WO2007110767A2

WO2007110767A2 - Interactive dressings for treatment of dermatological diseases

Info

Publication number: WO2007110767A2
Application number: PCT/IB2007/000803
Authority: WO
Inventors: Paola Perugini; Khaolé HASSAN; Claudia Colonna; Ida Genta; Tiziana Modena; Franca Pavanetto; Paolo Iadarola; Bice Conti
Original assignee: Universita' Degli Studi Di Pavia
Priority date: 2006-03-24
Filing date: 2007-03-22
Publication date: 2007-10-04
Also published as: WO2007110767A3; ITMI20060556A1

Abstract

Object of the invention is an interactive dressing the treatment of dermatological disorders. The invention comprises: a support, preferably a tissue non-tissue gauze, a hydrogel of a bioadhesive polymer like chitosan or hydroxyethylcellulose and liposomal systems containing an active principle of protein nature. It is a solid therapeutic system consisting of a dispersion of liposomes in hydrogel adsorbed on a tissue non-tissue gauze. The association of hydrogel with liposomal systems allows the site specific and prolonged release of active substances, through intracellular transfer of liposomes and therefore of the encapsulated drug. In addition to act as a storage form, liposomes protect the drug from enzymatic attacks, thus preserving its activity. The therapeutic system containing preferably the prolidase enzyme is used for the treatment, in human and mammals, of dermatological disorders with or without epithelial ulcers, preferably for treatment of ulcers in subjects affected by genetic prolidase deficiency.

Description

INTERACTIVE DRESSINGS FOR TREATMENT OF DERMATOLOGICAL

DISEASES

Field of the invention

The present invention relates to the field of dressings for topical use. State of the art Interactive dressings

The treatment of ulcers or sores, i.e. loss of substance lesions involving soft tissues due to external causes or endogenous pathological processes, with tendency to become chronic, involves elimination of necrotic tissue, exudate absorption and sore moisturizing in order to facilitate recovery.

For the management of this type of lesions, that often chronicize due to cell necrosis or infections, it is fundamental to keep a moisturized environment because, unlike wounds, scab formation is detrimental for ulcers and delays healing. For this purpose, several dressings present on the market have been developed (DuoDERM^®, Aquacel^®, Allevyn^®) which act both passively, through the absorption of the exudate, and actively by sore moisturizing, but do not contain liposomes or active principles of proteic nature.

Main components of interactive dressings are polymers such as alginates, gelatin, solid or hydrogel carboxymethylcellulose capable of absorbing large amounts of exudate.

Commercial products are represented by gel containing tubes or different forms of tissues composed by the primary components and possibly equipped with adhesive borders and waterproof film. Liposomes Liposomes are fatty vesicular structures consisting of a double phospholipid layer surrounding one or more aqueous compartments, used since a long time in the pharmaceutical field as therapeutic system for drug delivery. This type of delivery system provides several advantages such as the possibility to target the drug to the desired site, drug stabilization against endogenous degrading factors and, at last, the possibility of obtaining a prolonged effect (depot). Liposomes can have different physico-chemical characteristics such as: size, ranging from 30 nanometers to 10 micrometers; surface charge, fluidity of the phospholipid layer and membrane composition, all of which can affect delivery of the active principle. Prolidase and Prolidase deficiency Prolidase is an endothelial cytoplasmatic enzyme involved in protein catabolism, more precisely in the final step of collagen degradation. Production of a modified, inactive form of the enzyme causes a rare autosomal recessive disease termed prolidase deficiency (PD). About a hundred cases of this disease have been reported worldwide, prevalently in Italy. Disease severity varies from one subject to another. Generally, collagen is most affected: skin ulcers and scar formation (affecting especially the lower limbs), skin thickening associated with lymphedema, telangiectasia, photosensitivity and recurrent infections. Presently, there is no effective therapy for prolidase deficiency. Current conventional therapy involves:

S Topical application of cicatrizant and antibiotic ointments to patient's ulcers. S Topical application of Pro or Gly-Pro containing ointments to skin ulcers. S Blood transfusions from healthy individuals, containing erythrocytes with normal prolidase concentrations. This type of therapy is very expensive and requires hospitalization.

