WO2015181746A1

WO2015181746A1 - Gel form of a heparin sodium salt for dermal administration, and a method for its preparation.

Info

Publication number: WO2015181746A1
Application number: PCT/IB2015/053974
Authority: WO
Inventors: Krzysztof BILMIN; Paweł GRIEB; Piotr SZOPIŃSKI; Marek LAGNER; Magdalena PRZYBYŁO
Original assignee: Lipolek Sp Z Oo
Current assignee: Lipolek Sp Z Oo
Priority date: 2014-05-29
Filing date: 2015-05-27
Publication date: 2015-12-03
Anticipated expiration: 2016-11-29
Also published as: PL229532B1; PL408371A1

Abstract

The solution, according to the present invention, concerns the lipid nanostructural form of the heparin sodium salt for dermal applications, of a viscosity not less than 7000 cP, characterized by the fact that it consists of: heparin, or a pharmaceutically acceptable salt thereof, liposomes and/or hydrogel, proteolytic enzymes of plant origin or their stabilized derivative, preferably chitosan, positive polyions, organic solvents, buffer and gelling agents and optionally: preservatives, antioxidants, and a method for the preparation of a lipid nanostructural form of heparin sodium salt for dermal applications.

Description

Gel form of a heparin sodium salt for dermal administration, and a method for its preparation.

The invention relates to a gel form of a heparin sodium salt intended for dermal use as a medicinal product or cosmetic, and a method for its preparation.

Heparin is a highly water-soluble linear carbohydrate polymer of natural origin, characterized by a high content of sulfonic groups and a high negative charge in aqueous solution. This substance has been widely used in medicine, especially as an active ingredient of drugs and parapharmaceuticals with systemic or local effect, whose task is primarily to prevent blood clotting and the formation of blood clots in blood vessels.

In the European Union heparin as a substance intended for pharmaceutical purposes - the so-called porcine heparin API (abbreviation of Active Pharmaceutical Ingredient) - is obtained from porcine intestinal mucosa, which is a post-mortem waste, and then purified according to the pharmacopoeial requirements. Porcine heparin is a polydisperse polymer, whose chains have a molecular weight within the range of 3-30 kD, and the average molecular weight of 15.5-17.0 kD (Sommers CD., Ye H., Kolinski .E., Nasr M., Buhse L.F., Al-Hakim A., Keire D.A., Characterization of currently marketed heparin products: analysis of molecular weight and heparinase-l digest patterns, Anal. Bioanal. Chem. 2011;401:2445-2454). API heparin is most commonly found in the form of a sodium salt. Its pharmacological activity is expressed in international units (IU), wherein the activity of porcine heparin API can not be less than 180 lU/mg. Medical applications have also found low molecular weight heparins having an average molecular weight of <8.0 kD, obtained by a partial depolymerization of the heparin carried out chemically, physically or enzymatically. An example of a low molecular weight heparin is enoxaparin. In comparison to the effects of heparin, the impact of low molecular weight heparins on the coagulation system is modified, resulting in an improved benefit-risk ratio, especially after intravenous administration (Bhaskar U., Sterner E., Hickey A.M., Onishi A., Zhang F., Dordick J.S., Linhardt R.J., Engineering of routes to heparin and related polysaccharides, Appl. Microbiol. Biotechnol. 93:1-16, 2012).

The formulations of heparin and fractionated heparins intended for intravenous or subcutaneous injection are found in the trade in the form of drugs available on prescription, because their anticoagulant effect is strong and can sometimes be accompanied by serious and dangerous side effects, such as autosomal bleeding. Depending on the indication, there are various doses of heparin or its derivatives used. For example, in the treatment of recent myocardial infarction not less than 37000 IU of heparin is administered per day by continuous intravenous infusion, while in the prophylaxis of deep venous thrombosis and pulmonary embolism post-surgically 5000 IU is administered subcutaneously twice or three times a day (Hirsh J, Dalen J, Guyatt G; American College of Chest Physicians. The sixth (2000) ACCP guidelines for antithrombotic therapy for prevention and treatment of thrombosis, American College of Chest Physicians, Chest 2001;119 (1 Suppl):lS-2S.). Availability only via medical prescriptions and the need for intravenous or subcutaneous injection limit the scope of the use of heparin injection formulations in conditions requiring medical intervention.

