WO1996027373A1

WO1996027373A1 - Use of n-substituted l-phenylalanine esters for the treatment of eczema

Info

Publication number: WO1996027373A1
Application number: PCT/GB1996/000486
Authority: WO
Inventors: Karin Uta Schallreuter-Wood; John Martin Wood
Original assignee: Stiefel Laboratories, Inc.
Priority date: 1995-03-06
Filing date: 1996-03-01
Publication date: 1996-09-12
Also published as: GB9504444D0; IL117365A0; GB2299505A; ZA961785B; TNSN96037A1; AU4884796A

Abstract

Non-polar hydrolysable N-substitued L-phenylalanine esters are applied to the skin to treat eczema. The preferred ester is N-acetyl-L-phenylalanine ethyl ester.

Description

USE OF N-SUBSTITUTED L-PHENYLALANINE ESTERS FOR THE TREATMENT OF ECZEMA

The present invention relates to substances and compositions for the treatment of eczema.

N-acetyl-L-phenylalanine ethyl ester is a commercially available non-polar, hydrolysable, N-substituted phenyl- alanine ester hereinaf er referred to as "NALPA ester" . NALPA ester is known to be useful in the field of biochemical research where it has been used as an enzyme substrate, an inhibitor, a spectral standard, an enzyme protective agent and a starting reagent in the synthesis of other compounds. Examples of the use of the ester are listed below.

Pacaud, M. e_ς al. (1976) (European Journal of Biochemistry 69 (1) : 141-151) used NALPA ester as a test substrate in the characterisation of Protease I enzyme obtained from Escherichia coli.

Kobayashi, K. and Katunuma, N. (1978) (Journal of Biochemistry (Tokyo) 84 (1) : 65-74) used NALPA ester as a substrate in their characterisation of a serine protease enzyme extracted from the muscle layer of rat small intestine.

Bai, Y. and Hayashi, R. (1979) (Journal of Biological Chemistry 254(17) : 8473-8479) used NALPA ester as a substrate in their investigations of carboxypeptidase Y enzyme from bakers yeast.

Banno, Y. e£ aj,. (1979) (European Journal of Biochemistry 97(1) : 11-21) used NALPA ester as an enzyme substrate in the characterisation of serine protease from Morris hepatoma 8899. Doi, E. fit al. (1980) (Agricultural and Biological Chemistry 44 (1) : 85-92) used NALPA ester as a substrate in their characterisation of carboxypeptidase enzyme extracted from rice seeds.

Nihira, S. fit al (1983) (J. Biochem. (Tokyo) 94 (2) :565-73) and Tamura, S. fit al. (1984) (Biochemical and Biophysical Research Communications 119 (2 ) : 465-472) used NALPA ester to inhibit of the phagocytic activity of guinea pig peritoneal macrophages.

Tamura, S. fit al- (1984) (Biochemical and Biophysical Research Communications 119 (2) : 465-472) investigated the stimulation by insulin of the phosphorylation of the 95,000 dalton sub-unit of the insulin receptor. NALPA ester was tested for its inhibitory activity on the insulin- stimulated receptor phosphorylation.

Kumar, S. and Murray, R. . (1984) (Journal of the American Chemical Society 106(4) : 1040-1045) used NALPA ester as a substrate for benzophenone oxide. Benzophenone oxide oxidises the ester to the corresponding tyrosine derivative. The results of this oxidation reaction and others were used to predict the possible production of air- borne carcinogens from polycyclic aromatic hydrocarbons in polluted atmospheres.

Aso, K. (1989) (Agricultural and Biological Chemistry 53 (3) : 729-734) used NALPA ester as a substrate in the synthesis of a peptide sweetener,- N-acetyl-L-phenylalanyl- L-phenylalanyl-L-lysine.

Cuevas, L. ≤t al- (1990) (Phytochemistry 29 (5) : 1429- 1432) used NALPA ester to inhibit the 2,4-dihydroxy-7- methoxy-1, 4-benzoxazin-3-one (DIMBOA) inactivation of alpha-chymotrypsin enzyme. DIMBOA is a hydroxamic acid involved in the pest resistance properties of cereals. Adlercreutz, P. (1991) (European Journal of Biochemistry 199(3) .- 609-614) used NALPA ester as a substrate for the alpha-chymotrypsin enzyme in a study into the importance of the support materials used to support enzymes during enzymatic reactions carried out in organic media.

Vulfson, E.N. gt al. (1991) (Enzyme and Microbial Technology ______.: 123-126) used NALPA ester as a substrate for the subtilisin enzyme.

