Use of a hemoglobin for the preparation of dressings and resulting dressings

Abstract

The invention relates to the use of a haemoglobin for the preparation of dressings and to the resulting dressings.

Claims

1. A pharmaceutical composition comprising a extracellular Arenicola marina hemoglobin immobilized and stable in a physiologically acceptable hydrocolloid network, said hydrocolloid network forms a three-dimensional network defining pores, the pores forming a first population of large pores and a second population of pores wherein the size of the pores in the second population of pores is about 10 nm, without the release of the hemoglobin immobilized in said hydrocolloid network being greater than 10%, wherein the Arenicola marina hemoglobin is not crosslinked, and wherein the hemoglobin is contained in the second population of pores.

2. A pharmaceutical composition according to claim 1, which is in a form that can be administered topically at a rate of from 0.012 mg/d to 100 mg/d of active substance or in a form that can be administered orally at a rate of from 0.012 mg/kg/d to 100 mg/kg/d of active substance.

3. A cosmetic composition comprising extracellular Arenicola marina hemoglobin immobilized and stable in a physiologically acceptable hydrocolloid network, said hydrocolloid network forms a three-dimensional network defining pores, the pores forming a first population of large pores and a second population of pores wherein the size of is the pores in the second population of pores is about 10 nm, without the release of the hemoglobin immobilized in said hydrocolloid network being greater than 10%, wherein the Arenicola marina hemoglobin is not crosslinked, and wherein the hemoglobin is contained in the second population of pores.

4. A cosmetic composition according to claim 3, which is in a form that can be administered topically at a rate of from 10 mg/d to 20 mg/d.

5. A composition comprising extracellular Arenicola marina hemoglobin immobilized in a gelled hydrocolloid network, wherein the hydrocolloid network comprises from 0% to 98% by weight of water, and a first population of large pores and a second population of pores wherein the pores in the second population of pores have a size of about 10 nm; and the Arenicola marina hemoglobin is contained in the second population of pores; not crosslinked; and wherein the hemoglobin is present in an amount of about 0.1% w/w to about 60% w/w relative to the total dry weight of the hemoglobin and the hydrocolloid network.

6. The composition according to claim 5, wherein the composition is in the form of a dressing capable of covering a wound on skin.

7. A method for the external treatment of open, deep or chronic wounds, or of periodontal diseases, or of a gastric wounds or of tissues, the method comprising administering a pharmaceutical composition according to claim 1 to a wound or diseased tissue.

8. The method of claim 7, wherein the amount of hemoglobin, relative to the total dry weight of hemoglobin and of hydrocolloid network, is from 0.1% (w/w) to 60% (w/w).

9. The method of claim 7, wherein the percentage water content is from greater than 50%.

10. The method of claim 7, wherein said hydrocolloid network is based on chitosan, carrageenans, carboxymethylcellulose or alginates.

11. The method of claim 10, wherein said hydrocolloid network is based on calcium alginate, and wherein the amount of hemoglobin present in the hydrocolloid network, relative to the total weight of hemoglobin and of the hydrocolloid network is 55% (w/w) to 85%.

12. The composition according to claim 5, wherein the amount of Arenicola marina hemoglobin, relative to the total dry weight of Arenicola marina hemoglobin and of the hydrocolloid network, is from 15% w/w to 45% w/w.

13. The composition according to claim 5, wherein the water content is from 95% w/w to 98% w/w.

14. The composition according to claim 5, wherein the water content is less than 5% w/w.

15. The composition according to claim 5, wherein said hydrocolloid network comprises a hydrocolloid selected from the group consisting of chitosan, carrageenans, carboxymethylcellulose and alginates.

16. The composition according to claim 5, wherein the hydrocolloid network is based on an alginate and said alginate is calcium alginate, and wherein the amount of Arenicola marina hemoglobin relative to the total dry weight of hemoglobin and of the hydrocolloid network, is 40% w/w to 60% w/w.

17. The composition according to claim 5, wherein the hydrocolloid network comprises a gel-forming polysaccharide.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 represents an example of a chronic wound: diabetic patient suffering from ischemia in the foot.

(2) FIG. 2 represents an attack on the periodontium by anaerobic pathogens, creating pockets between the gum and the tooth organ.

(3) FIG. 3 represents the photograph of the scanning microscopy (JEOL JEM 1200=carried out on the external face of the matrix of calcium alginate containing Arenicola marina hemoglobin after gold shadowing. The resolution used here does not make it possible to visualize the hemoglobin.

(4) FIG. 4 represents the photograph of the scanning microscopy carried out on the internal face of the matrix of calcium alginate containing Arenicola marina hemoglobin (6 mg/ml) after gold shadowing and showing the porous appearance of the structure. The resolution used here does not make it possible to visualize the hemoglobin.

(5) FIG. 5 shows a Barcroft representation (10 mg/ml Arenicola marina hemoglobin, 15% Ca, 1% sodium alginate, Hepes buffer, pH 7.35) for determining the P.sub.50.