On the market are also present heparin formulations intended for dermal administration. They are in the form of creams, ointments or sprays and are available without prescription as parapharmaceuticals intended for self-treatment, used e.g. in the case of varicose veins of the lower limbs and the connected risk of superficial venous thrombosis, as well as swellings, pain and feeling of heaviness in the legs, or as dermocosmetics (medical cosmetics) which improve the skin condition. For some dermal preparations containing heparin, some clinical efficacy has been shown, although no systemic effects of reducing the clotting of circulating blood were observed. For this reason it is believed that the efficacy of heparin preparations for dermal administration in the prophylaxis of superficial venous thrombosis is significantly lower than the efficacy of intravenous or subcutaneous injections of heparin or low molecular weight heparins (Vecchio C, Frisinghelli A., Topically applied heparins for the treatment of vascular disorders: a comprehensive review, Clin. Drug Investig. 2008/28 603-614). An exception is the preparation in the form of an aerosol containing a liposomal form of heparin, whose composition is consistent with patent no. EP0704206 issued to the German company Mika-Pharma. Comparative studies of aerosols containing heparin according to the invention and subcutaneous administration of low molecular weight heparin (enoxaparin) have shown that two preparations are characterized by comparable efficacy in the prevention of lower limbs shallow vein thrombosis (Gorski G., Szopihski P., Michalak J., Marianowska A., Borkowski M., Geremek M., Trochimczuk M., Brotanek J., Sarnik S., Semenka J., Wilkowski D., Noszczyk W., Liposomal heparin spray: a new formula in adjunctive treatment of superficial venous thrombosis, Angiology 2005; 56:9-17; Katzenschlager ., Ugurlouglu A., Sipos G., Bihari I., Anyova E.B., Hirschl M., Maruszynski M., Noszczyk W., Rybak Z., Cencora A., Efficacy and tolerability of liposomal heparin spraygel as an add-on treatment in the management of superficial venous thrombosis, Angiology 2007; 58, Suppl 1:27S-35S; Katzenschlager R., Ugurluoglu A., Minar E., Hirschl M., Liposomal heparin- spraygel in comparison with subcutaneous low molecular weight heparin in patients with superficial venous thrombosis. A Randomized, controlled, open multicentre study, Journal fur Kardiologie - Austrian Jounal of Cardiology 2003; 10:375-378). However, the aerosol form of the formulation is its disadvantage, because the aerosol is reluctantly used by patients, being more associated with a cosmetic such as a deodorant rather than a drug or a medical cosmetic. In addition, spraying the preparation on the affected skin can be a source of an unpleasant feeling of cold, which is a result of the use of ethanol as an excipient.

The scientific literature also describes the liposomal forms of low molecular weight heparins (Song Y.K., Kim C.K., Topical delivery of low-molecular-weight heparin with surface-charged flexible liposomes, Biomaterials 2006; 27:271-280; Song Y.K., Hyun S.Y., Kim H.T., Kim C.K., Oh J.M., Transdermal delivery of low molecular weight heparin loaded in flexible liposomes with bioavailability enhancement: comparison with ethosomes, J. Microencapsul. 2011; 28:151-158). Low molecular weight heparins, due to the lower molecular weight of their carbohydrate chains, penetrate the skin better than unfractionated heparin (Betz G., Nowbakht P., Imboden ., Imanidis G., Heparin penetration into and permeation through human skin from aqueous and liposomal formulations in vitro, Int. J. Pharm. 2001; 228:147-159.). However, low molecular weight heparins are significantly more expensive than unfractionated heparin, which limits their practical applications in formulations for injections.

To increase the efficiency of active ingredients' penetration through the skin of dermatological and cosmetic preparations substances called permeability enhancers are often added. Mostly these are detergents, amphiphiles, terpenes or alcohols, which are used to destabilize the lipid portion of the horny part of the skin (Kanikkannan, N., Kandimalla, K., Lamba, S. S., and Singh, M. (2000) "Structure- activity relationship of chemical penetration enhancers in transdermal drug delivery" Curr Med Chem 7, 593-608). The use of such substances is often accompanied by skin irritation or the drying of the skin surface. When ethanol is used the patient will experience unpleasant cooling of the skin caused by the evaporation of alcohol. Cysteine-rich proteolytic enzymes of plant origin, such as papain and bromelain, have found numerous applications in cosmetology and cosmetic dermatology. For example, bromelain, an enzyme derived from pineapple, is used for cleaning up wounds caused by skin burns and for removal of scabs (Rosenberg L, Krieger Y, Silberstein E, Arnon O, Sinelnikov IA, Bogdanov-Berezovsky A, Singer AJ. Selectivity of a bromelain based enzymatic debridement agent: a porcine study. Burns 2012; 38:1035-1040). Papain is used dermally to improve the condition of the skin by removing or the transient destabilization of a protein intracellular matrix, which allows for an overall improvement of skin condition and removal of some of its heterogeneities or scars (S.J. Baik, B.Y. Kong, E.J. Kim, Cosmetic composition for exfoliating skin keratin, US Patent Application 2010/0254969 Al; Manosroi, A., Chankhampan, C, Manosroi, W., Manosroi, J. Transdermal absorption enhancement of papain loaded in elastic niosomes incorporated in gel for scar treatment, Eur J Pharm Sci 2013; 48: 474-483). Since these plant proteolytic enzymes are often not stable in cosmetics and forms of drug for dermal administration, various stable derivatives of these enzymes have been developed, for example papain conjugate with chitosan (Kilinc A., Onal S., Telefoncu A., Stabilization of papain by modification with chitosan. Turk. J. Chem. 2002; 26:311-316) and bromelain modified with anhydride groups [Y. Xue, C.-Y. Wu, C.J. Branford-White, X. Ning, H.-L. Nie, L.-M. Zhu, Chemical modification of stem bromelain with anhydride groups to enhance its stability and catalytic activity, Journal of Molecular Catalysis B: Enzymatic 63 (2010) 188-193). Proteolytic enzymes and their stabilized derivatives, although widely used in cosmetology and cosmetic dermatology, have never been used before as ingredients of heparin preparations for dermal administration.

