Pharmaceutical compositions containing hyaluronic acid and collagenase for the topical treatment of wounds, burns and ulcers

Abstract

The present invention concerns new compositions containing hyaluronic acid or the derivatives thereof in association with the proteolytic enzyme collagenase (and relative pharmaceutical formulations) for the preparation of a dressing for topical treatment of various kinds of wounds, burns of varying depth, pressure sores, vascular ulcers and diabetic foot ulcers as well as for the treatment of hypertrophic and keloid scars.

Claims

1. A method for treating Dupuytren's contracture which comprises administering to a subject having Dupuytren's contracture an injectable form of a pharmaceutical composition comprising hyaluronic acid salified with inorganic bases with a molecular weight of 50-730 kDa and the proteolytic enzyme collagenase, wherein said collagenase is from the non-pathogenic micro-organism belonging to the strain Vibrio alginolyticus sub. Iophagus; wherein the hyaluronic acid acts to reduce the rate of the proteolytic activity of the collagenase in the subject; wherein the concentration of the proteolytic enzyme collagenase ranges between 0.1 and 20 U/milligrams of said hyaluronic acid; and wherein the concentration of said hyaluronic acid is between 0.1 and 2% w/w.

2. The method according to claim 1, wherein said pharmaceutical composition further comprises an additional pharmacologically and/or biologically active sub stance.

3. The method according to claim 1, wherein the concentration of the enzyme collagenase is between 0.2 and 10 U/milligrams of said hyaluronic acid.

4. The method according to claim 3, wherein the concentration of said hyaluronic acid is between 0.2 and 0.4% w/w.

5. The method according to claim 1, wherein said composition further comprises maltose and/or carrageenan as a stabilizing agent.

6. A method for treating Dupuytren's contracture which consists of administering to a subject having Dupuytren's contracture an injectable form of a pharmaceutical composition comprising hyaluronic acid salified with inorganic bases with a molecular weight of 50-730 KDa and the proteolytic enzyme collagenase, wherein said collagenase is from the non-pathogenic micro-organism belonging to the strain Vibrio alginolyticus sub. Iophagus; wherein the hyaluronic acid acts to reduce the rate of the proteolytic activity of the collagenase in the subject; wherein the concentration of the proteolytic enzyme collagenase ranges between 0.1 and 20 U/milligrams of said hyaluronic acid; and wherein the concentration of said hyaluronic acid is between 0.1 and 2% w/w.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention describes and claims the association of HA and/or the derivatives thereof with the enzyme collagenase in pharmaceutical formulations of a lipophilic nature which, thanks to the presence of special excipients/stabilisers, allow the proteolytic enzyme to remain stable at room temperature for prolonged periods of time.

(2) Thanks to HA's special properties in tissue regeneration, enzymatic débridement by the new enzyme/HA composition substantially modifies the phases of eschar removal and new tissue regeneration that normally follow treatment with collagenase alone, permitting the establishment of new phases that determine proper wound healing without the formation of pathological scarring: the degradation/removal of eschar occurs at the same time as the formation of granulation tissue; the formation of new connective tissue determines a reduction in wound size after eschar removal (for the reasons given above) and, hence, a significant decrease in the risk of bacterial and/or viral infections; the healthy surrounding tissue is protected from the digestive action of collagenase thanks to HA; eschar removal can be performed painlessly.

(3) The claims of the new collagenase/HA formulations described above are possible because HA has proved capable of modulating the proteolytic activity of collagenase significantly slowing down its action during enzymatic débridement of the necrotic tissue, as demonstrated hereafter.

(4) The formulation of enzyme/HA is particularly indicated in the débridement of burns of varying depth, pressure sores, vascular ulcers and diabetic foot ulcers, wounds of various nature and of different sizes and depth and, moreover, in the treatment of hypertrophic and keloid scars with final patient compliance with the product that is decidedly greater than that with the enzyme alone.

(5) Furthermore, said composition is also claimed in the treatment of Dupuytren's contracture and glaucoma.

