Wound spray

Abstract

The present invention refers to a composition, comprising hemoglobin or myoglobin, wherein in at least 40% of said hemoglobin or myoglobin the oxygen binding site is charged by a non-O.sub.2 ligand, and at least one further ingredient, a method for preparing said composition and the use of hemoglobin or myoglobin charged with a non-oxygen ligand for external treatment of wounds.

Claims

1. A method for the external treatment of an open wound surface, comprising spraying on the open wound surface an aqueous composition comprising: (a) an oxygen carrier, wherein al least 40% of oxygen binding sites of said oxygen carrier are charged by a non-O.sub.2 ligand, such that the oxygen carrier is sprayed on the open wound surface with the oxygen carrier in its non-oxidized state in the spray and provides availability of oxygen to the open wound surface in the presence of oxygen partial pressure in air as opposed to a pressurized chamber of pure oxygen, and (b) at least one further ingredient selected from the group consisting of electrolyte(s), preservative(s), stabilizer(s), anti-flocculant(s), anticoagulant(s), pH buffering agent(s), solvent(s), antioxidant(s), film-forming agent(s) and crosslinking agent(s), wherein the oxygen carrier is hemoglobin and the non-O.sub.2 ligand is carbon monoxide (CO).

2. The method according to claim 1, wherein the wounds to be treated are selected from the group consisting of chronic wounds, operation wounds, injury wounds, wounds after trauma, open wounds, wounds with poor healing, hypoxic wounds, wounds arising from degeneration or stenosis of arterial blood vessels, diabetic wounds, chronic venous insufficiency wounds, decubitus ulcer wounds, burn wounds, chemical burns, freezing burns, and scalding wounds.

3. The method according to claim 1, wherein the composition administered is an organic solution.

4. The method according to claim 1, wherein at least 50% of the oxygen carrier is provided in non-O.sub.2 ligand-charged form.

5. The method according to claim 4, wherein at least 60% of the oxygen carrier is provided in non-O.sub.2 ligand-charged form.

6. The method according to claim 5, wherein at least 70% of the oxygen carrier is provided in non-O.sub.2 ligand-charged form.

7. The method according to claim 6, wherein at least 80% of the oxygen carrier is provided in non-O.sub.2 ligand-charged form.

8. The method according to claim 7, wherein at least 90% of the oxygen carrier is provided in non-O.sub.2 ligand-charged form.

9. The method according to claim 1, wherein the oxygen carrier is naturally occurring hemoglobin of human or animal origin.

10. The method according to claim 1, wherein the oxygen carrier is artificially-treated, crosslinked hemoglobin.

11. The method according to claim 1, wherein the composition administered is provided in sterilized form.

12. The method according to claim 1, wherein the composition administered is provided in an aerosol can.

13. The method according to claim 12, wherein the composition administered is provided in a pressure pack aerosol can.

14. The method of claim 1, wherein the ligand is replaced by oxygen when the spray is applied to the open wound surface due to oxygen partial pressure in the air.

15. A method for the external treatment of an open wound surface, comprising spraying from an aerosol on the open wound surface an aqueous composition comprising: (a) a hemoglobin oxygen carrier, wherein at least 40% of oxygen binding sites of the hemoglobin oxygen carrier are charged by carbon monoxide to render the hemoglobin oxygen carrier in a non-oxidized/non-activated status, such that the oxygen carrier is sprayed on the open wound surface with the oxygen carrier in its non-oxidized state in the spray and provides availability of oxygen to the open wound surface in the presence of oxygen partial pressure in air as opposed to a pressurized chamber of pure oxygen, and (b) at least one further ingredient selected from the group consisting of electrolyte(s), preservative(s), stabilizer(s), anti-flocculant(s), anticoagulant(s), pH buffering agent(s), solvent(s), antioxidant(s), film-forming agent(s) and crosslinking agent(s).

Description

FIGURES

(1) FIG. 1 shows the O.sub.2 saturation curve of hemoglobin dependent from the O.sub.2 surrounding partial pressure.

EXAMPLES

Example 1

(2) Hemoglobin was isolated from whole blood of pigs by separating the red blood cells from serum, lysing the collected red blood cells, pelleting cell debris, charging the hemoglobin with CO by introducing CO gas until saturation of the liquid sample is obtained, heating the solution, carrying out several filtration steps, including a virus filtration step and washing the obtained hemoglobin solution by adding twice a 2-fold volume of 0.9% saline and filtering the solution.

(3) A ready-to-use composition for wound treatment was prepared, comprising 10% of purified and stabilized hemoglobin, 0.05% N-acetyl cysteine and 0.7% phenoxy ethanol in 0.9% NaCl. The composition was charged again with CO gas, separated into 10 portions and packaged into an aerosol can, respectively.