The present invention is a gel containing heparin with an admixture of proteolytic enzymes, intended for use as a medicinal product or cosmetic, which is characterized by the durability required for such preparations, and at the same time increases the efficiency of heparin passing through the skin.

Heparin gel may contain excipients in the form of polymers (e.g. hydrogels) or lipid structures, whose function is to slow down the evaporation of water and to increase the efficiency of the active substance (heparin) passing through the skin. A suspension of neutral lipid aggregates in water has a tendency to aggregation and possible fusion, which significantly alters its properties and thus affects the release profiles of the active substance. To stabilize the suspension of lipid aggregates two solutions are used; placing the electrostatic charge on the aggregates surface and the use of polymers, which sterically stabilize the lipid aggregates suspension. These two modifications alone or in combination prevent an aggregation and possible fusion of liposomes ( Allen, T. M. and E. H. Moase: "Therapeutic opportunities for targeted liposomal drug delivery." Adv Drug Deliv Rev, 1996 21: 117-123; Barenholz, Y: "Liposome application: problems and prospects." Curr Opin Colloid & Interf. Sci, 2001, 6: 66-77; Heurtault, B., P. Saulnier, et al.: "Physico-chemical stability of colloidal lipid particles." Biomaterials, 2003 24: 4283-4300). Binding of strongly negatively charged heparin with lipid aggregates' surface allows for the electrostatic stabilization of the aggregates formed in this way. Lipid aggregates with bound heparin have a net negative surface charge, as shown in the accompanying examples (Fig. 1-5). Heparin's stabilizing effect on liposomes has also been observed previously by others (Han, H. D., A. Lee, et al.: "In vivo distribution and antitumor activity of heparin- stabilized doxorubicin-loaded liposomes." International J. Pharmaceutics 2006 313: 181-188). In addition, a spatial connection of heparin with lipid aggregates stabilizes the lipids' chemical integrity, thereby improving the chemical stability of the preparation of the present invention (Albertini, R., S. Rindi, et al.: "Heparin protection against Fe2+- and Cu2+-mediated oxidation of liposomes." Febs. +48 22 201 383 3 155-158). The addition to the formulation of the hydrogel, preferably 0.5%, further stabilizes the aggregates by their spatial separation (Mufamadi, M. S., V. Pillay, et al.: "A review on composite liposomal technologies for specialized drug delivery." J. Drug Delivery, 2011). In contrast to the present invention, the most effective liposome preparations containing heparin (e.g. Lipohep) on the market do not meet the criterion of physico-chemical stability, which significantly lowers their efficacy.

The penetration yield of active substances through the skin depends on two factors; the state of the stratum corneum and the concentration of active substance in the aqueous solution on the skin surface. The use of heparin alone results in a lack of its penetration into the dermis because it is not capable of penetrating the stratum corneum barrier. In addition, as a result of water evaporation it precipitates quickly on the skin surface, which results in a low functional stability of the preparation on the skin surface. For this reason preparations of this type have minimal efficacy. Another approach is a combination of heparin with a hydrophobic base, which limits the precipitation of the heparin on the skin surface, however, in this case, a low contact angle causes the preparation's contact surface with the skin to be limited, which reduces or even prevents the transport of heparin. The combination of heparin with a hydrogel slows down the evaporation of water and ensures good contact with the skin, however, both in this and previous solutions the stratum corneum barrier stays intact. This solution is also characterized by low efficacy resulting from a small stream of heparin penetrating the dermis. The combination of heparin with the lipid aggregates formed from natural lipids (as is the case of Lipohepie) beneficially influences the permeability of the stratum corneum, however, rapid evaporation limits the amount of heparin penetrating the dermis (Cevc, G., G. Blume, et al.: "The skin: A pathway for systemic treatment with patches and lipid-based agent carriers." Advanced Drug Delivery Reviews, 1996, 18(3): 349-378). Furthermore, the available liposomal preparation with the trade name LipoHep contains considerable amounts of ethanol (4-6%) in its composition, which may result in skin irritation.

