WO2002078687A2 - Delta-aminolevulinic acid for the therapeutical and cosmetic use - Google Patents

Abstract

The use of Δ-aminolevulinic acid in the treatment of cutaneous pigmentation disorders and as a cosmetic.

Description

DELTA-AMINOLEVULINIC ACID FOR THE THERAPEUTICAL AND COSMETIC USES

The present invention relates to the use of δ-aminolevulinic acid for both the treatment of alterations of cutaneous pigmentation, and for cosmetic purposes. δ-Aminolevulinic acid (ALA) has being used from about ten years for photodynamic therapy (PDT). This technique is based on the systemic administration or topical application of a photosensitizing agent, which, upon activation by light of a specific wavelength, induces a photochemical reaction, which destroys tissues. During the last years, a number of photosensitizing compounds, such as hematoporphyrin derivatives, have been used mainly through the injective route (1-9).

ALA is actively transported through plasma membranes, metabolized by cells and transformed into protoporphyrin IX that represents the real photosensitizer. Protoporphyrin IX absorbs light in the visible spectrum and induces a photodynamic reaction, which destroys the tissue in which such reaction takes place.

For dermatologic diseases PDT with ALA is widely used in the treatment of a number of cutaneous malignancies: basal cell carcinoma, squamous cell carcinoma, actinic keratoses, Bowen's and Kaposi's diseases (10-15). Moreover, photodynamic therapy with ALA has also been used in recent years for the treatment of inflammatory or infective dermatoses (16- 24). ALA-PDT has been experimented with encouraging results in patients affected by psoriasis, condylomata, plantar warts. Although the mechanism of action in the various dermatoses has not yet been completely elucidated, the experts share the conviction that photodynamic therapy with ALA may be beneficial whenever an alteration of the keratinic layer or accelerated epidermal turnover are present.

It has now been found that the topical administration of ALA induces PpIX formation also in epidermis non interested by those pathological phenomena involving alteration of the keratinic layer or accelerated epidermal turnover. In particular, it has been found that the topical administration of ALA to normal skin by a suitable "occlusive" medication, (which provides higher trans cutaneous penetration of the compound), and the subsequent irradiation with visible light (at lower doses than those conventionally used for the treatment of tumors), induce melanin pigmentation in the treated area. This pigmentary response induced on healthy human skin can persist for a time ranging from two weeks to two months.

Histological and histochemical features show an activation of epidermal melanocytes; colorimetric values change on all the treated areas, thus proving the changes of the epidermal color. No reactions are observed in control skin areas treated with: a) cream base not containing ALA, b) ALA without subsequent irradiation or c) visible light irradiation without previous ALA administration.

ALA, when administered on normal skin by occlusive medication, penetrates epidermis and superficial dermis in amounts that do not induce tissue necrosis upon irradiation. In all likelihood, ALA specifically acts on melanocytes, as proved in particular by immunohistochemical coloration which evidences a marked melanocytic activation. The cutaneous pigmentation effect observed after treatment with ALA allows to envisage its use in the treatment of cutaneous dermatoses characterized by hypopigmentation or for cosmetic purposes.

According to a first aspect, the invention therefore relates to the use of ALA for the preparation of a medicament for use in the photo activating treatment of hypopigmentation diseases. "Hypopigmentation diseases" herein means a series of congenital or acquired disorders consisting in an alteration of melanin synthesis. Examples of said pathologies are vitiligo, or post- inflammatory, post-burns or post-cutaneous infection hypopigmentations. According to a preferred embodiment, the invention relates to the treatment of vitiligo, a dermatological disorder characterized by sharply demarcated, achromatic areas with low or no melanocytes content.

According to a further aspect, the invention relates to the use of ALA as a cosmetic, in particular as a tanning agent.

For both therapeutical and cosmetic applications, ALA will be suitably formulated so as to guarantee an effective release of the active ingredient. Suitable administration forms include creams, ointments, pastes, emulsions, although other systems for the transcutaneous release may be used as well. An exhaustive treatise on said pharmaceutical forms and related excipients can be found in Remington's Pharmaceutical Sciences Handbook, Mack Pub. Co., NY, USA, XVII Ed. Most preferred are the creams containing a "fatty", "absorption" or "emulsion" base, able to provide a gradual occlusive effect. Fatty bases ensure optimal penetration of the active ingredient, but they are rather unpleasant to apply on the skin and are also difficult to remove. "Absorption" bases consist of a fatty hydrophobic base in which a water-in-oil emulsifier is included to increase hydrophilicity, whereas "emulsion" bases, compared with the first, also contain water. The latter are more suitable for the cosmetic uses, as they are easily applied to and readily removed from the skin. The topical preparations of the invention will have a content in active ingredient ranging from 0.1 to 30% based on the total weight of the preparation.

Photodynamic therapy with ALA for the treatment of the pigmentation disorders has to carried out under controlled conditions, using as the source of visible light a tungsten lamp or coherent light sources whose spectrum emission ranges between 400 and 700 nm, wavelength which is selectively absorbed by PpIX.

