polyricin esters and d) target additives to 100% with viscosity variations within the temperature range +4°C to +50°C between the highest and lowest viscosity of the skin protector having the value within the range 0 to ≤20000 mPa·s.

EFFECT: invention provides skin protection against cold; it is applied at min -18°C from a tube; herewith it remains paste-like or flowing and/or spread; it is stable and carries a load of freezing-thawing cycle for 3 times.

9 cl, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: tooth paste composition and a method for using said composition for eliminating dry mouth, preventing and/or reducing caries in an individual. The tooth paste composition contains an orally acceptable carrier the volume of which comprises silicone oil granules distributed. The tooth paste composition have a viscosity of min. 20000 centipoise.

EFFECT: use of the tooth paste composition provides superior cleanness of teeth, oral freshness and moisture.

11 cl, 2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to cosmetics. A "water-in-oil" emulsion composition wherein an oil phase preferentially making 20 to 55% of a composition base contains at least threshold portions both of silicone, and of oil having an effect on sensations, particularly specified in dicaprylyl ester and dicaprylyl carbonate; the weight relation of oil having an effect on sensations, and a polyatomic water-retaining substance preferentially makes 3:4 to 4:1. The similar compositions are especially to be mixed with a liquefiable propellant. The aerosol composition is applied locally on human skin.

EFFECT: group of invention provides balanced sensation properties, including humidity and lower adhesiveness.

30 cl, 12 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to a method for preparing an oral care composition containing a film or film fragments with the film containing an active ingredient. The method involves: (a) immersion of the film into a medium containing the active ingredient wherein the active ingredient contains a whitening agent and wherein the film contains a polymer specified in a group consisting of water-soluble, water-dispersed and water-insoluble polymers, and substantially does not contain the active ingredient; (b) transfer at least of a portion of the active ingredient from the medium into the film wherein the procedure is continued until the film contains such effective amount of the transferred active ingredient that makes the film acceptable for the use in the oral care composition wherein the amount of the transferred active ingredient found in the film after the procedure makes 0.5 wt % of film weight and higher with a transfer cycle making 30 and less minutes; and (c) application of the medium containing said film comprising the transferred active ingredient in an orally acceptable carrier.

EFFECT: preparing the oral care composition containing the film with the effective amount of the whitening agent in a relatively short time.

7 cl, 6 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular to method of obtaining form-preserving aggregates of gel particles, in which aggregates are held together by physical forces of non-covalent bonds, such as hydrophobic-hydrophilic interactions and hydrogen bonds. Method of obtaining form-preserving aggregates of gel particles includes introduction of preliminarily obtained suspension of gel particles in polar liquid, where gel particles have absolute electrochemical potential, into receiving medium, in which absolute electrochemical potential of gel particles decreases, which results in fusion of gel particles into form-preserving aggregate.

EFFECT: invention allows to obtain form-preserving gel aggregates in situ so that form of aggregate is determined by place of application.

49 cl, 35 ex, 11 tbl, 33 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, more specifically to biocompatible alginate systems with the delayed gelatinisation process. There are offered sets and compositions for making a self-gelatinised alginate gel containing sterile water-soluble alginate and particles of sterile water insoluble alginate with a gelling ion. There are offered methods for dosing self-gelatinised alginate dispersion for making the self-gelatinised alginate gel. The methods can include dosing the dispersion in an individual. There is offered the self-gelatinised alginate gel of the thickness more 5 mm and not containing one or more sulphates, citrates, phosphates, lactates, EDTA or lipids. There are offered implanted devices coated with the homogeneous alginate gel. There are offered methods for improving viability of pancreatic islets or other cell aggregate or tissue, after recovery and while stored and transported.

EFFECT: group of inventions provides creation of the alginate gelling system which contains alginate and the gelling ions with high biological compatibility; enables the gelatinisation process without pH variations, connected with the other systems, and requires minimum ingredients, thus provides variation of gelatinisation time and gel strength depending on the specific requirements.

62 cl, 11 dwg, 2 tbl, 27 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing biologically compatible gel which is thickened with cross-linked polymer by cross-linking a given amount of at least one biologically compatible natural polymer in a solution by adding a defined amount of cross-linking agent, an additional amount of polymer with molecular weight over 500000 dalton in a solution, in which the reaction mixture is diluted to reduce concentration of polymer in the solution, and the cross-linking reaction is stopped by removing the cross-linking agent.

EFFECT: gel and its use for separating, replacing or filling biological tissue or for increasing volume of such tissue, or supplementing or replacing biological fluid.

11 cl, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine. Particles of the viscoelastic material chosen from group, consisting of polysaccharides and their derivatives which are suitable for injection with gel particles having the size in a range from 1 to 5 mm at action of a physiological saline solution are described. An implant for increase of volume of the soft tissues, containing particles of the viscoelastic material chosen from group, consisting of polysaccharides and their derivatives where the basic volume of the specified particles represents the gel particles, suitable for injection and having the size in a range from 1 to 5 mm at action of a physiological saline solution, is described. The way of increase of volume of soft tissues at a mammal, including a human being, including subepidermal introduction in a place of a body of the specified mammal in which it is desirable to enlarge volume of soft tissues is described.

