COMPOSITION FOR DELIVERING NITRIC OXIDE TO SKIN

Abstract

A skin application composition comprising a first component in dry condition comprising a source of nitrite and a thiol and a second component comprising a source of water is provided.

Claims

1. A skin application composition comprising a first component in dry condition comprising a source of nitrite and a thiol and a second component comprising a source of water.

2. The skin application composition according to claim 1, wherein the skin application composition is a skin dressing.

3. The skin application composition according to claim 1, wherein the source of nitrite is a nitrite salt.

4. The skin application composition according to claim 1, wherein the nitrite and thiol are not in intimate contact with each other.

5. The skin application composition according to claim 1, wherein the first component is a solid material.

6. The skin application composition according to claim 5, wherein the solid material comprises a polymer material.

7. The skin application composition according to claim 6, wherein the polymer material comprises polyvinyl alcohol or polyvinyl pyrrolidone or a mixture thereof.

8. The skin application composition according to claim 5, wherein the solid material is in the form of a sheet, a layer, or a slab.

9. The skin application composition according to claim 1, wherein the first component comprises a non-aqueous liquid matrix comprising the nitrite and thiol dispersed therein.

10. The skin application composition according to claim 1, wherein the first component is provided in the form of particulate material.

11. The skin application composition according to claim 4, wherein the nitrite is kept at a pH of from 4.0 to 8.0.

12. The skin application composition according to claim 4, wherein the thiol is kept at a pH of from 1.0 to 4.0.

13. The skin application composition according to claim 1, wherein bringing the first and second components together into intimate contact results in a pH of less than 4.0.

Description

EXAMPLES

[0064] A number of model systems were prepared to determine the production and generation rate of S-nitrosothiols. The systems are detailed in tables at the start of each results section below. For each system, sample aliquots from the aqueous component were taken, at three time points after the system ‘activation’ (i.e. all components being brought together) to confirm the presence of S-nitrosothiol generation and release. The first time-point was always t=zero (i.e. measuring S-nitrosothiol in the aqueous component prior to being ‘activated’ with the dry/non-aqueous component to demonstrate there was no S-nitrosothiol at the start, which was the case in all systems), then t=2 hours after activation and finally t=6 hours after activation.

[0065] To demonstrate that the S-nitrosothiols are being generated by reaction between the nitrite and thiol, systems were prepared where (1) the nitrite and thiol are kept separate prior to use and (2) the nitrite and thiol are mixed together during manufacture together and pre-generate S-nitrosothiols prior to use.

Materials

[0066] Sodium nitrite (300 mM) in DI water [0067] Glutathione (300 mM) in DI water [0068] Thioglycerol (300 mM) in DI water [0069] Lactic acid (100 mM) in DI water—(adjusted to pH 4.0 with 0.2M NaOH) [0070] Sorbitol (1 M) in DI water [0071] Polyvinyl alcohol (7.5% w/w) in DI water—M.sub.w 31,000-50,000, 98-99% hydrolyzed—obtained from Sigma (363138) EDTA (disodium) (5 mM) in DI water [0072] Copper (2+) Nitrite (5 mM) in DI water

PVA Stock Solution Manufacture Procedure

[0073] 462.5 ml of DI water was measured out and heated on hot plate to constant temperature between 80-85° C.—controlled with digital thermometer. 37.5 g PVA powder was measured out and divided onto 5×7.5 g aliquots. Single aliquots of the PVA powder were added to the heated water which was being stirred (preventing PVA coagulation). Throughout the additions, the water/PVA temperature was maintained at 80-85° C.). Additions were repeated while maintaining the temperature of the water/PVA mix until the PVA is dissolved. After removal from the hotplate and cooling, the final volume was made up to 500 ml with DI water.

PVA Films Manufacture

[0074] PVA films were produced by mixing the PVA stock solution with active components and allowing the mixture to dry in a Petri plate at 40° C. 20 ml of each pre-prepared PVA solution comprising the active components was poured into 10×10 cm Petri plates and left to dry overnight in an incubator at 40° C. The PVA films were produced with the active components in two separate films to be brought together each comprising a nitrite or a thiol (PVA1, PVA2 and PVA3) or the nitrite and thiol included together in a single film (PVA4 and PVA5). In both cases the formed films are to be brought together with an aqueous system to activate release of S-nitrosothiols. The composition of the PVA mixtures prior to drying is shown in Table 1.

[0075] In the case of PVA4 and PVA5 it is assumed that the nitrite and thiol react together to form S-nitrosothiol during manufacture and prior to formation of the PVA film, and are included as comparative examples.