(6) The HA derivatives that can be used in the new formulations that are the subject of the present invention are listed below: 1. HA salified with organic and/or inorganic bases with a molecular weight of 50-730KDa (EP0138572 B1) or with a high molecular weight of 750-1230 KDa, (EP 535200 B1); 2. Hyaff®: esters of HA with alcohols of the aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic or heterocyclic series, with a percentage of esterification that may vary according to the type and length of the alcohol used, between 1 and 100%, preferably between 50 and 75% (EP 216453 B1); 3. Hyadd™: amides of HA with amines of the aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic series, with a percentage of amidation of between 1 and 10%, preferably 4% (EP 1095064 B1); 4. O-sulphatated derivatives of HA up to the 4th degree of sulphatation (EP 0702699 B1); 5. ACP®: inner esters of HA with a percentage of inner esterification between 0.5 and 10% and preferably 5% (EP 0341745 B1); 6. Deacetylated HA derivatives: derived from the deacetylation of the N-acetyl-glucosamine fraction with a percentage of deacetylation preferably between 0.1 and 30%, while all the carboxy groups of HA can be salified with organic and/or inorganic bases (EP1313772 B1); 7. Hyoxx™: percarboxylated derivatives of HA obtained from the oxidation of the primary hydroxyl of the N-acetyl-glucosamine fraction with a degree of percarboxylation of between 0.1 and 100% and preferably between 25 and 75%. All the carboxy groups of HA can be salified with organic and/or inorganic bases (patent application No. EP1339753).

(7) The HA used in the present invention as such or in the preparation of its derivatives may be derived from any source, for example it may be obtained by extraction from rooster combs (EP0138572 B1), by fermentation, or by technological means and its molecular weight may vary between 400 and 3×10.sup.6 Da, in particular between 1×10.sup.5 Da and 1×10.sup.6 Da, and more particularly still between 50,000 and 200,000 Da.

(8) The concentration of the collagenase enzyme to be used in association with HA or the derivatives thereof may vary between 0.01 U and 100 U/milligram of HA, preferably between 0.1 and 20 U and more preferably still between 0.2 and 10 U/mg of HA.

(9) The final concentration of HA or the derivatives thereof in the final pharmaceutical composition may vary between 0.01 and 5% weight/weight of the final product, ranging preferably between 0.1 and 2% w/w, and more preferably still between 0.2 and 0.4% w/w.

(10) One unit of collagenase is defined as the quantity of enzyme that hydrolyses 1 nmole of PZ-Pro-Leu-Gly-Pro-D-Arg in one second at pH equal to 7.1 and at 37° C. (PZ=4-phenyl-azobenzyloxycarbonyl) (Wunsch E. et al, Physiol Chem, 1963, 333:149-151).

(11) The collagenase preferably used or to be used in the compositions that are the subject of the present invention is that produced from the non-pathogenic micro-organism belonging to the Vibrio alginolyticus sub. Iophagus strain, NCIMB (National Collection of Industrial and Marine Bacteria), strain number 11038, equivalent strain: LMG 3418.

(12) It is a gram negative micro-organism that produces a collagenase with a molecular weight of from 90,000 to 110,000 Daltons, (IUBMB nomenclature: EC 3.4.24.3), stable in a pH range of 4.0 and 11.0 but with an optimal pH of between 6.0 and 8.0, stable at temperatures varying between 4 and 40° C. with an optimal temperature of 37° C.

(13) The enzyme is defined as a metalloendopeptidase because it breaks up the collagen into peptide fragments, acting directly on its triple-chain protein structure. The specific activity of the enzyme is inhibited by the silver and copper salts and also by chelating agents such as EDTA, which bind the calcium ion necessary to collagenase activity.

(14) Collagenolytic Activity of collagenase from Vibrio alginolyticus Versus the Activity of the Same Enzyme Associated with Hyaluronic Acid

(15) The aim of the study was to compare the activity of the enzyme as such with that of the new composition of collagenase/HA, to observe the influence of hyaluronic acid on the proteolytic activity of the same enzyme.

