GB2267437A - Treatment of allergic contact dermatitis with hydrogen peroxide - Google Patents

Abstract

A formulation for use in the manufacture of a medicament for the treatment of allergic contact dermatitis, wherein the formulation is capable of inducing oxidative stress and a heat shock response. The active agent is preferably hydrogen peroxide or a precursor thereof such as chitin, chitosan, gelatin, pectin or derivatives thereof.

Description

Allergic Contact Dermatitis The present invention is concerned with a formulation for the treatment of allergic contact dermatitis.

Mild heat shock may be induced in skin as a result of the skin temperature rising to about 45"C and during conditions of oxidative stress and leads to induction of heat shock protein (or stress protein) formation. The heat shock response is regarded as a survival strategy which serves to protect living cells against temperature and other stresses.

It is known that hydrogen peroxide is able to induce the heat shock response (Burdon RH, Gill V, & Rice Evans C. Active oxygen species and heat shock protein induction. In: Stress Proteins. Induction and function. pp. 19-25. Schlesinger MJ, Santoro MG, & Garaci E (Eds).

Springer-Verlag, Berlin, 1990) and also to generate oxidative stress. It is also known that prostaglandins can induce heat shock protein formation (Santoro MG, Garaci E, & Amici C.

Induction of HSP70 by prostaglandins. In: Stress Proteins. Induction and function. pp.

27-44. Schlesinger MJ, Santoro MG, & Garaci E (Eds). Springer-Verlag, Berlin, 1990).

Oxidative stress is a perturbation of redox homeostasis in favour of higher levels of oxidizing species relative to reducing species and is essentially a shift in the thiol/disulfide status of tissue biochemistry in favour of higher levels of disulfides.

Materials for inducing heat shock, such as hydrogen peroxide and polymeric materials capable of generating hydrogen peroxide, have previously been used for wound healing. In non-healing wounds, high levels of hydrogen peroxide (in the range of 10.6 M to about 1 M) may serve to initiate the inflammatory phase of wound healing during which wound debris is removed. Low levels of hydrogen peroxide (around 10.8 M to 10.6 M) are capable of stimulating fibroblast proliferation during later reconstructive stages of wound healing.

The allergic contact dermatitis reaction is however distinct from the wound healing reaction. Allergic contact dermatitis is regarded essentially as a response arising initially from the activity of antigen-presenting cells and subsequently from the activities of T cells when altered (or foreign) protein is detected by the immune system. Although this may lead to a visible inflammatory and eczematous or bullous reaction, soft tissue breakdown id ulcer formation to form a wound is an occasional but not an inevitable outcome. In contrast, a wound may be regarded as a lesion arising directly from soft tissue breakdown.

We have now developed a formulation suitable for use in the treatment of allergic contact dermatitis.

According to the present invention therefore, there is provided a formulation for use in the manufacture of a medicament for the treatment of allergic contact dermatitis, wherein said formulation is capable of inducing oxidative stress and the heat shock response.

The oxidative stress and heat shock response induced by the formulation is preferably such, that the formulation is capable of converting the allergic reaction of the allergic contact dermatitis to an irritant reaction.

It is thought that the oxidative stress induces the heat shock response and converts the allergic reaction of the dermatitis to an irritant reaction, the latter having a shorter time span than the former. It is further thought that the heat shock inhibits the enzyme NADPH oxidase.

The formulation is preferably in a form suitable for application to a patient's skin such as a cream, gel, sheet, spray, or the like for treatment of allergic contact dermatitis.

The present invention has particular applicability in the treatment of allergic dermatitis induced by poison ivy or the like, and therefore provides a remedy for a prevalent skin complaint.

The formulation according to the invention preferably comprises a hydrogen peroxide source; preferably the latter comprises a hydrogen peroxide precursor, although it is envisaged that the source may initially comprise hydrogen peroxide per se.

It is further preferred that the formulation is capable of generating and/or releasing hydrogen peroxide at a level of about 1.0 to 100 x 10-7 M over a 24 hour period as measured by a horseradish peroxidase-scopoletin assay (as described below) using a sample concentration of 0.05 to 0.50% w/v, so as to be capable of permeating the skin in sufficient quantity to enhance the level of oxidative stress and induce the heat shock response.