TABLE-US-00001 TABLE 1 Film ID Film Components PVA1 PVA (5% w/w) Sodium nitrite (30 mM) PVA2 PVA (5% w/w) Glutathione (30 mM) Lactic acid (5 mM) at pH 4.0 PVA3 PVA (5% w/w) Thioglycerol (30 mM) Lactic acid (5 mM) at pH 4.0 PVA4 PVA (5% w/w) Glutathione (30 mM) Sodium nitrite (30 mM) Lactic acid (5 mM) at pH 4.0 PVA5 PVA (5% w/w) Glutathione (30 mM) Sodium nitrite (30 mM) Lactic acid (5 mM) EDTA (0.05 mM) at pH 4.0

Powder Manufacture

[0076] Powders comprising nitrite or thiol were produced by mixing the nitrite or the thiol with a bulking agent (sorbitol), followed by drying the mixture. For each powder, 20 ml of each pre-prepared solution was poured into 10×10 cm Petri plates and left to dehydrate for 24 hours in an incubator at 40° C., followed by a thorough desiccation. Once in powder form, the formulations were dispersed in neat Propylene Glycol (0.1 g of powder to 1 ml Propylene Glycol).

[0077] The composition of the aqueous mixtures for powder preparation prior to drying is shown in Table 2.

[0078] Powders were activated for S-nitrosothiol release, once brought into contact with an aqueous system (such as a hydrogel). Powders were produced with the active components, in two separate powders to be brought together each comprising a nitrite or a thiol (P1 and P2) or the nitrite and thiol included together in a single powder (P3 and P4).

[0079] In the case of P3 and P4 it is assumed that the nitrite and thiol react together to form S-nitrosothiol during manufacture and prior to formation of the powder and are included as comparative examples.

TABLE-US-00002 TABLE 2 Powder ID Powder Components P1 Sorbitol (500 mM) Sodium nitrite (30 mM) P2 Sorbitol (500 mM) Glutathione (30 mM) Lactic acid (5 mM) at pH 4.0 P3 Sorbitol (500 mM) Glutathione (30 mM) Sodium nitrite (30 mM) Lactic acid (5 mM) at pH 4.0 P4 Sorbitol (500 mM) Glutathione (30 mM) Sodium nitrite (30 mM) Lactic acid (5 mM) EDTA (0.05 mM) at pH 4.0

Aqueous Components

[0080] The pre-prepared PVA films and Powders require contact with an aqueous component to activate S-nitrosothiol generation and release. The various aqueous components are shown below in table 3.

TABLE-US-00003 TABLE 3 AQ ID Aqueous Component AQ1 Sheet hydrogel AQ2 Sheet hydrogel imbibed with 5 mM Copper Nitrite Solution (50 μL of Cu(NO.sub.3).sub.2 per 1 cm.sup.2 hydrogel) AQ3 Amorphous Hydrogel AQ4 Amorphous Hydrogel (50 μL of 5 mM Cu(NO.sub.3).sub.2 per 1 cm2 hydrogel) AQ5 DI water AQ6 0.2 mM Cu(NO.sub.3).sub.2 Solution (in DI water)

[0081] Details of the sheet hydrogel and amorphous hydrogel materials are shown in Table 4.

TABLE-US-00004 TABLE 4 Component Name Manufacturer Details Sheet ActiformCool Activa 70% H.sub.2O: 30% Acrylic hydrogel Polymer (Taurate derivative). Phenoxyethanol as preservative. Used in moderate to heavily exuding wounds Amorphous ActivHeal Advanced Medical Hydrogel with high water Hydrogel Solutions content - 85%. Used in nil to low exudate wounds Foam ActivHeal Advanced Medical Non-adhesive absorbent Solutions Polyurethane. Used in moderate to heavily exuding wounds. Total Fluid Handling ca. 24 g/10 cm.sup.2 1

S-Nitrosothiol Measurement

[0082] The presence of generated S-nitrosothiols was measured by an Absorbance reading at 490 nm using the Griess reagent method described below. S-nitrosothiol concentration can be calculated from the absorbance measurement using the extinction coefficient of ca. 10,000 M.sup.−1 cm.sup.−1. Absorbance measurement was carried out using Fisherbrand™ Digital Colorimeter Model 45.

[0083] Two different methods are required to calculate generated S-Nitrosothiol concentrations depending on whether a Hydrogel (AQ1 to AQ4) or a Solution (AQ5 and AQ6) are utilised as the aqueous component.