(16) Material:

(17) collagen extracted from bovine skin at a concentration of 1 mg/ml (of buffer prepared as described hereafter) as a substrate for enzymatic activity; collagenase extracted from Vibrio alginolyticus tested at three different concentrations of 0.33, 0.66 and 1.32 enzyme units; collagenase extracted from Vibrio alginolyticus tested at the same three concentrations of 0.33, 0.66 and 1.32 U but in association with hyaluronic acid at the following ratios: 0.16 U/mg of HA, 0.33 U or 0.66 U/mg of HA (hence, 0.33, 0.66 and 1.32 U are associated with 2 mg of HA). In all three cases, the concentration of the polysaccharide was kept constant.
Method of Enzymatic Digestion:

(18) the collagen degradation process was performed at 37° C. for a time period initially set at 90 minutes. Subsequently, the process was repeated at the same concentrations and at the same temperature, but the reaction was halted 4 and 12 hours after it was begun.

(19) The digestion reaction occurred in Tris-HCL buffer, 0.05 M, containing CaCl.sub.2 0.01M at pH=7.4 to which the reaction substrate had been added, either collagenase itself or the enzyme associated with set concentrations of HA as described above.

(20) Results:

(21) the enzymatic digestion mixture obtained (enzyme and relative collagen degradation products) was analysed on 7% polyacrylamide gel (electrophoresis: SDS-PAGE; Laemmli U. K., Nature, 1970, 680-685), to map the various protein fragments separated according to their molecular weight, (MW) and stained with Comassie blue.

(22) After the electrophoresis run, the collagen separates into subunits with a MW of about 100,000 Daltons with further peptide fragments with a MW of about 33,000 Daltons.

(23) All the gels were loaded with molecular weight standards, with undigested collagen as positive control, with collagen degraded by collagenase as such and relative enzyme separately loaded and, lastly, with collagen degraded by collagenase associated with HA and enzyme alone.

(24) FIG. 1 clearly shows that 0.33 U enzymes only partially degrade collagen in 90 minutes but, above all, that the presence of HA in the collagenase/HA composition (at the same concentration of enzyme) modifies the collagenolytic action of the enzyme, reducing its activity.

(25) FIGS. 2 and 3 further show the modulatory action exercised by hyaluronic acid on the enzyme: after 90 minutes 0.66 and 1.32 U of collagenase completely degrade the collagen while, especially at 0.66 U, HA slows down the collagenolytic action of the enzyme. The influence of said polysaccharide on the enzyme is even more evident with 1.32 U of collagenase.

(26) FIGS. 4 and 5 show the situation after 4 and 12 hours: after 4 hours of incubation of the enzyme (0.66 U) with collagen it is still possible to observe the action of HA on collagenase because the undigested protein fractions of collagen are visible, albeit faintly; after 12 hours (again with 0.66 U of enzyme) the modulatory action of the polysaccharide is no longer evident and the collagen is completely degraded.

(27) For purely descriptive purposes, and without being limited by the same, we report hereafter some examples of how the new formulations that are the subject of the present invention can be prepared:

(28) Method of Producing Collagenase/HA Lipogel

(29) Preparation of the Functional Components of the Lipogel:

(30) An initial aqueous solution or a buffer solution at pH 7.1 (TrisHCL 25 mM, CaCl.sub.2 10 mM) is prepared with 82.7 U/ml of collagenase; 45 ml of this solution is freeze-dried with the following excipients: Maltose (18 g) as diluent/stabiliser, Carrageenan, possibly purified (0.54 g) as stabiliser and water 45 g. 18.6 g of freeze-dried product is obtained (dry product), composed as follows: maltose 95% w/w carrageenan 2.8% w/w collagenase 0.9% w/w

(31) The freeze-dried product is then micronised.

(32) At the same time, HA obtained by fermentation and with a MW of 160 KDs is also micronised.