Example 2

(4) A first portion of the composition of Example 1 was used immediately after preparation to treat a female patient (Diagnosis: Diabetes, Hypertension) with a chronic wound at the right foot (72 cm). The wound was treated once daily by spraying the composition onto the wound. After 3 weeks first signs of healing were visible. The next weeks the healing process was pronounced and complete closing of the wound was obtained after 18 weeks.

Example 3

(5) A second portion of the composition of Example 1 was stored at 4 C. for 2 years after preparation and thereafter used to treat a chronical wound of a male patient suffering from diabetes mellitus type II. The composition was applied to a chronic superficial ulcer (43 cm) at the left tibia of the patient. The wound was treated once daily. After two weeks a healing was appearing, after 4 weeks the wound was healed more than 50% of the baseline. The next weeks a fast healing was observed and complete closing of the wound was obtained after 8 weeks.

Example 4

(6) A third portion of the composition of Example 1 was stored at 10 C. for 2 years. Thereafter it was used for treatment of a chronic superficial ulcer (42 cm) at the right leg of a male patient suffering from diabetes mellitus type II. The wound was treated once daily. After 2.5 weeks healing was visible. The next weeks a fast healing was observed and complete closing of the wound was obtained after 12 weeks.

Example 5

(7) A portion of the composition of Example 1 was used to treat a male patient after contact with hot boiling water and steam. The diagnosis was 1.sup.st and 2.sup.nd degree burns on his face.

(8) Initial treatment with cool gel and Neosporin for one week showed no improvement and by the tending physician the application of skin grafts on the nose and other parts of the face, like eyelids was proposed.

(9) Instead the patient was treated with the composition of example 1 for 7 weeks. The composition was applied to the skin by spraying three or four times per day. A fast improvement and healing of the skin was observed and the patient was discharged from the hospital after seven weeks. No skin grafts were required.

Example 6: Comparison of Charged/Non-Charged Hemoglobin

(10) A) A charged hemoglobin-spray was prepared according to Example 1

(11) A ready-to-use composition for wound treatment was prepared, comprising 10% of purified and stabilized hemoglobin, 0.05% N-acetyl cysteine and 0.7% phenoxy ethanol in 0.9% NaCl. The composition was charged again with CO gas, separated into 20 portions and packaged into an aerosol can, respectively.

(12) The composition can be stored between 4 C. and room temperature for months to years.

(13) B) A second portion of the pig whole blood was treated as in Example 1 with the exception that no CO charging was carried out. During the heating step a considerable amount of hemoglobin precipitated. The following filtration steps were difficult to carry out. The yield of purified hemoglobin per liter whole blood decreased to less than 30% of the yield when the CO charging is carried out before heating. This shows that charging the samples with CO during preparation stabilizes the hemoglobin in a way that during a heating step considerably less hemoglobin precipitates.

(14) A ready-to-use composition for wound treatment was prepared, comprising 10% of purified hemoglobin, 0.05% N-acetyl cysteine and 0.7% phenoxy ethanol in 0.9% NaCl. The composition was portioned and packaged into aerosol cans, respectively.

(15) C) A further portion of hemoglobin was isolated from whole blood of pigs by a method as described in Example 1 of WO2003/077941 without charging the hemoglobin during preparation: the hemoglobin was freed from plasma and cellular membrane constituents without heating by means of centrifugation and ultrafiltration, and was purified.

(16) A ready-to-use composition for wound treatment was prepared, comprising 10% of purified hemoglobin, 0.05% N-acetyl cysteine and 0.7% phenoxy ethanol in 0.9% NaCl. The composition was separated into 10 portions and packaged into an aerosol can, respectively.

(17) A such prepared non-charged hemoglobin spray was freshly prepared during the treatment period as often as needed (all three days) and stored at 4 C. maximal for one day before use.

(18) Treatment of Patients:

(19) Several male patients (age 65-75) with Diabetic foot ulcer at the lower leg were treated for three months with hemoglobin spray according to A) (stored at 10 C. for up to 6 months) or a freshly prepared hemoglobin spray according to C) (storage at 4 C. for 3 days max., i.e. all three days a fresh composition was used) until complete wound healing was obtained at all of the patients. According to the attending dermatologists, the wound healing obtained with the compositions according to A) showed in all cases less secretion, less incrustation and/or less suppuration in comparison to wounds treated by the hemoglobin spray according to C) and in particular in comparison to wounds treated conventionally. Due to the minor problems the wounds treated with the charged hemoglobin composition according to A) showed a faster healing per cm.sup.2 than the wounds treated with the non-charged hemoglobin spray C) and much faster than the conventionally treated wounds. The patients treated with composition A) furthermore reported less discomfort.

Example 7

(20) Samples of compositions prepared according to Example 6A), Example 6B) or Example 6C), respectively, were examined considering the O.sub.2 charge of the samples as well as the production of methemoglobin, a species which is unable to bind O.sub.2. Furthermore methemoglobin affects hemoglobin molecules in the immediate vicinity in a way that these can still bind O.sub.2, but cannot release it no more.