Mach, H. fit al. (1992) (Analytical Biochemistry 200 (1) : 20-26) used NALPA ester to provide near ultraviolet second-derivative spectra. The spectra were then employed in a method for detecting proteins and phenol in deoxyribonucleic acid (DNA) samples by second-derivative absorption spectroscopy.

JP-A-59082310 discloses the topical use of N-acyl-L- phenylalanine amides or N-acyl-L-phenylalanine esters in a pack cosmetic to remove excess sebum from the skin.

JP-A-59212417 discloses the topical use of L-phenyl- alanine and hydrolysable derivatives such as C₁ -C₂₀ alkyl esters to inhibit tyrosinase and thereby provide a skin- lightening and melanin sedimentation-preventing effect after sun-burn.

JP-A-60084209 discloses the topical use of condensates of an L-phenylalanine alkyl ester and a protein as a hair or skin cosmetic.

JP-A-03181417 discloses the use of metal salts of N- long chain acyl-L-phenylalanines as topical antibacterial agents. The preferred acyl groups are stearoyl, lauroyl, myristoyl and palmitoyl. JP-A-6032727 discloses the use of topical compositions containing certain beta-branched phenylalanine derivatives (Formula 1) to inhibit tyrosinase activity and hence the production of melanin. These active derivatives include the ethyl ester of beta-methylphenylalanine but are unsubstituted at the amino group. There is a reference to N-acetyl intermediates having an additional acid or ester group on the ct-carbon atom (relative to the amino group) (see Formula 5) in the manufacture of the active derivatives.

It was stated in Schwenke et al (Dermat. Wschr. 151 (1965) 890-892) that it is unlikely that phenylalanine metabolism plays a significant role in the pathogenesis of atopic eczema.

Atopic eczema is a pruritic inflammatory skin disease exhibiting a characteristic cutaneous morphology. The disease is found generally amongst genetically predisposed individuals and it is believed to arise as the result of a complex interaction of environmental, physiological and genetic factors, e.g. food antigens, psychological stress, temperature and humidity. Related eczematous disorders include dermatitis, seborrhoeic dermatitis and allergic eczema.

Topical corticosteroids have been found to be particularly useful in the treatment of acute severe eczematous disorders. Examples include compounds such as beclomethasone dipropionate, betamethasone, clobetasol propionate, desonide, diflucortolone valerate, fluclorolone acetonide, fluocinolone acetonide, fluocinonide, fluo- cortolone, flurandrenolone, halcinonide, hydrocortisone- butyrate and triamcinolone acetonide. All of the above compounds simply suppress various components of the inflammatory reaction caused by eczema. When treatment with corticosteroids is discontinued a rebound exacerbation of the eczema may occur. Local side effects may arise from repeated and uncontrolled topical use of corticosteroids. These include the spread and worsening of untreated local infection, a thinning of the skin, increased hair growth, acne, mild skin depigmentation and after a long period of treatment irreversible striae atrophicae.

Weeping eczemas can be treated using corticosteroids and wet dressings of 0.01% (w/v) potassium permanganate solution.

Coal tar is known to provide a useful treatment for subacute eczema. The particular formulation of coal tar to be used and its strength depends on the patient's tolerance to coal tar and the severity of the condition. Coal tar paste or zinc and coal tar paste have been found to be generally suitable in most cases when applied directly to the skin. However, they suffer from the drawbacks of an unpleasant smell and an unpleasant appearance when in use on the skin.

Coal tar pastes cannot safely be used to treat eczema on a patient's face. The preparations can also give rise to side effects such as skin irritation, skin photo- sensitisation and acne-like eruptions. Coal tar-based compositions tend to stain skin, hair and fabric.

Less acute forms of eczema can be treated with ichthammol. This has a milder action than coal tar and can be applied in the form of an ointment, optionally in combination with zinc paste. A drawback with ichthammol is that it cannot be applied to broken or inflamed skin. Like coal tar ichthammol has also been found to give rise to skin irritation and skin sensitisation.

In eczematous disorders where there is a marked thickening of the skin and pronounced scaling, the application of a keratolytic such as salicylic acid followed by ichthammol or coal tar has been found to provide an effective treatmen .

Emollients such as aqueous creams and basic ointments are known to be useful in the treatment of skin dryness in patients who do not exhibit the symptoms of eczema. The emollients are also useful in the treatment of dry, fissured, scaly lesions of the skin.

The present inventors have found that N-acetyl-L- phenylalanine ethyl ester and other non-polar, hydrolysable, N-substituted L-phenylalanine esters provide an effective treatment for eczema.

As used in this application, the term eczema includes eczematous disorders such as dermatitis, seborrhoeic dermatitis, allergic eczema and, especially, atopic eczema.