(33) Dispersion of the Functional Components in the Lipophilic Base:

EXAMPLES 1 AND 2

(34) The enzyme collagenase and HA prepared as described above are evenly dispersed in a lipophilic base constituted by the following excipients: hydrogenated castor oil in powder form (with gelling action) cetylstearylic alcohol (as agent of consistence) stringy Vaseline (as lipophilic phase) light Vaseline oil (as lipophilic phase)
Preparation of the Lipophilic Base:

(35) dissolve and solubilise at about 88-90° C. the stringy Vaseline and cetylstearylic alcohol in the Vaseline oil.

(36) Once a smooth, melted mass has been obtained, dissolve and solubilise the hydrogenated castor oil powder in the newly formed lipophilic phase.

(37) Once the mass is evenly melted, cool to 25-30° C.

Lipogel: Example 1A

(38) TABLE-US-00001 % w/w Function (weight/weight) Main components Micronised HA 0.2 Freeze-dried, 1.8 U/g micronised collagenase Excipients Hydrogenated castor Gelling 1.0 oil agent Cetylstearylic alcohol Agent of 5.0 consistence Stringy Vaseline Lipophilic 36.5 phase Light Vaseline oil Lipophilic Q b a 100 phase

Lipogel: Example 1B

(39) TABLE-US-00002 % w/w Function (weight/weight) Main components Micronised HA 0.2 Freeze-dried, 1.5 U/g micronised collagenase Excipients Hydrogenated castor Gelling 1.0 oil agent Cetylstearylic alcohol agent of 5.0 consistence Stringy Vaseline Lipophilic 36.5 phase Light Vaseline oil Lipophilic Q b a 100 phase

Lipogel: Example 1C

(40) TABLE-US-00003 % w/w Function (weight/weight) Main components Micronised HA 1 Freeze-dried, 1.8 U/g micronised collagenase Excipients Hydrogenated castor Gelling 1.0 oil agent Cetylstearylic alcohol agent of 5.0 consistence Stringy Vaseline Lipophilic 36.5 phase Light Vaseline oil Lipophilic Q b a 100 phase

Lipogel: Example 1D

(41) TABLE-US-00004 % w/w Function (weight/weight) Main components Micronised HYAFF (50% 0.2 esterified with benzyl alcohol) Freeze-dried, 1.8 U/g micronised collagenase Excipients Hydrogenated castor Gelling 1.0 oil agent Cetylstearylic alcohol agent of 5.0 consistence Stringy Vaseline Lipophilic 36.5 phase Light Vaseline oil Lipophilic Q b a 100 phase

Lipogel: Example 2A

(42) TABLE-US-00005 Function % w/w Main component Micronised HA 0.2 Freeze-dried, 1.8 U/g micronised collagenase Excipients Hydrogenated castor Gelling 1.0 oil agent Cetylstearylic alcohol agent of 10.0 consistence Stringy Vaseline Lipophilic 34.5 phase Light Vaseline oil Lipophilic Q b a 100 phase

Lipogel: Example 2B

(43) TABLE-US-00006 Function % w/w Main component Micronised HA 0.2 Freeze-dried, 2 U/g micronised collagenase Excipients Hydrogenated castor Gelling 1.0 oil agent Cetylstearylic alcohol agent of 10.0 consistence Stringy Vaseline Lipophilic 34.5 phase Light Vaseline oil Lipophilic Q b a 100 phase

(44) Dispersion of the Functional Components in the Lipophilic Base:

EXAMPLE 3

(45) Incorporate and solubilise light Vaseline oil in Jojoba Glaze while slowly stirring at 70-75° C. (lipophilic gelling system based on styrene copolymers-propylene-butylene in Jojoba oil) until a smooth mass is obtained. Cool to room temperature and then add, while stirring, Micronised HA and micronised, lipophilic Collagenase as previously described, until the powders have been completely amalgamated in the lipogel.