(21) All the samples of Table 1 were diluted 1:1 with a 0.9% NaCl solution to imitate the conditions of a wound treatment. Samples 7.1 to 7.8 were measured immediately after dilution (storing of samples 7.5 to 7.8 was at room temperature before dilution). Gas treatment was as follows: 6 ml of said diluted solution were transferred into a 30 ml glass flask each. Gas treatment was carried out by filling the glass flask completely with the respective gas, closing the flask and pivoting the sample for 30 sec. All the samples were stored for the mentioned time period at 30 C. The samples were treated according to the conditions described in Table 1 and the total amount of hemoglobin (hemoglobin+methemoglobin), the oxygen content, the methemoglobin content and optionally the CO content was measured.

(22) TABLE-US-00001 TABLE 1 totalHb Example sample treatment g/dL O.sub.2 % CO % MetHb % 7.1 Freshly prepared comp. none 5.5 1.7 96.3 3.9 according to Example 6A) 7.2 Freshly prepared comp. none 5.7 26.8 5.1 According to Example 6B) 7.3 Freshly prepared comp. none 5.6 27.0 4.7 According to Example 6C) 7.4 Composition of Example 6A), none 5.9 1.8 96.5 3.8 stored for 3 month (10 C.) 7.5 Composition of Example 7.2 Stored for 24 h 5.6 26.9 11.2 7.6 Composition of Example 7.2 Stored for 48 h 5.5 27.0 17.0 7.7 Composition of Example 7.3 Stored for 24 h 5.7 26.5 10.5 7.8 Composition of Example 7.3 Stored for 48 h 5.6 26.7 16.2 7.9 Composition of Example 7.4 0.5 h O.sub.2 5.8 13.7 84.7 4.4 7.10 Composition of Example 7.4 1 h O.sub.2 5.7 19.6 79.1 4.2 7.11 Composition of Example 7.4 2 h O.sub.2 5.8 25.7 73.1 4.3 7.12 Composition of Example 7.4 3 h O.sub.2 5.9 30.7 68.1 4.2 7.13 Composition of Example 7.4 24 h O.sub.2 5.7 33.5 59.6 9.8 7.14 Composition of Example 7.4 48 h O.sub.2 5.8 32.5 55.9 15.5 7.15 Composition of Example 7.4 72 h O.sub.2 5.7 31.7 55.8 16.0 7.16 Composition of Example 7.4 0.5 h CO.sub.2 5.8 4.0 91.8 5.7 7.17 Composition of Example 7.4 1 h CO.sub.2 5.7 6.0 90.0 5.7 7.18 Composition of Example 7.4 2 h CO.sub.2 5.9 9.2 86.7 5.8 7.19 Composition of Example 7.4 3 h CO.sub.2 5.7 9.6 85.1 6.9 7.20 Composition of Example 7.4 24 h CO.sub.2 5.6 4.9 77.5 19.7 7.21 Composition of Example 7.4 48 h CO.sub.2 5.8 8.2 68.2 27.4 7.22 Composition of Example 7.4 72 h CO.sub.2 5.7 9.4 65.9 28.3 7.23 Composition of Example 7.4 0.5 h air 5.8 9.2 89.0 4.5 7.24 Composition of Example 7.4 1 h air 5.7 12.4 85.9 4.4 7.25 Composition of Example 7.4 2 h air 5.9 18.0 80.5 4.3 7.26 Composition of Example 7.4 3 h air 5.6 20.7 77.9 4.3 7.27 Composition of Example 7.4 24 h air 5.8 24.3 69.3 9.1 7.28 Composition of Example 7.4 48 h air 5.7 25.4 64.3 13.9 7.29 Composition of Example 7.4 72 h air 5.7 26.8 62.2 14.3

(23) As can be seen from the results in Table 1 the compositions of the invention, wherein the oxygen carrier is charged with CO, not only can be stored for a long time without forming methemoglobin, but further are able to replace the bound CO by O.sub.2 when it is offered to the charged oxygen carrier. If exposed to 100% O.sub.2 (examples 7.9 to 7.15) the O.sub.2 saturation of the hemoglobin increases very fast.

(24) If the composition is exposed to CO.sub.2, representing the situation inside of mammalian pathway-active tissue, an increased amount of methemoglobin is formed (examples 7.16 to 7.22).

(25) The O.sub.2 partial pressure in air is only about 21%, thus according to the O.sub.2 saturation curve of hemoglobin shown in FIG. 1, the maximum possible O.sub.2 saturation of hemoglobin with oxygen under air is about 29%. Considering Examples 7.23 to 7.29 the surprising result is that within 3 hours the composition exposed to air is charged with 20.7% oxygen, but still has a very low methemoglobin content. When external wounds are treated, the composition is sprayed to the (cleaned) wound surface and remains in contact with air.

(26) These results show that a composition according to the invention comprises a stabilized oxygen carrier which after several months of storing provides high oxygen transport when it is in contact with air.