The solution used in the present invention is characterized by the fact that the heparin may be adsorbed on the surface of stable liposomes or contained in a hydrogel matrix, which makes the process of water evaporation slow down considerably (Fig. 7). Additionally, excipients, such as lipids in the form of liposomes, a hydrogel, or both together, in each case together with a proteolytic enzyme act synergistically on the stratum corneum temporarily increasing its permeability for hydrophilic substances including heparin. This fact is illustrated in Fig. 9 where the penetration of hydrophilic substances through the skin is illustrated by the example of carboxy-fluorescein. (OECD-# 428) Liposomes modify the lipid fraction of the skin while the proteolytic enzymes destabilize the protein fraction of the epidermis. All this causes the penetration of heparin into the dermis to increase. (Fig. 8 and Fig 10), Preparation of the invention (Example 4) provides a much higher stream of heparin through the skin, measured on an in vitro model. Fig. 8 and Fig. 10 show cumulative amounts of heparin in the trans range measured on the Franz model for the preparation of the invention, and the most effective so far on the market, Lioton (OECD- #428).

The essence of the solution according to the invention is the form of the heparin sodium salt for dermal applications, having a viscosity of less than 7000 cP, characterized by the fact that it consists of: heparin, or a pharmaceutically acceptable salt thereof; a hydrogel or liposomes, or both together; a proteolytic enzyme of plant origin or its stabilized derivative; and also chitosan, positive polyions, organic solvents, buffer and optionally: preservatives, antioxidants and gelling agents.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the overall concentration of the positive polyions is between 0 and 10 % by weight.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the overall concentration of the chitosan is between 0 and 10 % by weight.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the overall concentration of heparin in the formulation, ensuring the activity of a substance in the bloodstream as a result of dermal administration is between 0.1 and 10 % by weight, preferably 2.4 % by weight.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the building block of the liposomes is phosphatidylcholine, preferably 1.2-palmitoyl-sn-glycero-3- phosphatidylcholine.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the overall concentration of 1.2-palmitoyl-sn-glycero-3-phosphatidylcholine is between 0 and 30 % by weight, preferably 10 % by weight.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the building block of liposomes is phosphatidylcholine mixed with the quaternary ammonium compound from the group of tensides, preferably with benzalkonium chloride.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the overall concentration of cationic substance is between 0 - 80 mol% relative to phosphatidylcholine, preferably 40 %.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that it contains single-layer liposomes.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that it contains multi-layer liposomes.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that it contains a mixture of single-layer and multi-layer liposomes. Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the heparin is in an unbound form with liposomes.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the heparin is in a bound form with liposomes.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the heparin is in a bound and an unbound form with liposomes.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the factor supporting penetration through the skin is a cysteine-rich proteolytic enzyme of plant origin, papain or bromelain, or a stabilized derivative of this enzyme.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the proteolytic enzyme or stabilized derivative thereof is in an aqueous phase outside the liposomes.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the proteolytic enzyme is located both inside and outside the liposomes.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the concentration of the proteolytic enzyme is in the range of 0.01 - 10 % by weight.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the organic solvents are compounds from the group of dihydroxyl alcohols.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the organic solvents are compounds from the group of short chain alcohols, preferably with a chain length of c2-c4.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the overall concentration of the dihydroxyl alcohol is between 0 and 30 % by weight, preferably 10 %. Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the organic solvents are compounds from the group of alkyl halides.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the overall concentration of organic solvent is not more than 0.5 % by weight of the formulation.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the buffer substance is 4-2-hydroxyethyl-l-piperazineethanesulfonic acid. Preferably, the lipid nanostructural form of heparin sodium salt form is characterized by the fact that the pH of the HEPES buffer (4-2-hydroxyethyl-l-piperazineethanesulfonic acid) is in the range of 6.0 to 8.0.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the antioxidant is a disodium edetate.

Preferably, the lipid nanostructural form of heparin sodium salt form is characterized by the fact that the preservative is a methyl parahydroxybenzoate.

Preferably, the lipid nanostructural form of heparin sodium salt form is characterized by the fact that the gelling agent is a copolymer of ammonium acrylamidomethylpropanosulfate and vinylpyrrolidone.