(6) FIG. 6 represents a photograph of the syringe for producing a dressing in situ. The syringe is constituted of two compartments containing, firstly, the sodium alginate (Satialgine™ US 61) in aqueous solution with the hemoglobin and, secondly, an aqueous solution of calcium chloride for the polymerization of the alginate in situ, at the syringe outlet.

(7) FIG. 7 represents the dialysis polymerization on a “Millipore” Minicell support.

(8) FIG. 8 represents the diffusion-chamber polymerization.

(9) FIG. 9 represents the release of Arenicola marina hemoglobin (HbA) contained in a dressing prepared with Satialgine™ US551 alginate (Cargill) at 1% as a function of time.

(10) FIGS. 10, 11 and 12 represent HPLC chromatograms at two wavelengths (280 and 414 nm) obtained on samples taken, respectively, at 1 h30, 8 h and 24 h, from a solution which is isoionic with respect to human blood (see example 5) in which a dressing containing Arenicola marina hemoglobin has been placed.

(11) FIG. 13 represents the control HPLC chromatogram at the two wavelengths (280 and 414 nm), obtained with a dressing without hemoglobin at 24 h.

EXPERIMENTAL SECTION

Example 1

Purification of Hemoglobin from Arenicola marina

(12) The worms originate from a SeaBait breeding farm. These worms are frozen at −80° C., which causes a hemorrhagic shock and rupture of the wall of the worm; hemoglobin extraction is thus facilitated.

(13) The worms, once thawed for 24 h at 4° C. in the presence of the extraction buffer (400 mM NaCl, 2.95 mM KCl, 32 mM MgSO.sub.4, 11 mM CaCl.sub.2, 50 mM Hepes, 5 mM ascorbic acid, 10 mM reduced glutathione, pH 7.5, filtered through 0.2 μm) in a proportion of 0.2 ml/g, are centrifuged (4500 g, 4° C., 15 min). The supernatant is recovered and the worm pellet is redispersed in 0.2 ml/g of extraction buffer and centrifuged again, this being carried out 4 times. The combined supernatants are filtered under pressure (2 bar) through a 5 μm filter and then a 0.1 μm filter (Pall filters).

(14) The filtrate can be treated in two ways: First method: the filtrate is diafiltered against 5 diavolumes of storage buffer (90 mM NaCl, 23 mM sodium gluconate, 27 mM sodium acetate, 5 mM KCl, 1.5 mM MgCl.sub.2, 2.5 mM CaCl.sub.2, pH 7.35, filtered through 0.2 μm) on a Pellicon XL-1000 kDa ultrafiltration cassette (Millipore), at 4° C. The retentate is finally concentrated on this same cassette to ˜100 mg/ml before sterilizing filtration through a 0.2 μm filter and storage at −80° C. Second method: the 5 μm filtrate is precipitated at the isoelectric point of Arenicola marina hemoglobin by adding 50% by volume of a 0.5N solution of sodium acetate/acetic acid, pH 4.15. After stirring for 30 minutes at 4° C., the solution is centrifuged (4500 g, 4° C., 15 min). The supernatant is removed and the pellet (containing the Arenicola marina hemoglobin) is washed twice against the same volume equivalent of ultrapure water (4500 g, 4° C., 5 min). The rinsed pellet is redispersed in the same volume equivalent of the storage buffer with stirring for 1 h at 4° C. The solution is centrifuged so as to remove the debris (4500 g, 4° C., 15 min). The supernatant is filtered under pressure through a 0.1 μm filter (Pall) and then diafiltered against 2 diavolumes on a Pellicon XL-1000 kDa cassette to a final concentration of ˜100 mg/ml before sterilizing filtration through a 0.2 μm filter and storage at −80° C.

Example 2

Preparation of the Liquid Intermediate Composition

(15) Two types of sodium alginate sold by Cargill: Satialgine™ US 61 and Satialgine™ US 551 EP were used. They conform to European pharmacopeia standards and are used as an additive, by way of a texturing: thickening and/or gelling agent, for many therapeutic applications.

(16) They are used at a concentration of from 1 to 3% (w/v) depending on the type of application and the final texture desired. The sodium alginate powder is diluted, with magnetic stirring, in MilliQ (MQ) water to the desired concentration. The higher the alginate concentration, the more difficult it is to dissolve.

(17) Furthermore, Satialgine™ US 551 EP is more viscous than Satialgine™ US 61. It is therefore sometimes necessary to heat (˜50° C.) in order to improve the dissolution. Once the solution is homogeneous, after a few hours, it is cooled in ice, before adding the Arenicola marina hemoglobin thereto.

(18) The Arenicola marina hemoglobin is prepared as described previously and stored at −80° C. before use, at a concentration of 100 mg/ml in a physiological buffer, termed storage buffer, which is calcium-free.

(19) The Arenicola marina hemoglobin is thawed at 4° C. and dissolved in the sodium alginate solution at the concentration of 6 mg/ml. The solution is then homogenized with magnetic stirring.

Example 3

Preparation of the Dried Intermediate Composition

(20) The solution obtained in example 2 is then dried under an air vacuum and in the presence of silica gel for between 12 and 24 h so as to obtain a dried intermediate composition.