EFFECT: augmentation of volume of soft tissues at a mammal.

24 cl, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine. Claimed is composition with hyaluronic acid (HA), which includes gel particles of bound water-insoluble hydrated HA. HA includes bindings, represented with the following structural formula: HK'-U-R2-U-TK'. Where each group HA' represents the same or other molecule of bound HA'; each U independently represents optionally substituted 0-acylisourea or N-acylurea; and R2 represents optionally substituted alkyl, alkenyl, alkinyl, alkoxy, cycloalkyl, cycloalkenyl, cycloalkinyl, aryl, heteroaryl, heterocyclic radical, cycloaliphatic alkyl, aralkyl, heteroaralkyl or heterocyclolalkyl. Also claimed is method of developing tissues in individual, including introduction of needle into individual in place where development of tissues is necessary, needle is connected to syringe filled with composition with HA, and applying force to syringe in order to supply composition with HA to individual. Method of obtaining composition with HA includes formation of water-insoluble dehydrated particles of bound HA, separating insoluble in water particles by their average diameter, selection of subset of particles by average diameter and hydration of subset of dehydrated particles by means of physiologically compatible water solution. Other method of obtaining composition with bound HA includes binding precursor of bound HA by means of bis-carbodiimide in presence of pH buffer and dehydration of bound HA. Also included is method of developing tissues in individual that needs tissue development. Method of stabilisation of bound HA includes hydration of water-insoluble dehydrated bound HA by means of physiologically compatible water solution which includes local anesthetic, so that value of elasticity module G' for stabilised composition constitutes not less than approximately 110% from value G' for non-stabilised composition.

EFFECT: claimed composition of hyaluronic acid and method of preparation and application of HA composition are efficient for development of tissue and/or drug delivery.

27 cl, 22 ex, 2 tbl, 7 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely to reconstructive surgery, traumatology-orthopedy, maxillofacial surgery, stomatology and can be applied at osteo-plastic operations. For delivery of medical products immediately in a zone of defect and their prolonged influence in the centre of a lesion medicinal preparations are dissolved in a normal saline solution in a dose providing local effect, collagen-containing component is added to a solution in the ratio 9-20 g: 100 ml of a solution also admix with the carrier from dispersed allotransplants in the ratio of 1:1-3.

EFFECT: method allows lowering a dose necessary for reception of medical effect in 10 times, and also allows accelerating reparative processes in a defect zone.

3 dwg

FIELD: medicine.

SUBSTANCE: method of antibiotics fixation within porous implants is described. Result of method application lies in possibility of reliable fixation of antibiotic solution within porous implant and arrangement of favourable conditions for haemostasis in operative wound due to application of 10% gelatine solution as antibiotic carrier. Specified result is achieved by filling porous implants with antibiotic solution in liquid gel. For this purpose implant is dipped in solution by 3/4. Filling occurs under the influence of capillary forces. After solution cooled to form dense gel, antibiotic is fixed in implant pores and gradually released after installation to bone defect area.

EFFECT: reliable fixation of antibiotic solution within porous implant and arrangement of favourable conditions for haemostasis in operative wound.

3 cl, 1 ex

FIELD: medicine-destination polymers.

SUBSTANCE: invention relates to biologically stable hydrogels to be employed as endoprosthesis consisting essentially of following components: polyacrylamide including acrylamide, crosslinked methylene-bis-acrylamide, wherein acrylamide and methylene-bis-acrylamide are linked at molar ratio from 150:1 to 1000:1. Hydrogel is rinsed with water or physiologic solution so that it contains about 0.5-3.5% polyacrylamide and less than 50 ppm acrylamide and methylene-bis-acrylamide monomers, while modulus of elasticity of hydrogel is approximately 10 to 700 Pa and its complex viscosity about 2 to 90 Pa*sec. Rinsing stage allows removal of nearly all amounts (even trace amounts) of above-indicated monomers resulting in lower toxicity and higher stability of hydrogel. Biologically stable hydrogel is used as injectable prosthesis to fill soft tissues and also to treat or prevent urinary incontinence or anal incontinence. Hydrogel, obtained in a few stages including combining acrylamide and methylene-bis-acrylamide, initiating radical polymerization, and rinsing with apyrogenic water or physiologic solution, is also useful in treatment or prevention of bladder-ureter reflux in mammalians. In all these cases biologically stable hydrogels contain between 0.5 and 25% polyacrylamide.

EFFECT: enlarged resource for manufacturing endoprostheses.

10 cl, 3 dwg, 7 tbl

The invention relates to medicine, in particular to plastic surgery
The invention relates to medicine, namely to a method for producing compositions for injection, for use in reconstructive and cosmetic surgery

FIELD: medicine.

SUBSTANCE: present invention refers to medicine, more specifically to use of a polymer containing thiol groups, for producing a tissue extension implant where a base polymer is polysaccharide.

EFFECT: polymers containing thiol groups have an antioxidant effect, and are characterised by longed stay on an application site.

13 cl, 4 dwg, 7 ex

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