It is preferred that the hydrogen peroxide precursor comprises a polymeric material capable of generating hydrogen peroxide. Preferred polymeric materials include chitin, chitosan, gelatin, pectin, and derivatives of these materials, or a material such as that commercially available under the trade mark Granuflex (a material comprising hydrocolloid tangles comprising gelatin, sodium carboxymethylcellulose and pectin). The hydrogen peroxide generating capacity of the latter material has been ascribed to the gelatin and pectin.

The polymeric material may therefore typically comprise a composition comprising gelatin and pectin.

A formulation for use according to the present invention is typically a hydrogel or hydrocolloid. The hydrogel or hydrocolloid may be in the form of a paste or sheet, suitable for topical application typically under a semi-occlusive tape such as that commercially available under the trade mark Pharmaclusive. Alternatively, the formulation may be impregnated in a protective covering such as a film, foam, or pad which may be applied onto the affected skin of a patient. The formulation may further comprise prostaglandins which further contribute to the induction of the heat shock response.

The following Examples 1 to 4 illustrate assay of hydrogen peroxide-generating activity of various materials.

EXAMPLE 1 A hydrogen peroxide assay was carried out as follows: Reagents Catalase (from bovine liver, ammonium sulphate and thymol free), scopoletin, horseradish peroxidase (type I), and other chemicals of high purity were obtained from Sigma Co. Ltd., Poole, Dorset, U.K. The chitin/chitosan samples were supplied by Protan Biopolymers, Drammen, Norway. The degree of deacetylation of these materials was estimated using infra-red spectroscopy as previously described by Miya M, Iwamoto R, Yoshikawa S, and Mima S (I.R. spectroscopic determination of CONH content in highly deacylated chitosan; International Journal of Biological Macromolecules, 1980, volume 2, pp. 323-324) and by Sannan T, Kurita K. Ogura K, and Iwakura Y. (Studies on chitin: 7.

I.r. spectroscopic determination of degree of deacetylation; Polymer, 1978, volume 19, 458459). Other polysaccharides and polysaccharide-based materials were commercial products obtained from various suppliers.

A procedure adapted from that previously described by Boveris A, Martino E, & Stoppani AOM (Evaluation of the horseradish peroxidase-scopoletin method for the measurement of hydrogen peroxide formation in biological systems. Analytical Biochemistry, 1977, volume 80, pp. 145-158) was used.

The reaction mixture contained, in a final volume of 6 millilitres, 5 x 10-7 moles/litre scopoletin, test material (0.05 - 0.50% w/v) and 50 millimoles/litre potassium dihydrogen orthophosphate / potassium hydroxide buffer pH 7.4.

The reaction was started by adding 60 microlitres of horseradish peroxidase (HRP, 80 units/millilitre). The mixture was agitated and kept in the dark at 37"C. The change in fluorescence of the mixture was monitored at time intervals (excitation: 320 nm; emission: 460 nm).

The catalase control consisted of 5 x 10-7 moles/litre scopoletin, test material (0.05 - 0.50% w/v), 400 units/millilitre catalase, 0.8 units/millilitre HRP and 50 millimoles/litre potassium dihydrogen orthophosphate / potassium hydroxide buffer pH 7.4 in a final volume of 6 millilitres. The scopoletin control consisted of 5 x 10-7 moles/litre scopoletin, test material (0.05 - 0.50% w/v) and 50 millimoles/litre potassium dihydrogen orthophosphate / potassium hydroxide buffer pH 7.4.

The level of hydrogen peroxide formed is related to the difference in the fluorescence of the reaction mixtures of the test materials with and without catalase. The level of hydrogen peroxide generated by the test materials expressed in moles/litre was derived from a standard curve constructed with known concentrations of standard hydrogen peroxide (10-6 - 10-8 moles/litre). A known amount of standard hydrogen peroxide was added to the reaction mixture containing the test material, scopoletin and HRP to obtain a correction factor for its inhibitory effect on the assay. Thus, the level of hydrogen peroxide detected without and with correction for the inhibitory effect is termed the "lower limit" and the "upper limit" respectively.The actual levels of hydrogen peroxide released by the samples lie between the upper and lower limits.

EXAMPLE 2 Protein chitin/chitosan samples (0.05-0.50% w/v) were assayed using the technique described in Example 1.

Hydrogen peroxide was generated by the samples. The level of hydrogen peroxide generated was related to the concentration and degree of deacetylation as shown in Table 1.