Example 4

Polymerization

(21) The dried intermediate composition, which is in the form of a thin film, is immersed in 10 ml of a 1% (w/v) solution of calcium chloride. The calcium solution is buffered with 10 mM Hepes (Sigma) at pH 7.0. This step enables the polymerization of the alginate solution and the immobilization of the Arenicola marina hemoglobin in the alginate matrix.

(22) Several techniques were used to polymerize the solution of sodium alginate containing hemoglobin. Depending on the method used, it is possible to obtain various forms of dressing and therefore to envision the treatment of various types of wounds or of periodontal infections linked to the presence of anaerobic pathogens.

(23) 4.1: In Situ Polymerization

(24) Double syringes (Plas-pak) are used for this application. One compartment of the syringe (A) is filled with a solution containing sodium alginate (Satialgine™ US 61) at 1% and hemoglobin at 5 mg/ml.

(25) The other compartment (B) is filled with a 1% solution of CaCl.sub.2 in a 10 mM Hepes buffer, pH 7.0.

(26) Pressure exerted on the plunger makes it possible to bring the solutions of the two compartments into contact at the end (C) of the syringe and to polymerize the alginate in solution containing the hemoglobin (D).

(27) 4.2: Dialysis Polymerization

(28) The principle of the polymerization is shown in detail in FIG. 7.

(29) The solution of sodium alginate containing the hemoglobin is deposited onto a porous membrane (A) (Minicell, 0.4 μm, Millipore) (HbAm=Arenicola marina hemoglobin).

(30) The solution can be degassed and dried under vacuum and in the presence of silica gel (B), before polymerization, for between 12 h and 24 h, or polymerized as it is.

(31) The polymerization is carried out by immersing the porous membrane (C) in a bath containing a solution containing 1% CaCl.sub.2, 10 mM Hepes, pH 7.0, with stirring (D). The calcium diffuses through the membrane during the 12 h. The polymerization is carried out at 4° C.

(32) The polymer obtained is then thoroughly rinsed with MilliQ H.sub.2O (E) and then stored at 4° C. (F). The gel obtained was analyzed by scanning microscopy (FIGS. 3 and 4).

(33) It is possible to dry the gel under vacuum by evaporation or by lyophilization, so as to conserve it and to rehydrate it before use (F).

(34) 4.3: Diffusion-Chamber Polymerization

(35) The polymerization (FIG. 8) takes place on either side of the aqueous solution of sodium alginate (1%) and of Arenicola marina hemoglobin (A) which is maintained between two porous membranes (1 μm) in a bath containing a solution containing 1% CaCl.sub.2, 10 mM Hepes, pH 7.0, and with stirring (B).

Example 5

Hemoglobin Release

(36) The dressing, prepared according to one of the methods described above, is immersed in 10 ml of a solution that is isoionic with respect to human blood (145 mM NaCl, 4 mM KCl, 2 mM MgCl.sub.2, 10 mM Hepes, 2.5 mM CaCl.sub.2, pH 7.35) and the whole is incubated at 34° C. in order to simulate the physiological medium of the wound.

(37) At regular time periods, an amount of solution (0.5 ml) is removed in order to assay the released hemoglobin with Drabkin's reagent, reading being carried out on a spectrophotometer at 540 nm (colorimetric assay). The concentrations thus obtained in the volume of 0.5 ml are converted to a total amount of hemoglobin released by the dressing.

(38) FIG. 9 shows the results obtained: After 4 h in solution, the dressing has released only 4% of hemoglobin. After 20 h, the dressing has released only 6% of hemoglobin. After 24 h, only 8% of the hemoglobin contained in the dressing has been released.

(39) These three measurements confirm that the hemoglobin remains immobilized in the matrix for more than 24 hours.

(40) An analysis of the structure of the hemoglobin released, by size exclusion chromatography (superose 6 column, 0.5 ml/min), was carried out. Samples were taken at various times (1 h30, 8 h and 24 h) and the optical density was measured at 280 nm and 414 nm (FIGS. 10, 11, 12).

(41) FIGS. 10, 11 and 12 give the chromatograms obtained and show that the hemoglobin released is a degraded hemoglobin (in dodecamer form, peak at 30 minutes), since the absorption peak for native hemoglobin, which is at 21 minutes, is nonexistent.

(42) Consequently, the small percentage of hemoglobin which is released (less than 10%) is degraded hemoglobin, thus confirming that the Arenicola marina hemoglobin remaining in the dressing is stable (otherwise it would be released).

(43) FIG. 13 gives the chromatogram obtained with a “control” dressing without hemoglobin, thus giving the absorption due only to the dressing.

Example 6

Comparison with the Preparation of Application US2003/0180365

(44) The preparation of application US2003/0180365 was prepared as described, by inserting therein 6 mg/ml of Arenicola marina hemoglobin.

(45) The Arenicola marina hemoglobin release tests were carried out as described in example 5.

(46) The formulation thus obtained has the appearance of a very viscous gel and, once immersed in the solution which is isoionic with respect to human blood, as described in example 5, complete liquefaction of the gel after 12 h and therefore total release of the Arenicola marina hemoglobin are observed.