Preferably, the lipid nanostructural form of heparin sodium salt is characterized by the fact that the overall concentration of the copolymer of ammonium acrylamidomethylpropanosulfate and vinylpyrrolidone is between 0 and 5 % by weight.

The method for the preparation of the lipid nanostructural form of heparin sodium salt for dermal applications is characterized by the fact that the heparin, or its pharmaceutically acceptable salts, is partially complexed in a two-phase system with an organic solvent in the presence of cationic polyions in a system with/without chitosan, and is stabilized with a lipid system in an organic solvent and with the elimination of the solvent there occurs a transfer of a stabilized aggregate to an aqueous phase. The water portion, which is of low viscosity, by means of high pressure processes, different from the process of homogenization, turns to a high viscosity phase of not less than 7000 cPs. In addition, the proteolytic enzyme, an antioxidant, a preservative and a surplus portion of heparin are added, giving a product with a viscosity of not less than 7000 cPs, suitable for dermal administration in the form of a gel. The mixture of thus prepared liposomes is mixed with a gelling agent.

Preferably, the method for the preparation of the lipid nanostructural form of heparin sodium salt is characterized by the fact that the acquisition of its final, stable form does not require the use of gelling agents, and is provided by a suitable qualitative, quantitative composition and the manufacturing process.

Preferably, the method for the preparation of lipid nanostructural form of heparin sodium salt is characterized by the fact that the complexation of heparin with lipid aggregate is carried out using positive polyions.

Preferably, the method for the preparation of lipid nanostructural form of heparin sodium salt is characterized by the fact that the complexation of heparin with the lipid aggregate is carried out using chitosan. Preferably, the method for the preparation of lipid nanostructural form of heparin sodium salt is characterized by the fact that the complexation of heparin with the lipid aggregate is carried out using both chitosan ions and positive polyions.

Preferably, the method for the preparation of lipid nanostructural form of heparin sodium salt is characterized by the fact that the process of complexation of heparin with the lipid aggregate occurs as a result of the thermodynamic phase separation process.

The use of lipid nanostructures in dermal gel composition increases the bioavailability of heparin, and also the nanostructures used form a thin lipid film, modifying the lipid portion of the epidermis and protecting the ointment from drying too quickly, extending at the same time the absorption time of heparin and increasing its efficiency. In addition, the presence of a proteolytic enzyme destabilizes the protein components of the epidermis aiding the absorption process of heparin.

Nanostructures produced according to the following examples were subjected to standard size measurement using a dynamic light scattering method showing their homogeneity, and the sign and value of the surface charge is determined via the measurement of the zeta-potential.

The measurement of the value and zeta-potential of nanostructures was performed in the aqueous phase prior to the addition of a gelling substance.

As a result of carrying out the above procedure a nanostructure was obtained, the construction of which is schematically shown in Fig. 6. Negatively charged heparin is condensed on the surface of the chitosan. Due to the anisotropic nature of charge distribution on the polymer (heparin) surface after binding to chitosan, the aggregate still shows a negative charge (Fig. 4). In order to neutralize and stabilize the aggregate in the next step a lipid from an organic solvent with a positive net charge is added, stabilizing the nanoaggregate. Transferring the aggregate to the aqueous phase takes place in the vicinity of positively charged polyions, stabilizing the nanoaggregate in the presence of neutral amphiphilic particles, ensuring a uniform distribution of the complex. The outer layer is formed of a neutral lipid with a result that the nanostructure is readily dissolved in water and shows a uniform size distribution (Fig. 3, Fig.5).

The invention is illustrated by the following examples and the corresponding drawing, in which:

Fig. 1 contains a left panel showing the size distribution of chitosan in a buffer with pH 6.5 and a right panel showing the fitting of correlation function to the experimental data for a solution of chitosan in a buffer with pH 6.5.

Fig. 2 contains a left panel showing the size distribution of heparin condensed on the surface of chitosan in a buffer with pH 6.5 and a right panel showing the fitting of correlation function to the experimental data for a solution of heparin with chitosan in a buffer with pH 6.5. Fig. 3 contains a left panel showing the size distribution of heparin condensed on the surface of chitosan in envelopes from a lipid bilayer in a buffer with pH 6.5 with the addition of calcium ions a nd a right panel showing the fitting of the correlation function to the experimental data for a solution of heparin with chitosan in envelopes from a lipid bilayer in a buffer with the addition of calcium ions with pH 6.5.

Fig. 4 contains a left panel showing a zeta-potential distribution of heparin aggregates with chitosan in aqueous solution (the clearly negative (-40Mv) average value of the zeta potential is visible) and a right panel showing a phase diagram corresponding to the distribution from the left panel.