A liposomal system for systemic delivery of prolidase enzyme has been proposed recently by the authors of the present invention. The results have shown that liposomes can transfer the enzymatic activity to in vitro cultured fibroblasts (P.Perugini et a/. Journal of Control. ReI. 102, 181-190, 2005) that are genetically deficient in prolidase enzyme. A user-friendly formulation for topical use, capable of releasing liposomes directly on ulcerous tissues, thereby promoting an effective direct action, was not yet devised. Summary of the invention The invention relates to an interactive dressing comprising: a) a support, b) a hydrogel carrier consisting of polysaccharide polymers, acrylic polymers, proteins or high molecular weight polyhydroxyl compounds. c) liposomes containing an active principle consisting of an active principle of protein nature.

According to a preferred embodiment, said active principle is an enzyme selected among: prolidase, collagenase, hialuronidase, lipase, amylase or is a growth factor and the hydrogel b) of polysaccharidic nature is selected from the group consisting of: cellulose and its derivatives, alginates or derivatives, pectin or derivatives, chitosan or its derivatives, chondroitin or derivates, chondroitin sulphate, vegetable rubber or derivatives. Liposomes c) comprise lipids selected among: phospholipids and/or distearoylphosphatidylcholine and/or sphingolipids (ceramides) and/or cholesterol, where phospholipids are preferably selected among: phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine or their derivatives and derivatized compounds, where distearoylphosphatidylcholine, if present, is preferably derivatized with polyethylene glycol. The dressing preferably containing a liposomal composition containing the enzyme prolidase is preferably sterile and can be in lyophilized form. Its use is claimed for direct topical application on the lesion site or ulcer, especially in subjects affected by genetic prolidase deficiency. Description of the figures Figure 1 - TEM (Transmission electron microscopy) photomicrograph of prolidase containing liposomes.

Figure 2 - SEM (Scansion Electronic Microscope) photomicrograph of fibroblasts after 6 days of contact with chitosan-based interactive dressing (the fusion of liposomes with cellular membranes is visible). Detailed description of the invention

The invention is represented by solid, semi-solid or flexible supports capable of delivering, by means of a hydrogel, liposomes loaded with an active principle of protein nature, preferably the enzyme prolidase, that can be directly applied to the skin. In the present invention the applicant has solved the problem of an effective delivery of liposomes containing the active principle through the skin, by mixing said liposomes with a hydrogel, making topical application possible, convenient and easy.

In fact the system, as proposed, can be directly applied by the patient to the skin without intervention of health professionals and no need for hospitalization. Furthermore, in the specific case, it can be stored for a long time in sterile and/or lyophilized form.

In the dressing according to the invention, the support can be cotton, in the form of tissue or gauze or tissue non-tissue (TNT). A hydrogel composed of polysaccharidic or polyacrylic polymers, generally endowed with bioadhesive and biocompatible properties, is adhered to said support by pouring or spreading. Particularly preferred are polysaccharidic polymers such as: chitosan, cellulose, cellulose derivatives, hyaluronic acid, chondroitin sulphate, alginates. Among those, even more preferred is chitosan and/or its salts or derivatives. Said polymers, in hydrated or re-hydrated form, deliver liposomes containing a proteinaceous substance such as an enzyme with pharmacological or cosmetic activity (e.g. prolidase, collagenase, hyaluronidase, growth factors, lipase, amylase). Particularly preferred is the enzyme prolidase.

The enzyme prolidase (Myara I. et al, Life Sciences, 34, 1985-1998, 1984) is a dimeric molecule; human prolidase is composed of 492 amino acids and has a molecular weight of 110000 Daltons; 2 subunits of 55000 Daltons are produced by degradation. The presence of the divalent cation Mn²⁺ in the enzyme active site is essential for enzyme activity