,XAMPLE 3 The following polysaccharides were investigated for their hydrogen peroxide generating capacity - chitosan lactate (Protan), chitin (Sigma), pectin (SquibbDerm), gelatin (Courtaulds), sodium carboxymethylcellulose (SquibbDerm) and Ca/Na alginate (Courtaulds).

The results are shown in Table 2.

EXAMPLE 4 A selection of polysaccharide-based wound dressings, as shown in Table 3, were also investigated for their hydrogen peroxide generating capacity. As can be seen in Table 3, the material commercially available under the trade mark Granuflex Granules, was the only test dressing which generated hydrogen peroxide.

The following Example illustrates the present invention.

EXAMPLE 5 Two 10 microlitre applications of a known skin allergen helenin (0.15% w/v) in ethanol were made to the left forearm of an individual previously sensitized to this allergen.

A further 10 microlitre application of helenin was made to the right forearm and was left untreated as a control. The applications were left uncovered and untreated for 16 hours. During this time, a pruritic skin reaction (1+) began to evolve.

Treatment of the reactions on the left forearm was then initiated. To one reaction was applied a hydrogen peroxide generating hydrocolloid paste containing gelatin and pectin. To the second was applied a square of poly(ethylene oxide) hydrogel which does not generate hydrogen peroxide. The whole area was covered with a semi-occlusive adhesive tape. After 12 hours, the treatments were removed.

Both reactions had evolved into slightly oedematous erythematous papulovesicular eruptions (2+) of 2 centimetre diameter. Pruritus was perceptible but not uncomfortable. The reaction treated with the hydrogen peroxide generating hydrocolloid paste was clearly more intense than that treated with the hydrogel sheet; both were more intense than the control reaction on the right forearm. The whole area of the left forearm was then placed under hot running water at 50"C for 1 minute, dried, and the treatments renewed by exchanging the hydrocolloid mixture and hydrogel applications.

After a further 12 hours, the treatments were removed and the area examined.

The hydrocolloid-treated reaction again appeared noticeably more erythematous than the hydrogel treated reaction. The overall intensity (2+) of the reactions was otherwise unchanged. As before, the whole area was then placed under hot running water for 1 minute, dried, and the treatments renewed, both reactions being covered with the hydrogel.

After a further 12 hours the treatments were again removed, heat treated for 1 minute, and covered with hydrogel for another 12 hours prior to final heat treatment.

After a total of about 72 hours all swelling and itching had subsided from the hydrocolloid treated forearm, and reactions had essentially resolved with only slight reddening still evident on the sites of helenin application. In addition, there was little evidence of post-inflammatory hyperpigmentation (when observed at 7 days).In contrast, the untreated skin reaction on the right arm took about 3 weeks to resolve, was at times almost intolerably pruritic, and resulted in distinct post-inflammatory hyperpigmentation. Table 1 Formation of hydrogen peroxide by chitin/chitosan samples 1 - 6 (0.05-0.50% w/v) in 50 mM potassium hposphate / KOH buffer pH 7.4at37 C quantified by a scopoletin - HRP assay at 24 hours Conc., Hydrogen peroxide, x 10-7M/24 hours (% w/v) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample6 LOWERLIMIT 0.05 0.61#0.07 0.61#0.04 0.26#0.01 2.61#0.31 1.98#0.02 2.72#0.26 0.10 1.19#0.03 1.13#0.12 0.31#0.03 ## 2.14#0.03 3.29#0.12 0.25 1.25#0.16 1.01#0.15 0.27#0.02 ## 1.79#0.06 2.77#0.12 0.50 1.86#0.22 0.62#0.06 0.15#0.02 ## 1.72#0.03 2.36#0.08 UPPER LIMIT 0.05 1.67#0.36 0.70#0.05 0.36#0.02 ND 3.76#0.21 7.60#0.71 0.10 7.37#0.41 1.31#0.15 0.44#0.05 ND 3.68#0.09 13.73#0.60 0.25 10.37#0.91 1.16#0.16 0.45#0.06 ND 3.63#0.05 50.94#8.54 0.50 12.96#1.73 0.73#0.06 0.27#0.03 ND 17.52#1.60 ND Mean#s.e.m.;n=6; ##:Exceeds detection range;ND:Not determined Increasing order of deacetylation:sample3(~55% deacetylation) < sample 2(~65% deacetylation) < sample 1(~65% deacetylation) < sample 5 (~80% deacetylation) < sample 6(~80% deacetylation)#sample 4(~100% deacetylated, oligosaccharide units); extent of depolymerization not determined for any of the samples.