Accordingly, the present invention provides the use of a non-polar, hydrolysable, N-substituted L-phenylalanine ester in the manufacture of a topical medicament for the treatment of eczema.

In a second aspect, the present invention provides a method of treating eczema comprising the topical application to a patient of an effective amount of a non- polar, hydrolysable, N-substituted L-phenylalanine ester. For the treatment of at least some forms of atopic eczema, it appears that it may be necessary to limit the amount of ester used so that the in vivo hydrolysis thereof produces L-phenylalanine at a concentration of less than 100 micromolar.

The invention also provides topical compositions for the treatment of eczema comprising a non-polar, hydrolysable, N-substituted L-phenylalanine ester, preferably at a bioavailable concentration equivalent to less than 100 micromolar free L-phenylalanine in a pharmaceutically acceptable topical vehicle. Preferably, the ester bioavailable concentration in the composition is equivalent to 50 to 80 micromolar free L-phenylalanine. By "bioavailable", we mean that amount of an active substance which is available to the patient in vivo.

The esters used in the invention have the following structure:-

wherein X and Y are hydrolysable groups which are removed by in vivo hydrolysis following application of the compound to the skin and its absorption thereby to release phenylalanine.

X is preferably an acyl group (R'CO-) . Usually, R' will be a saturated aliphatic group although it may be an unsaturated aliphatic group such as an alkene or an alkyne or an aromatic group, e.g. benzene. It is particularly preferred that X is a C₂-C₆, especially C₂-C₄, alkanoyl group, especially acetyl or propyl.

Y is preferably a saturated aliphatic group although it may be an unsaturated aliphatic group such as an alkene or an alkyne, or an aromatic group e.g. benzene. It is particularly preferred that Y is a C₂-C₆, especially C₂-C₄, alkyl group, especially ethyl or propyl.

Presently, the preferred esters are N-propyl-L- phenylalanine ethyl ester, N-acetyl-L-phenylalanine propyl ester and, especially N-acetyl-L-phenylalanine ethyl ester ("NALPA ester") .

It is believed the esters used in the invention undergo hydrolysis in the skin to yield L-phenylalanine and byproducts corresponding to the ester and N-substituent. When the ester is NALPA ester, the products are L- phenylalanine, ethanol and acetic acid. L-phenylalanine is an essential amino acid present in the body and acetic acid and ethanol are known to be present in the skin under normal conditions in low concentrations. Furthermore, ethanol is known to be metabolised in the skin. Accordingly, the use of NALPA ester presently is particularly preferred because of the low risk of side effects.

The ester will be applied to the skin in a pharmaceutically acceptable vehicle. The vehicle is preferably a cream, gel, ointment or paste but can be, for example, a lotion, dusting powder, application, collodion, liniment or paint.

The invention will now be described with reference to the following examples.

EXAMPLE 1

A composition comprising 100 x 10^"6 M N-acetyl-L- phenylalanine ethyl (NALPA) ester (23.5 mg/lOOOg) in Neribas^* (Schering A.G.) was prepared. Neribas^* is a cream vehicle containing Macrogol stearate 2000; stearyl alcohol; liquid paraffin,- white soft paraffin,- polyacrylic acid; sodium hydroxide; disodium EDTA (i.e. ethylenediaminetetra- acetic acid disodium salt); methyl and propyl Paraben (i.e. 4-hydroxybenzoic acid methyl and propyl esters) ; and water.

Fifteen patients suffering severe atopic eczema were instructed to apply the NALPA ester cream to the parts of their bodies affected by eczema. The average application by each patient was in the range four to eight times a day. The patients were assessed 24 hours, 48 hours, 72 hours, one week and three weeks after the start of the treatment using the Severity Scoring of Atopic Dermatitis (SCORAD) Index (Dermatology 186: 23-31 (1993)).

After 24 hours of treatment there was a significant improvement of skin symptoms in all patients. The treatment first reduced erythema (redness) followed by a decrease in the amount of scaling, oozing and lichenification. After four to five days the treatment had eliminated clinical signs of eczema from the patients.

EXAMPLE 2

Ten male patients with atopic eczema were treated with the NALPA ester cream referred to in Example 1 above. The areas of the body exhibiting eczema in each patient is set out in Table 1 below. All ten patients had severe eczema at the time of first treatment. The mean age of the patients was 21.5 years. At the start of the treatment, the mean serum IgE level was 4432 kU/1. Two patients were found to have normal IgE levels of less than <100 kU/1. The mean of the peripheral eosinophiles was 7.3%. Three patients were in the normal range of <5%. The NALPA ester cream was applied to the affected areas three to five times each day. Seven out of ten of the patients exhibited eosinophilia and so all the patients were also treated with one tablet of lOmg ceterizine dihydrochloride (sold under the trade name Zyrtec by UCB Chemie) each day. Ceterizine is known to provide an effective anti-pruritic treatment possibly by stabilising the eosinophil cells.