Said enzyme is usually extracted from natural sources, primarily pig kidney, and is commercially available from Biozyme Laboratories (Blaevon, GB). Alternatively, the human recombinant enzyme is preferably used. Prolidase is classified as an essential enzyme because of the unique ability to cleave imino peptides at the C-terminus of Pro (proline) or Hyp (hydroxyproline), a fundamental step for degradation and synthesis of new collagen. In addition to human, prolidase is present also in other animals and in bacteria. In the human body, the highest prolidase activity is found in kidney, intestinal mucosa and erythrocytes; the activity is also present in other organs such as liver, brain, heart, uterus, and in cells like leukocytes and fibroblasts. Production of a modified, inactive form of the enzyme causes a rare autosomal recessive disease termed prolidase deficiency (PD), with different severity from one subject to another. Case studies have shown that the PD phenotype is very heterogeneous even though the consequences on collagen are most common, leading to spontaneous skin ulcers and scar formation (affecting especially the lower limbs), skin thickening associated with lymphedema, telangiectasia, photosensitivity and recurrent infections.

Thus, one of the preferred embodiments according to the invention comprises the use of supports according to the invention, wherein liposomes contain the enzyme prolidase, for topical therapy of ulcers in patients affected by prolidase deficiency. Liposomes are vesicular structures primarily composed of lipids. Different methods for preparation of liposomes are available. In the specific case, involving delivery of active enzymes, conditions for liposome preparation were optimized in order to take into account the parameters affecting chemical stability (deamination, hydrolisis, B-elimination, disulphide bridges) and physical stability (denaturation, precipitation, aggregation) of the proteinaceous material.

According to a preferred embodiment, liposomes are prepared as described in Perugini et al. J. Controlled Release, 2005, 102: 181-190, dissolving the lipid components, preferably phospholipids and cholesterol, in a molar ratio preferably close to 1 :1 , in an organic solvent, preferably chloroform. Liposomes used in the invention have preferably the following weight composition: phospholipids (preferably comprising phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine or their derivatives and derivatized products) between 60 and 80%, cholesterol between 20 and 40%. Briefly, liposomes are prepared by evaporating the organic solvent from the phospholipid containing solution, preferably at a temperature above 25°C and reduced pressure, even more preferably at 40° C, for at least 3O'-1 hour, then liposomes are treated with a nitrogen flow at 0.01 atm for at least 30' (1 hour). The lipid film is subsequently hydrated (for 1 hour at room temperature) with a buffer solution, preferably Tris pH 8 comprising a reducing agent, for instance 0.1 mM glutathione and 1.2 mM manganese chloride (TMG) and the enzyme prolidase at concentrations of 0.1-5 mg protein/ml, more preferably 0.2- 1 mg protein/ml and bovine serum albumin (BSA) at concentrations lower or equal to 2%, preferably lower than 1 %, even more preferably at a concentration of 0.5% (w/v). Optionally liposomes are purified for example by centrifugation preferably carried out at 16400 rpm, 15°C, for 20 minutes.

In the liposomes of the invention, together with cholesterol, sphingolipids can be used as an alternative to phospholipids (ceramids).

However, according to a preferred embodiment, liposomes are composed of phospholipids, cholesterol and distearoylphosphatidylcholine derivatized with polyethylene glycol with molecular mass comprised between 1000 and 7500 Dalton or preferably comprised between 1000 and 5000D, even more preferably comprised between 1500 and 3000D or 2000 D.

Distearoylphosphatidylcholine derivatized with polyethylene glycol, in which polyethylene glycol is covalently linked to phospholipid molecules when used in the formulation of liposomes leads to a modification of the liposomal membrane giving rise to a class of liposomes termed "stealth". These liposomes are not detected by cells of the immune system and remain in circulation for much longer than conventional liposomes, and thus are better directed to their sites of action. The primary effect of these molecules is to create around the liposome a hydrophilic cloud that reduces the uptake by cells of the reticuloendothelial system (RES). Therefore, the realization of stealth liposomes is particularly preferred for the preparation of the dressing. According to this last embodiment, liposomes have the following weight composition: phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine) in variable molar ratios corresponding to concentrations in weight comprised between 25 and 70 %, cholesterol between 10 and 25%, distearoylphosphatidylcholine derivatized with polyethylene glycol between 20 and 50%.