Fig. 5 contains a left panel showing a zeta-potential distribution of heparin aggregates with chitosan in aqueous solution (it is visible that the clearly increased value of the zeta-potential has shifted towards positive values (-6.8mV) average value of the zeta-potential) and a right panel showing a phase diagram corresponding to the distribution from the left panel.

Fig. 6 shows a block diagram of the lipid nanoaggregates "elastosome" containing condensed heparin on chitosan stabilized with positive polyions and a constant charge on the surface of the lipid.

Fig. 7 shows evaporation curves obtained for the heparin formulation of liposomal form, having the composition described in example 1 ( - A -) in comparison to the commercial preparation LipoHep (-♦-

)^■

Fig. 8 shows cumulative amounts of heparin in a trans range measured on the Franz model for the preparation of the invention (Example 4) and the market equivalent (LipoHep)

Fig. 9 shows cumulative amounts of hydrophilic fluorescent marker carboxy-fluorescein in a trans range measured on the Franz model for the preparation of the invention (P2 - Formulation of Example 4) and the market equivalent (Lioton) The obtained result shows increased penetration through a skin model (for the formulation from Example 4; P2) of hydrophilic substances in the presence of a proteolytic enzyme (5 % by weight of papain).

Fig. 10 shows cumulative amounts of heparin in a trans range measured on the Franz model for the preparation of the invention (Example 4) and the market equivalent (Lioton) The obtained result shows increased penetration through a skin model by heparin in the presence of a proteolytic enzyme (5 % by weight of papain). The graph also shows that increasing the amount of heparin (P3 - 2400 units of heparin) also increases its amount in the trans range.

EXAM PLE 1

Heparin (Shenzhen Hepalink) was dissolved at room temperature in purified water, followed by the addition of 2-[4-(2-hydroxyethyl)-l-piperazinyl] ethanesulfonic acid giving a buffer with pH = 6.5. A cationic lipid (DOTAP) (Avanti Lipids, USA) was dissolved in chloroform and added to the aqueous solution of heparin and chitosan. As a result, two immiscible phases were formed. Methanol was added to the sample so as to form one homogeneous phase.

The mixture was stirred at a temperature of about 25 °C until a homogeneous solution was obtained. Purified phosphatidylcholine dissolved in chloroform was added to a solution containing heparin, chitosan and a cationic lipid, then all was topped up with a HEPES buffer to the final volume (5 ml). This was all centrifuged in order to accelerate phase separation. The lower phase contained mostly chloroform with an excess of purified phosphatidylcholine and methanol. The upper phase containing nanostructures and the buffer was separated, taking care that it was not contaminated by the lower phase in order to determine the size distribution of nanostructures. The remaining methanol in the buffer phase was filtered using a MilliPore system. Finally, a proteolytic enzyme was added to the aqueous phase.

Table 1

The composition of the ready formulation according to example 1

Table 2

The composition of the ready formulation according to example 1

EXAMPLE 2

Heparin together with chitosan was dissolved at room temperature in a purified water, followed by the addition of 2-[4-(2-hydroxyethyl)-l-piperazinyl] ethanesulfonic acid and calcium chloride giving a buffer with pH = 6.0. Benzalkonium chloride was dissolved in chloroform and added to the aqueous solution of heparin, resulting in the formation of two immiscible phases. Methanol was added to the sample so as to form one homogeneous phase.

The mixture was stirred at a temperature of about 25 °C until a homogeneous solution was obtained. Purified phosphatidylcholine dissolved in the chloroform was added to a solution containing heparin, chitosan, calcium chloride and benzalkonium chloride, then the whole mixture was topped up with purified water to the final volume. The whole mixture was centrifuged in order to accelerate phase separation. The lower phase contained mostly chloroform with an excess of purified phosphatidylcholine. The upper phase containing mostly water was separated, taking care that it was not contaminated by the lower phase in order to determine the size of the nanostructures. The remaining methanol in the buffer phase was filtered using a MilliPore system. The aqueous portion was subjected to the gelation process involving gelling agents.

Table 3

The composition of the ready formulation according to example 2

Table 4

The composition of the ready formulation according to example 2 ACTIVE INGREDIENTS:

Heparin 5.0

EXCIPIENTS:

Purified Phosphatidylcholine 10.00

Benzalkonium chloride 8.00

Chitosan 10

2-[4-(2-hydroxyethyl)-l-piperazine]ethanesulfonic acid 0.15

(HEPES)

CaCI₂ 10.0

Papain 5

Purified water 51.85

SUM 100.00

EXAMPLE 3

Heparin and chitosan were dissolved at room temperature in purified water. Then 2-[4-(2- hydroxyethyl)-l-piperazinyl] ethanesulfonic acid and calcium chloride were added giving a buffer with pH = 8.0. Dimethyldioctadecylammonium bromide salt and l,2-palmitoyl-sn-glycero-3- phosphatidylcholine were dissolved in chloroform. After evaporation of the solvent to the formed dry lipid film, a heparin and chitosan buffer solution was added.