Table 2 Formation of hydrogen peroxide by various polysaccharides (0.05-0.50% w/v)in 50 mM potassium phosphate/KOH buffer pH 7.4 at 37 C quantified by a scopoletin - HRP assay at 24 hours Conc., Hydrogen peroxide, x 10-7 M/24 h Gelatin Sodium carboxy- Ca/Na alginate %w/v Chitosan lactate Chitin Pectin (Courtaulds) methylcellulose (Courtaulds) (Protan) (Sigma) (SquibbDerm) (SquibbDerm) LOWERLIMIT 0.05 2.53#0.21 0.68#0.07 0.32 0 * 0 * 0 * 0.10 3.93#0.05 0.67#0.03 0.70 0.47#0.06 0 * 0 * 0.25 4.88#0.36 1.10#0.16 1.45#0.03 2.43#0.32 0 * 0 * 0.50 4.28#0.05 1.58#0.07 0.37#0.05 6.65#0.12 0 * 0 * UPPER LIMIT 0.05 3.29#0.28 0.85#0.08 0.80 0 * 0 * 0 * 0.10 7.43#0.08 0.87#0.05 2.70 0.79#0.10 0 * 0 * 0.25 8.74#0.65 2.08#0.31 11.15#0.22 7.11#0.76 0 * 0 * 0.50 6.94#0.07 3.47#0.14 3.40#0.41 27.73#0.51 0 * 0 * Mean#s.e.m.;n=6; * :Below detection limit Table 3 Formation of hydrogen peroxide by various polysaccharide-based wound dressings (0.05 - 0.50% w/v) in 50 mM potassium phosphate/KOH buffer pH 7.4 at 37 C quantified by a scopoletin - HRP assay at 24 hours Conc., Hydrogen peroxide, x 10-7 M/24 hours %w/v Granuflex Comfeel Debrisan Bard Kaltostat Sorbsan Granules Powder Granules Dressing Dressing Dressing LOWER LIMIT 0.05 0.70 0 * 0 * 0 * 0 * 0 * 0.10 1.35 0 * 0 * 0 * 0 * 0 * 0.25 1.45#0.01 0 * 0 * 0 * 0 * 0 * 0.50 1.43#0.02 0 * 0 * 0 * 0 * 0 * UPPER LIMIT 0.05 5.83 0 * 0 * 0 * 0 * 0 * 0.10 10.07 0 * 0 * 0 * 0 * 0 * 0.25 11.15#0.23 0 * 0 * 0 * 0 * 0 * 0.50 15.93#0.18 0 * 0 * 0 * 0 * 0 * Mean#s.e.m.;n=6; * :Below detection limit

Claims (10)

1. Claims: 1. A formulation for use in the manufacture of a medicament for the treatment of allergic contact dermatitis, wherein said formulation is capable of inducing oxidative stress and the heat shock response.
2. 2. A formulation according to claim 1, which comprises hydrogen peroxide.
3. 3. A formulation according to claim 1, which comprises a hydrogen peroxide precursor.
4. 4. A formulation according to claim 3, wherein said precursor comprises a polymeric material capable of generating hydrogen peroxide.
5. 5. A formulation according to claim 3 or 4, wherein said precursor comprises any of chitin, chitosan, gelatin, pectin, and derivatives of these materials.
6. 6. A formulation according to any of claims 3 to 5, wherein said precursor comprises gelatin and pectin.
7. 7. A formulation according to any of claims 3 to 5, wherein said precursor comprises hydrocolloid granules comprising gelatin, sodium carboxymethylcellulose and pectin.
8. 8. A formulation according to any of claims 2 to 7, which is capable of generating and/or releasing hydrogen peroxide to an extent as measured by a horseradish peroxidase-scopoletin assay in the range 1.0 to 100 x 10-7 M from a sample concentration of 0.05 to 0.50% w/v over a 24 hour period.
9. 9. A formulation according to any of claims 2 to 8, wherein said formulation is in a form suitable for application to a patient's skin.
10. 10. A formulation according to any of claims 1 to 9, which includes at least one prostaglandin.