Liposome mediated delivery of pharmacologically active proteins is a well known technique that offers the following advantages: liposomes stabilize the active principle, make possible its prolonged release over time and deliver the active principle to the cell through a mechanism of fusion with cellular membranes. In addition to the pharmacologically active protein, excipients, diluents, stabilizers or proteins devoid of pharmacological activity (e.g. serum albumin) can be carriered within the liposome; moreover diluents and/or excipients or ions can be present in a buffer system that is included in the liposome, together with the active principle, by means of liposome hydration. In the case of the enzyme prolidase, the buffer system is preferably Tris.

According to a preferred embodiment, the enzyme prolidase is present in the interactive dressing at a support concentration not lower than 1 μg/cm², more preferably comprised between 5 and 20 μg/cm², with an enzymatic activity not lower than 3 IU/cm² or more preferably comprised between 2.5 and 15 IU/ cm² (IU, 1 International Unit is defined as the amount of enzyme that hydrolizes 1 μmole of Gly-Pro in 1 minute at 37 ⁰C) . The finished therapeutic system can be lyophilized, and in this case will appear as a solid system, while the therapeutic system that is not lyophilized will appear as hydrated formulation. Would the dressing need to be sterile, as for instance for application to open ulcers, aseptic conditions are produced by using sterile raw materials and liposomes sterilized by filtration. The association of hydrogel with liposomal systems in the interactive dressing of the invention makes possible a site specific and prolonged release of active substances by intracellular transfer of the liposome, hence of the encapsulated drug or enzyme. In addition to act as a form of storage, liposomes protect the enzyme from degradation and preserve its activity. Moreover, the presence of hydrogel is indispensable to keep the ulcer moisturized, thus avoiding scab formation and therefore accelerating healing.

The aim of the proposed therapeutic system, consisting of a combination of hydrogel and liposomes, is the treatment, in human and mammals, of dermatological disorders that show epithelial ulcers. Said system achieves the following objectives:

- it is able to deliver pharmacologically active proteinaceous substances, keeping their activity unchanged and stabilizing them;

- it is able to release therapeutic concentrations of said proteinaceous substances directly at the cellular level; - it optimizes cellular penetration of the active principle,

- it keeps a moisturized environment suitable for tissue repair.

According to a further aspect, the invention relates to a process for the preparation of dressings, involving the preparation of liposome-containing hydrogels performed by mixing in the cold a solution containing the polymer, preferably chitosan and/or its salts or derivatives, at a concentration comprised between 0.5-2% w/w, with a suspension of liposomes at a concentration comprised between 0.2 mg/ml and 1.2 mg/ml, more preferably between 0.3 and 0.9 mg/ml. Said mixture is distributed by pouring or spreading on a solid support, so that the final concentration of liposome suspension in the hydrogel is not lower than 0.4 μmol lipids/mg of polymer and never higher than 10 μmol lipids/mg of polymer or, even better, is comprised between 0.5 and 2 μmol lipids/mg of polymer or, even more preferably, the concentration of vesicles is comprised between 0.4 and 1.25 μmol lipids/mg of polymer, optimal for stabilization of the finished product.

According to a preferred aspect, liposomes are prepared by a method of hydration of the lipid film followed by an extrusion process. Hydration is preferably carried out at temperatures not higher than 45°C for a time comprised between 30' and 2 hours, more preferably 1 hour. The preferred buffer for hydration of liposomes in presence of the enzyme prolidase is a Tris buffer pH 8 containing 0.1 mM glutathione and 1.2 mM manganese chloride (TMG). The extrusion phase which makes possible to obtain unilamellar liposomes of the desired size can be performed by using polycarbonate filters of different porosity of 50, 100, 200 nm, preferably 100 nm. It is indispensable to use the nitrogen flow at a pressure not higher than 5 atmospheres.

Moreover, the method may comprise an optional step of lyophilization of the finished dressing. The proposed interactive dressing is preferably directed to local application on open ulcers: the support facilitates system application, the bioadhesive gel optimizes persistence in-place of the system and serves as vehicle for liposomes. Liposomes are the vehicle of the active principle, stabilize the active principle, make possible its prolonged release over time and deliver the active principle in the cell by a mechanism of fusion with cellular membranes. According to what has been determined for the specific realization concerning prolidase, the dressing prepared according to the invention can release active prolidase for at least 6 days.