The whole mixture was stirred at a temperature of about 25 °C until a homogeneous suspension was obtained. The resulting suspension was extruded at 60°C under a pressure of 150 psi. As a result of the extrusion a gel with a viscosity of 9000 cPs was obtained.

Table 5

The composition of the ready formulation according to example 3

Table 6

The composition of the ready formulation according to example 3

Table 7

The composition of the ready formulation according to example 3 ACTIVE INGREDIENTS:

Heparin 0.01

EXCIPIENTS:

l,2-palmitoyl-sn-glycero-3-phosphatidylcholine 1.00 lecithin 15

Bromine salt of dimethyldioctadecylammonium 3.00

Chitosan 0.10

2-[4-(2-hydroxyethyl)-l-piperazine]ethanesulfonic acid 0.15 (HEPES)

CaCI₂ 10

Papain 5 copolymer of ammonium 5 acrylamidomethylpropanosulfate and vinylpyrrolidone

Purified water 60.74

SUM 100.00

EXAMPLE 4

Sample P2 with papain. Fosfolipon 90 G was added to propylene glycol. The whole mixture was stirred at a temperature of 50°C until complete dissolution of the lipid. Heparin was added to purified water and dissolved at room temperature. The thus prepared solutions were mixed together and full hydration of the lipid portion was allowed to occur, stirring the whole mixture at temperature of 30°C till a homogeneous suspension was obtained. The resulting suspension was extruded at 40°C under a pressure of 150 psi. As a result of the extrusion a gel with a viscosity of 10 200 cP was obtained. Then, papain and/or the fluorescent marker carboxyfluorescein was added to the thus obtained liposome suspension as an internal standard. For the final liposome gel a copolymer of ammonium acrylamidomethylpropanosulfate and vinylpyrrolidone was added.

Table 8

The composition of the ready formulation according to example 4

Table 9

The composition of the ready formulation according to example 4

EXAMPLE 5

Sample P3 with papain. Heparin was added to purified water and dissolved at room temperature. To the prepared solution was added 1 g of ammonium acryloyldimethyltaurate and polyvinylpyrrolidone copolymer and it was stirred at 25°C until complete crosslinking of the gel. To the thus prepared gel was added 3 g of papain and it was stirred until the enzyme dispersed in the gel structure.

Table 11 The composition of the ready formulation according to example 5

Claims

1. Heparin sodium salt form for dermal applications, characterised in that its viscosity is not less than 7000 cP, it consists of: heparin, or a pharmaceutically acceptable salt thereof, liposomes, and/or hydrogel, proteolytic enzyme of plant origin, and optionally chitosan, positive polyions, organic solvents, buffer, preservatives, anti-oxidants and gelling agents.

2. Heparin sodium salt form according to claim 1, characterised in that the overall concentration of positive polyions is of between 0 and 10 % by weight.

3. Heparin sodium salt form according to claim 1, characterised in that the overall concentration of chitosan is of between 0 and 10 % by weight.

4. Heparin sodium salt form according to claim 1, characterised in that the overall concentration of heparin in the formulation is of between 0.01 and 10 % by weight, preferably 2.4 % by weight.

5. Heparin sodium salt form according to claim 1, characterised in that the building block of liposomes is phosphatidylcholine, preferably l,2-palmitoyl-sn-glycero-3-phosphatidylcholine.

6. Heparin sodium salt form according to claim 5, characterised in that the overall concentration of 1,2- palmitoyl-sn-glycero-3-phosphatidylcholine is of between 0 and 30 % by weight, preferably 10 % by weight.

7. Heparin sodium salt form according to claim 1, characterised in that the building block of liposomes is phosphatidylcholine mixed with the quaternary ammonium compound from the group of surfactants, preferably with benzalkonium chloride.

8. Heparin sodium salt form according to claim 1, characterised in that the overall concentration of cationic substance is of between 0 - 80 mol% relative to phosphatidylcholine, preferably 40 mol%.

9. Heparin sodium salt form according to any one of claims 1 to 8, characterised in that its composition may contain single-layer liposomes.

10. Heparin sodium salt form according to any one of claims 1 to 8, characterised in that its composition may contain multi-layer vesicles.

11. Heparin sodium salt form according to any one of claims 1 to 8, characterised in that its composition may contain a mixture of single-layer and multi-layer liposomes.

12. Heparin sodium salt form according to any one of claims 1 to 11, characterised in that the heparin is in the unbound form with liposomes.