After 24 hours treatment, significant changes in the eczematous regions of each patient were observed. Erythema was reduced dramatically within thirty minutes of treatment. The patients were treated and observed for three weeks. At the end of the treatment acute eczematous skin reaction was completely absent.

Two patients also were treated on their arms twice daily with Nerisona^* cream (Schering A.G.) and on the rest of their body with the NALPA cream. Nerisona cream comprises 0.1% (w/w) diflucortolone valerate in water- miscible base and provides potent corticosteroid treatment for severe acute eczema. No significant difference in the results of the two treatments was observed.

Tablβ 1

π-mπm-ry of ten patients treated as described in Example 2

Patient Age in Serum IgE Blood Eczematous Number years (kU/1) eosinophiles areas of body

(%)

1 27 <19,000 18.1 face, neck, arms

2 10 527 7.2 arms, legs

3 12 66 3.0 arms, legs, penis, scrotum

4 10 340 6.0 face, neck, upper anterior (front) chest, arms legs

5 22 48 9.9 face, neck, arms, legs

6 25 2,592 8.1 total body

7 26 247 4.1 eyelids, arms, legs

8 24 1,500 8.2 eyelids, perioral, arms, neck

9 26 1,000 3.0 total body

10 22 <19,000 11.0 total body mean 21.5 4,432 7.26 EXAMPLE 3

To test the effect of L-phenylalanine on the beta-2- adrenoceptor density in atopic eczema, cell cultures of epidermal keratinocytes were established from suction blister roofs of two patients, one mother of a patient and from two healthy controls. An analysis of beta-2- adrenoceptor numbers/cell was conducted after saturation binding with (-) [³H]CGP-12177 radioligand in subconfluent cell cultures of epidermal undifferentiated keratinocytes in serum-free MCDB 153 medium, containing 0.1 x 10^"3 M calcium and different concentrations (30, 50, 70, 90, 120 & 150 x 10^"6 M) of L-phenylalanine. The results from atopic eczema revealed a maximum receptor number at 70 x 10^"β M L- phenylalanine. The maximum receptor numbers for the mother and controls were at 90 x 10^"6 M yielding a broader range in receptor homeostasis compared to the patients. Keratinocytes from atopic eczema showed a significantly more narrow range for receptor homeostasis (i.e., 30-90 x 10^~6 M L-phenylalanine) compared to controls (i.e., 30-150 x 10^~6 M) , whereas optimum receptor expression of the mother was between 30-120 x 10^"6 M.

Claims

1. The use of a non-polar, hydrolysable, N-substituted L- phenylalanine ester in the manufacture of a topical medicament for the treatment of eczema.

2. A use as claimed in Claim 1, wherein the ester is an N- (C₂-C₆ alkanoyl) -L-phenylalanine (C₂-C₃ alkyl) ester.

3 A use as claimed in Claim 1, wherein the ester is an N-acetyl-L-phenylalanine (C₂-C₆ alkyl) ester.

4. A use as claimed in Claim 1, wherein the ester is N- acetyl-L-phenylalanine ethyl ester.

5. A use as claimed in Claim 1, wherein the medicament contains the ester in a bioavailable amount equivalent to less than 100 micromolar free L-phenylalanine.

6. A topical composition for the treatment of eczema comprising a non-polar, hydrolysable, N-substituted L- phenylalanine ester in a pharmaceutically acceptable topical vehicle.

7. A composition as claimed in Claim 6, wherein the ester is present at a bioavailable concentration equivalent to less than 100 micromolar free L-phenylalanine.

8. A composition as claimed in Claim 6, wherein the ester is an N- (C₂-C_fi alkanoyl) -L-phenylalanine (C₂-C₆ alkyl) ester.

9. A composition as claimed in Claim 6, wherein the ester is an N-acetyl-L-phenylalanine (C₂-C₆ alkyl) ester.

10. A topical composition for the treatment of eczema comprising N-acetyl-L-phenylalanine ethyl ester in a pharmaceutically acceptable topical vehicle.

11. A composition as claimed in Claim 10, wherein the ester is present at a bioavailable concentration equivalent to less than 100 micromolar free L-phenylalanine.

12. A method of treating eczema comprising the topical application to a patient of an effective amount of a non- polar, hydrolysable, N-substituted L-phenylalanine ester.

13. A topical composition for use in the treatment of eczema comprising a non-polar, hydrolysable, N-substituted L-phenylalanine ester in a pharmaceutically acceptable topical vehicle.