The proposed interactive dressing is directed to therapies of dermatological disorders, as for instance ulcers that occur in subjects affected by prolidase deficiency. In addition to this type of application, the dressing can be used for moisturized treatment of wounds with healing problems (pressure sores, venous ulcers) or for cosmetic treatments, as for instance deep enzymatic peeling for the treatment of acne. In the mentioned cosmetic treatments, the dressing can deliver for instance lipase and amylase as active substance for peeling or collagenase for treatment of cheloids. In the case of other pharmacological treatments, the dressing can deliver growth factors with cicatrizant action.

According to a further aspect, the invention relates to the use of liposomes containing the enzyme prolidase for the preparation of dressings where said liposomes are present in a hydrogel preferably composed of polymers of polysaccharidic or proteic nature, of acrylic polymers or of high molecular weight polyhydroxyl compounds. According to a preferred embodiment, the polymer of polysaccharidic nature is selected from the group consisting of: cellulose and its derivatives, alginates or derivatives, pectin or derivatives, chitosan or its derivatives, chondroitin or derivates, chondroitin sulphate, vegetable rubber or derivatives. ,

EXPERIMENTAL PART

Example 1. Preparation of liposomes

The solution of lipids in chloroform, containing 0.013 mM phosphatidylcholine

(EPC), 0.013 mM cholesterol (Choi) and 0.0026 mM distearoylphosphatidylcholine derivatized with polyethylene glycol 2000 (DSPE-PEG), was desiccated in a rotating evaporator at 40° C and reduced pressure, for 1 hour, at a rotation speed of 110 rpm, followed by treatment with nitrogen flow at 0.01 atm for 1 hour. The hydration of the lipid film was carried out for 1 hour at room temperature with Tris buffer pH 8 containing 0.1 mM glutathione and 1.2 mM manganese chloride (TMG) containing prolidase at concentrations of 0.266 - 1.064 mg protein/ml and 0.5 % bovine serum albumin (BSA). Liposomes were purified by centrifugation at 16400 rpm, 15°C, for 20 minutes. At last, the suspension was subjected to 10 extrusion cycles through a 0.1 μm polycarbonate membrane in an extruder, using a nitrogen flow at a pressure of 5-6 atmospheres.

The extruded unilamellar liposomes, homogeneous in size, were filter sterilized trough a 0.22 μm millex membrane and collected in a bacteriological tube in order to ensure sterility.

The same procedure was also used to prepare placebo liposomes, having the same composition described for liposomes but not containing the enzyme prolidase. The morphology of liposomes was characterized by transmission electron microscopy and their size was analyzed by a light diffraction device. Moreover, liposomes were characterized for their lipid content by HPLC analysis, for their total protein content by spectrophotometric analysis and, in the case of prolidase- containing liposomes, the amount of encapsulated active enzyme was determined by HPLC.

Liposomes of homogenous shape and size were obtained (Fig. 1), where their size was equal to: d₅o 0,122 μ, d_go 0,144μ. The production yield in terms of lipids was always higher than 60%, the content in total protein and active protein varied with the theoretical ratio (w/w) between prolidase and BSA used for liposome preparation. The best results were obtained with liposomes containing a theoretical ratio prolidase/BSA of 1 :5 (lot a), which were found to have a protein content of 167.69 μg/μmol lipids, a protein encapsulation efficiency of 72.08% and an enzymatic activity of 8.67 units/μmol lipids, where 1 unit is defined as the amount of enzyme that hydrolizes 1 μmole of Gly-Pro in 1 hour at 37 ⁰C. The so obtained liposomes showed good morphological characteristics with respect to shape, size and loading. The preparation method turned out to be advantageous because the stability of the delivered protein was not negatively affected and the method can be easily transferred on industrial scale. Example 2. Preparation of the interactive dressing The gel-forming polymer chitosan glutamate was dissolved in water at a concentration of 1.5%, in the cold and under magnetic stirring, and the solution obtained was autoclave sterilized before use. Liposomes prepared as explained in example 1 were dispersed in suspension at a concentration of 0.315mg/ml and then the two solutions were mixed. The liposomal suspension in chitosan was dispensed on pieces of TNT gauze to obtain a final concentration of suspension of 1.5 mg/cm²; the so prepared gauzes were subjected to lyophilization before use.