13. Heparin sodium salt form according to any one of claims 1 to 11, characterised in that the heparin is in the bound form with liposomes.

14. Heparin sodium salt form according to any one of claims 1 to 11, characterised in that the heparin is in the bound and unbound form with liposomes.

15. Heparin sodium salt form according to any one of claims 1 to 14, characterised in that it consists of a proteolytic enzyme of plant origin, preferably papain or bromelain, or a stabilised derivative of such an enzyme.

16. Heparin sodium salt form according to any one of claims 1 to 14, characterised in that the overall concentration of the proteolytic enzyme or its stabilised derivative is of between 0.01 and 10 % by weight.

17. Heparin sodium salt form according to any one of claims 1 to 16, characterised in that the proteolytic enzyme or its stabilised derivative is found on the outside of the liposomes.

18. Heparin sodium salt form according to any one of claims 1 to 16, characterised in that the proteolytic enzyme or its stabilised derivative is found on the inside of the liposomes.

19. Heparin sodium salt form according to any one of claims 1 to 16, characterised in that the proteolytic enzyme or its stabilised derivative is found on the inside and outside of the liposomes.

20. Heparin sodium salt form according to claim 1, characterised in that the organic solvents are compounds from the group of dihydroxyl alcohols

21. Heparin sodium salt form according to claim 1, characterised in that the organic solvents are compounds from the group of short chain alcohols, preferably of chain length of c2-c4.

22. Heparin sodium salt form according to claim 20, characterised in that the overall concentration of dihydroxyl alcohol is of between 0 and 30 % by weight, preferably 10 %.

23. Heparin sodium salt form according to claim 1, characterised in that the organic solvents are compounds from the group of alkyl halides.

24. Heparin sodium salt form according to claims 20 to 23, characterised in that the overall concentration of organic solvent is not more than 0.5 % by weight of the formulation.

25. Heparin sodium salt form according to any one of claims 1 to 24, characterised in that the buffer substance is 4-2-hydroxyethyl-l-piperazineethanesulfonic acid.

26. Heparin sodium salt form according to claim 25, characterised in that the pH of HEPES buffer (4-2- hydroxyethyl-l-piperazineethanesulfonic acid) is in the range of between 6.0 and 8.0.

27. Heparin sodium salt form according to any one of claims 1 to 26, characterised in that the antioxidant is disodium edetate.

28. Heparin sodium salt form according to any one of claims 1 to 27, characterised in that the preservative is methyl parahydroxybenzoate.

29. Heparin sodium salt form according to any one of claims 1 to 28, characterised in that the gelling agent is a copolymer of ammonium acrylamidomethylpropanosulfate and vinylpyrrolidone.

30. Heparin sodium salt form, according to claim 29, characterised in that the overall concentration of the copolymer of ammonium acrylamidomethylpropanosulfate and vinylpyrrolidone is of between 0 and 5 % by weight.

31. Method for the preparation of the lipid nano-structured form of the heparin sodium salt for dermal applications, characterised in that the heparin or its pharmaceutically acceptable salts, is partially complexed in a two-phase system with organic solvent in the presence of cationic polyions in a system with/without chitosan, is stabilised with the lipid system in an organic solvent and in the process of solvent elimination a stabilised aggregate is carried out to the aqueous phase, wherein the aqueous fraction with a low viscosity by means of high pressure processes, other than the process of homogenisation, is carried out to a phase of high viscosity of not less than 7000 cP, moreover the remaining organic solvents, proteolytic enzyme, antioxidant, preservative and surplus portion of heparin are being added, giving a product with a viscosity of not less than 7000cP, suitable for dermal supply in the form of a gel, and additionally the mixture of liposomes is mixed with the gelling agent.

32. Method for the preparation of the lipid nano-structured heparin sodium salt form according to claim 31, characterised in that the obtaining of its final, stable form does not require the use of gelling agents, and is provided by a suitable qualitative and quantitative composition and the appropriate manufacturing process.

33. Method for the preparation of the lipid nano-structured heparin sodium salt form according to claim 30, characterised in that the stabilisation of heparin with lipid aggregate is carried out using positive polyions.

34. Method for the preparation of the lipid nano-structured heparin sodium salt form, according to claim 30, characterised in that the stabilisation of heparin with lipid aggregate is carried out using chitosan.

35. Method for the preparation of the lipid nano-structured heparin sodium salt form, according to claim 31, characterised in that the stabilisation of heparin with lipid aggregate is carried out using both chitosan ions and positive polyions. Method for the preparation of the lipid nano-structured heparin sodium salt form, according to claim 31, characterised in that the process of heparin stabilisation with lipid aggregate is the result of the thermodynamic process of phase separation.