Reagents for S-Nitrosothiol Measurement

[0084] Reagent 1: Na-phosphate buffer (pH 7.4, 0.1 M). [0085] Reagent 2: Griess reagent: 20 mg of N-(1-Naphthyl)ethylendiamine dihydrochloride (NADD)+500 mg of sulphanilamide dissolved in 2 mL of DMSO. [0086] Reagent 3: Mercuric chloride (10 mM) in DMSO (13.58 mg of HgCl2 in 5 mL of DMSO).

Procedure to Measure S-Nitrosothiol Concentration in Gels

[0087] 1. Dispense 25 mL of Reagent 1 and 825 μL of Reagent 2 into a 250 ml pot [0088] 2. Weigh accurately 300 mg of the hydrogel and immerse it in the reagent mix. Incubate while shaking mildly for 30 min. [0089] 3. Transfer 2.6 ml of the reagent mix from the pot into a plastic cuvette [0090] 4. Add 25 μl of Reagent 3 [0091] 5. Read absorbance of the resulting mixture at 490 nm in 10 min

Procedure to Measure S-Nitrosothiol Concentration in Solutions

[0092] 1. Dispense 1.5 mL of Reagent 1 into a plastic cuvette [0093] 2. Add 200 μL of the sample [0094] 3. Add 1.17 μL of DI water [0095] 4. Add 100 μL of Reagent 2 [0096] 5. Add 30 μL of Reagent 3 and mix thoroughly [0097] 6. Read absorbance of the resulting mixture at 490 nm in 10 min

Results

S-Nitrosothiol Measurements in PVA Film Systems

[0098] Generation and release of S-nitrosothiol from PVA film systems comprising separate sources of nitrite and thiol, following activation by contact with aqueous systems is shown in Table 5. In each case the measurements were repeated, and the results are shown in mAU.

TABLE-US-00005 TABLE 5 PVA PVA Aqueous Absorbance Absorbance Absorbance film 1 film 2 component at t = 0 at 2 hrs at 6 hours PVA1 PVA2 Aq1 0 70 160 0 60 140 PVA1 PVA2 Aq2 0 40 0 0 50 50 PVA1 PVA2 Aq3 0 50 110 0 40 110 PVA1 PVA2 Aq4 0 10 70 0 0 70 PVA1 PVA3 Aq1 0 0 0 0 0 0 PVA1 PVA3 Aq2 0 0 0 0 0 0 PVA1 PVA3 Aq3 0 0 30 0 10 60 PVA1 PVA3 Aq4 0 0 40 0 0 30

[0099] The following combinations, comprising pre-mixed nitrite and thiol were tested as comparative examples, as shown in table 6.

TABLE-US-00006 TABLE 6 PVA Aqueous Absorbance Absorbance Absorbance film component at t = 0 at 2 hours at 6 hours PVA4 Aq1 0 10 60 0 10 50 PVA4 Aq2 0 0 60 0 0 40 PVA4 Aq3 0 70 90 0 70 80 PVA4 Aq4 0 20 40 0 20 50 PVA5 Aq1 0 10 20 0 0 20 PVA5 Aq2 0 20 30 0 10 30 PVA5 Aq3 0 40 80 0 40 90 PVA5 Aq4 0 20 100 0 20 90

S-Nitrosothiol Measurements in Systems Based on a Non-Aqueous Liquid Component

[0100] Generation and release of S-nitrosothiol from powder based non-aqueous systems comprising separate sources of nitrite and thiol, following activation by contact with aqueous systems is shown in Table 7. In each case the measurements were repeated, and the results are shown in mAU.

TABLE-US-00007 TABLE 7 Non-aqueous Non-aqueous Aqueous Absorbance Absorbance Absorbance component 1 component 2 component at t = 0 at 2 hrs at 6 hours P1 P2 Aq3 0 20 10 0 10 10 P1 P2 Aq4 0 10 10 0 10 10 P1 P2 Aq5 0 370 290 0 370 300 P1 P2 Aq6 0 450 420 0 450 420

[0101] The following combinations comprising pre-mixed nitrite and thiol were tested as comparative examples, as shown in table 8.

TABLE-US-00008 TABLE 8 Non-aqueous Aqueous Absorbance Absorbance Absorbance component 1 component at t = 0 at 2 hrs at 6 hours P3 Aq3 0 30 50 0 30 50 P3 Aq4 0 40 30 0 30 30 P3 Aq5 0 80 50 0 80 50 P3 Aq6 0 70 0 0 70 0 P4 Aq3 0 10 40 0 0 30 P4 Aq4 0 0 0 0 0 0 P4 Aq5 0 80 60 0 70 60 P4 Aq6 0 50 0 0 30 0

CONCLUSIONS

[0102] Generation and release of S-nitrosothiol from non-aqueous components based on PVA films and powder based systems was demonstrated on contact with an aqueous component.