TNT gauzes, containing liposomal suspensions in highly viscous hydroxyethylcellulose, were prepared using the same procedure and concentrations. The so prepared gauzes were subjected to lyophilization before use. The homogeneity of protein distribution within the gauze was evaluated. Each 1.5 cm² area of the gauze was dissected and separately analysed for its protein content, after exposure to a 1% Triton TX 100 solution in TRIS buffer. Protein amount was determined by spectrophotometric analysis at 562 nm, exploiting the colorimetric reaction that takes place with Bicinchoninic acid (BCA protein assay). Results showed an average protein concentration of 9 ±1 μg/cm².

The interactive dressing was found to be homogeneous with respect to morphology, thickness and distribution of the protein.

Example 3. Evaluation of toxicity of materials composing the interactive dressing on fibroblast cultures. Placebo liposomes, at concentrations ranging between 0.4 and 12 μmol lipids/ml, were incubated with fibroblasts obtained from skin biopsies from patients affected by prolidase deficiency. Cells in 3.5 cm² diameter Petri dishes were grown in Dulbecco modified Eagle's medium (DMEM). After incubation for 5 and 10 days in presence of different concentrations of liposomes, cells were trypsinized, centrifuged and resuspended in a small aliquot of medium (about 2 ml). The suspension was used to count the cell number by use of the Neubauer chamber.

The assessment of cell growth as function of the concentration of polymer (chitosan or hydroxyethylcellulose) in contact with fibroblasts was made by a cell viability test that makes use of methylthiazoldiphenyltetrazolium bromide (MTT) as cell viability revealing agent. With respect to liposomes, the best results were obtained with vesicle concentrations ranging between 0.4 and 1.25 μmol Iipids/ml. Results of the cell viability test carried out on polymers showed that the maximum tolerated concentration of hydroxyethylcellulose was 140 μg/10000 cells, corresponding to 15 μg/10000 cells for the chitosan derivative used in this study. The results obtained made possible to determine the concentrations of individual materials to be used for preparation of the interactive dressing. Said concentrations were comparable to those expected for a dressing of 10 cm diameter. Example 4. Ex-vivo assessment of prolidase activity on fibroblast cultures. Fibroblasts obtained from skin biopsies from patients affected by prolidase deficiency (PD) were placed in suitable culture flasks in presence of Dulbecco modified Eagle's medium (DMEM).

Series of fibroblast cultures from PD patients were set up in each experiment: one was treated with samples containing liposomes loaded with prolidase (A series), one was incubated with samples of dressings based on chitosan gel containing liposomes loaded with prolidase (Ai series), one was incubated with samples of dressings based on hydroxyethylcellulose gel containing liposomes loaded with prolidase (A₂ series), while the other untreated series was used as control (B series). After 24 hours the initial medium was respectively replaced with: DMEM containing 1 % of lTS+3 serum substitute for the B series; and the same medium containing also samples with prolidase for A, A1 and A2 series. Using this procedure, 12 cell cultures were set up: 4 for the A series, 4 for the A1 series, 4 for the A2 series and 4 for the B series, with different incubation times ranging from 24 hours to 10 days for A and B series, and of 6 days for Ai and A₂ series, respectively.

At defined times, the medium was removed and dishes were washed with PBS buffer to remove traces of culture medium. The PBS washing buffer was used to analyze prolidase activity that was determined by capillary electrophoresis. Fibroblasts were at the same time treated with PBS solution containing KCI, protease inhibitors, TX-100, agitated for 30 minutes at room temperature, denatured at 80° C for 15 minutes and centrifuged for 30 minutes at 8000 rpm, 4° C. The supernatant was concentrated by a microcon 3000 filtration system for 5 minutes at 5000 rpm and at room temperature, and this material was the cell extract used for determination of prolidase activity by capillary electrophoresis. Maximum enzymatic activity was detected after 6 days, but about 30% of the enzymatic activity was still present after 10 days. Table 1 shows the results of quantitative determination of prolidase activity in fibroblasts after 6 days of incubation with prolidase-containing liposomes or with dressings containing liposomes carriered in chitosan or cellulose gel. No enzymatic activity was detected in the culture medium.