Lipogel: Example 3A

(46) TABLE-US-00007 Function % w/w Main component Micronised HA 0.2 Accessory component Freeze-dried, 1.8 U/g micronised collagenase Excipients Jojoba Glaze LV Lipophilic 80.0 phase viscous agent Light Vaseline oil Lipophilic Q.s a 100 phase

Lipogel: Example 3B

(47) TABLE-US-00008 Function % w/w Main component Micronised HA 0.2 Accessory component Freeze-dried, 1.5 U/g micronised collagenase Excipients Jojoba Glaze LV Lipophilic 80.0 phase viscous agent Light Vaseline oil Lipophilic Q.s a 100 phase

(48) Said formulations may contain pharmacologically and/or biologically active substances such as, for example, antibiotics, antivirals, wound healers, cytostatic/cytotoxic agents, anticancer drugs, hormones, steroid and non-steroid anti-inflammatory drugs, trophic factors and cytokines of various nature.

(49) The invention being thus described, it is clear that these methods can be modified in various ways. Such modifications are not to be considered as divergences from the spirit and purpose of the invention and any modification that would be evident to an expert in the field comes within the scope of the following claims.

EXPLANATIONS OF THE FIGURES

(50) FIG. 1: Collagen digestion time of 90 minutes MWS=molecular weight standards represented by 6 bands: 205 KDs, 116 KDs, 97 KDs, 66 KDs, 45 KDs, 29 KDs; CP=Collagen as such, Positive control; Coll. 1=Collagenase at a concentration of 0.33 U+collagen; Coll. 2=Collagenase at a concentration of 0.33 U; Coll./HA 1=Collagenase at a concentration of 0.33 U+collagen in the presence of HA; Coll./HA 2=Collagenase at a concentration of 0.33 U in the presence of HA.

(51) FIG. 2: Collagen digestion time of 90 minutes MWS=molecular weight standards represented by 6 bands: 205 KDs, 116 KDs, 97 KDs, 66 KDs, 45 KDs, 29 KDs; CP=Collagen as such, Positive control; Coll. 1=Collagenase at a concentration of 0.66 U+collagen; Coll. 2=Collagenase at a concentration of 0.66 U; Coll./HA 1=Collagenase at a concentration of 0.66 U+collagen in the presence of HA; Coll./HA 2=Collagenase at a concentration of 0.66 U in the presence of HA.

(52) FIG. 3: Collagen digestion time of 90 minutes MWS=molecular weight standards represented by 6 bands: 205 KDs, 116 KDs, 97 KDs, 66 KDs, 45 KDs, 29 KDs; CP=Collagen as such, Positive control; Coll. 1=Collagenase at a concentration of 1.32 U+collagen; Coll. 2=Collagenase at a concentration of 1.32 U; Coll./HA 1=Collagenase at a concentration of 1.32 U+collagen in the presence of HA; Coll./HA 2=Collagenase at a concentration of 1.32 U in the presence of HA.

(53) FIG. 4: Collagen digestion time of 4 hours MWS=molecular weight standards represented by 6 bands: 205 KDs, 116 KDs, 97 KDs, 66 KDs, 45 KDs, 29 KDs; CP=Collagen as such, Positive control; Coll. 1=Collagenase at a concentration of 0.66 U+collagen; Coll. 2=Collagenase at a concentration of 0.66 U; Coll./HA 1=Collagenase at a concentration of 0.66 U+collagen in the presence of HA; Coll./HA 2=Collagenase at a concentration of 0.66 U in the presence of HA

(54) FIG. 5: Collagen digestion time of 12 hours MWS=molecular weight standards represented by 6 bands: 205 KDs, 116 KDs, 97 KDs, 66 KDs, 45 KDs, 29 KDs; CP=Collagen as such, Positive control; Coll. 1=Collagenase at a concentration of 0.66 U+collagen; Coll. 2=Collagenase at a concentration of 0.66 U; Coll./HA 1=Collagenase at a concentration of 0.66 U+collagen in the presence of HA; Coll./HA 2=Collagenase at a concentration of 0.66 U in the presence of HA.