In cell extracts a very high enzymatic activity was detected, corresponding to all the active protein loaded in liposomes.

Together with the absence of enzymatic activity in the cell medium, this result makes possible to conclude that prolidase enters fibroblasts in an active form. This result was confirmed by SEM analysis (Figure 2): this analysis showed a process of fusion of liposomal vesicles with fibroblast membranes that could be responsible for transfer of the enzyme inside the cells.

Table 1 - Enzymatic activity determined in cellular extracts after 6 days of incubation in presence of prolidase-containing liposomes, interactive dressing based on chitosan, interactive dressing based on ethylcellulose.

(1) Healthy fibroblasts, mean value estimated on 20 determinations

(2) Fibroblasts of patients affected by Prolidase Deficiency (P. D.), mean value estimated on 7 determinations.

(3) PD fibroblasts + prolidase containing liposomes, mean value estimated on 7 determinations. (4) P. D. fibroblasts + interactive dressing based on chitosan, mean value estimated on 3 determinations.

(5) P. D. fibroblasts + interactive dressing based on ethylcellulose, mean value estimated on 3 determinations. *1 unit is defined as the amount of enzyme that hydrolizes Iμmole of Gly-Pro in 1 hour at 37 ⁰C

Results showed that the interactive dressings of the present invention were able to transfer the active enzyme inside enzyme-deficient fibroblasts. In particular, the best results were obtained using chitosan as the polymer, which appears to be best suited to deliver liposomes while keeping prolidase activity practically unaltered.

As further evidence of the efficiency of transfer of the active principle, it was also observed that diseased fibroblasts acquired a normal appearance after exposure to the interactive dressing, indicating the recovery of cellular activity.

Claims

1) Interactive dressing comprising: a) a support, b) a hydrogel carrier consisting of polymers of polysaccharide or protein nature, acrylic polymers, high molecular weight polyhydroxyl compounds. c) liposomes containing an active principle consisting of one or more enzymes or growth factors.

2) The dressing according to claim 1 wherein said support a) consists of gauze, tissue or tissue non-tissue. 3) The dressing according to claim 2 wherein said support is made of cotton or cellulose.

4) The dressing according to claim 1 wherein liposomes c) contain an enzyme selected among: prolidase, collagenase, hyaluronidase, growth factors, lipase, amylase. 5) The dressing according to claim 4, wherein said enzyme is prolidase.

6) The dressing according to claims 1-5, wherein the hydrogel b) of polysaccharidic nature is selected from the group consisting of: cellulose and its derivatives, alginates or derivatives, pectin or derivatives, chitosan or its derivatives, chondroitin or derivates, chondroitin sulphate, vegetable rubber or derivatives.

7) The dressing according to claim 6 wherein the hydrogel is chitosan and/or its salts or derivatives.

8) The dressing according to claim 1 wherein the hydrogel of proteic nature is gelatin. 9) The dressing according to claim 1 wherein the hydrogel of acrylic nature is a polyacrylic acid or a polymetacrylate.

10) The dressing according to claim 1 wherein said high molecular weight polyhydroxyl compounds are selected among polyalkylene-glycol or its derivatives, polyvinylpirrolidone or its derivatives. 11) The dressing according to claims 1-10 wherein said liposomes c) comprise lipids selected among: phospholipids or their derivatives, phospholipids derivatized with polyethilene-glycol and/or sphingolipids (ceramides) and/or cholesterol. 12) The dressing according to claim 11 wherein liposomes comprise phospholipids in a proportion from 60 to 80 % and cholesterol from 40 to 20%.

13) The dressing according to claims 11-12 wherein said phospholipids are selected from the group consisting of phosphatidylcholine and/or phosphatidylserine and/or phosphatidylethanolamine or their derivatives and in which a phospholipid is derivatized with polyethilene-glycol.

14) The dressing according to claim 13 wherein polyethilene-glycol has a molecular mass comprised between 1000 and 7500 Dalton or preferably comprised between 1000 and 5000D, even more preferably of 2000 D. 15) The dressing according to claim 14 wherein the phospholipid derivatized with polyethilene-glycol is distearoylphosphatidylcholine.

16) The dressing according to claim 5 and 11-15 wherein the active principle contained in liposomes is in aqueous solution.

17) The dressing according to claim 16 wherein said aqueous solution is a Tris (Tris(hydroxymethyl)aminomethane) buffer with a pH value equal to 7 or higher.

18) The dressing according to claim 17 wherein said buffer solution comprises in addition glutathione and manganese chloride.

19) The dressing according to claims 13-18 wherein liposomes have the following weight composition: phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine in weight ratios ranging between 25 and 70 %, cholesterol in weight ratios ranging between 10 and 25%, distearoylphosphatidylcholine derivatized with polyethylene glycol in weight ratios ranging between 20 and 50%.

20) The dressing according to claims 5 and 13-21 comprising as enzyme or growth factor the enzyme prolidase at a concentration not lower than 1 μg/cm² and with an activity not lower than 2.5 IU/cm² of support.

21) The dressing according to claim 22 wherein the concentration of said enzyme is comprised between 5 e 20 μg/cm² of support and wherein the enzymatic activity is comprised between 3 and 15 IU/cm² of support. 22) The dressing according to claims 1-21 in lyophilized form.

23) The dressing according to claims 1- 22 in sterile form.

24) Method for the preparation of the interactive dressings according to claims 1- 21 , comprising: i) preparation of a liposome suspension at a concentration comprised between 0.2 mg/ml and 1.2 mg/ml, more preferably between 0.3 and 0.9 mg/ml, and of a polysaccharide polymer solution; ii) optional sterilization of the liposome suspension by filtration; iii) mixing of the liposome suspension with the solution comprising the polysaccharide polymer iv) pouring or spreading of the mixture on a solid support, v) drying and gelatinization of the mixture, vi) optional lyophilization and/or sterilization.

25) The method according to claim 24 wherein said polymer of polysaccharidic nature is chitosan and/or its salts or derivatives.

26) The method according to claims 24-25 wherein the mixing at step iii) is carried in the cold. 27) The method according to claims 24-26 wherein liposomes used in solution at point i) of the procedure are prepared by hydration of the lipid film followed by an extrusion process.

28) The method according to claim 27 wherein said hydration is carried out at temperatures not higher than 45°C for a time comprised between 30' and 2 hours. 29) The method according to claims 24-28 wherein liposome preparation is carried out by dissolving the lipid component in an organic solvent, solvent evaporation, treatment with a nitrogen flow, hydration in a buffer solution comprising an enzyme and/or an active principle.

30) The method according to claim 29 wherein said enzyme is prolidase and hydration occurs in a solution comprising 0.1-5 mg/ml prolidase and bovine serum albumin (BSA) at a concentration lower than 2% (w/v) and the solution further comprises reducing agents.

31) The method according to claims 24-30 wherein liposome extrusion is made by use of polycarbonate filters with porosity comprised between 50, 100, 200 nm, preferably 100 nm, using a nitrogen flow at a pressure not higher than 5 atmospheres.

32) The method according to claims 24 - 31 wherein the mixture resulting at step iii) has a final concentration of liposomes in the hydrogel comprised between 0.4 μmol and 10 μmol lipids/mg polymer, even more preferably between 0.5 and 2 μmol lipids/mg polymer.

33) The method according to claims 24-31 wherein steps a)-e) are carried out under aseptic conditions.

34) Dressing according to claims 5-23 for treatment of ulcers.

35) Dressing according to claim 34 in subjects affected by prolidase deficiency.

36) Dressing according to claims 1-5 for cosmetic use.

37) Use of the dressing according to claims 1-23 for topical application. 38) Cosmetic use of the dressing according to claims 1-23.

39) Use of liposomes comprising the enzyme prolidase in combination with a hydrogel carrier composed of polymers of polysaccharidic nature or acrylic polymers or proteins or high molecular weight polyhydroxyl compounds for the preparation of dressings for topical use.