On the other hand, controlled conditions are not necessary to obtain a tanning stimulating cosmetic effect, as the periodical application of the formulation based on ALA and the subsequent exposure of the skin to sunlight are sufficient.

The following examples further illustrate the invention. Experimental section Materials and methods Patients

5 Healthy volunteers (4 male and 1 female), of age from 38 to 49, skin type 111 and IV according to Fitzpatrick, gave their written informed consent and entered the study. Subjects were not taking any systemic or topical drug and were in good physical condition. Materials

The ALA used was purchased from Sigma (SL Louis, MO) and was prepared in 5%, 10% and 20% concentrations in an oil-in-water emulsion (Eucerin cream base).

A slide projector equipped with a 150 W tungsten lamp whose spectrum emission ranged between 400 and 700 nm with a peak at 630 nm was used as source of visible light. The irradiance was 35 mW/cm at 20 cm distance from the lamp.

A spectrocolorimeter X-Rite 968 was employed to obtain chromometric data from the ALA-PDT treated areas. The spectrocolorimeter detects reflected light in the visible spectrum (range 400-700 nm) but it also works as a chromometer recording colors in a three-dimensional space.

In the system named L*a*b*, L* expresses the relative brightness of the color ranging from black to white; a* and b* represent the color hue, ranging respectively from red to green and from blue to yellow. The real skin color is a mix of all the above mentioned values.

In this study both a*, which is the value that allows a real assessment of erythema changes, and L*, which shows pigmentation changes, have been considered.

Optical microscopy

Tissue were fixed with formalin and included in a paraffin block from which 5 microns thick serial sections were cut. A hematoxylin-eosin section of each biopsy was examined using a Leitz Leborlux K microscope (Leica Imaging System, Inc., Cambridge, England) in order to assess the relative histologic features. Immunohistochemistry

Two serial sections were fixed, rehydrated through alcohol and treated with 3% hydrogen peroxide for 5 minutes to inactivate endogenous peroxidases and then washed in distilled water.

Incubation with the anti-SlOO (SI 00 DAKO, dil. 1 : 1000) and with anti HM-B45 (DAKO, MO 634 monoclonal, dil. 1 : 100) antibodies was carried out overnight at 4°C in a moist chamber.

The conventional ABC (Avidin-Biotin Complex) procedure was then applied (28).

Dyaminobenzidine was used as chromogen, and hematoxylin for nuclear counterstaining. Electron microscopy

Biopsy specimens were fixed with 2.5% glutaraldehyde. Tissue was then rinsed with 0.1 M sodium cacodylate buffer (pH 7.4) and post- fixed with 2% osmium tetroxide.

Specimens were dehydrated in graded ethanol solutions and embedded in Epon. Sections were obtained using an ultramicrotome UM-4 LKB; they were counterstained con uranyl acetate and lead citrate. The electron- microscope used was the EM 109 Zeiss. Treatment

On each subject, five square areas named A, B, C, D, E (2 x 2 cm) were defined on the upper part of the arms. Two areas acted as controls (A area: only 20%) ALA application without VIS irradiation and B area: VIS irradiation after application of the vehicle without ALA).

C, D and E areas received ALA- containing cream at concentrations of 5, 10 and 20%, respectively, and 4 hours later they were irradiated with a fixed dose of 40 J/cm of visible.

After ALA application, the skin areas were covered by the occlusive chambers (Finn chamber test) which are usually utilized for skin patch tests.

Clinical and colorimetric skin assessments were made before the ALA or vehicle applications and 30 minutes 1, 2, 3, 7 and 14 days after the irradiative session. During the skin measurements, room temperature was under control (23 °C). Volunteers were at rest for 10 minutes before each measurement.

2.5 mm diameter, full-thickness punch biopsies were taken from the two control sites and from the 20% ALA-treated site, 3 and 7 days after irradiation. Punch biopsies were studied by light and transmission electron microscopy (TEM). In vivo fluorescence: the experimental apparatus

The excitation source was a dye laser (PTI model PL 201).

The probe was composed by the heads of fifty-six quartz fibers, twenty-seven of which delivered the excitation radiation from the laser to the patient's skin. The remaining twenty-eight fibers, whose heads are located alternatively with respect to the excitation ones in the probe, delivered

AutoFluorescence radiation from irradiated tissues to spectrograph entrance slit. The instrument for light analysis was the scanning spectrograph- monochromator Chromex 500 IS/SM (asymmetrical Czerny Turner configuration, focal length = 0.5 m), equipped with toroidals collimator and camera mirrors. In our measurements the excitation radiation was obtained by using in the dye laser the PLD 500 dye (PTI Canada Inc.) that upon excitation at 337 nm produces a well peaked fluorescence emission at 500 nm.

The spectral measurements were performed by averaging the spectra recorded on all the rows of the CCD matrix. We averaged fluorescence radiation from all the different cutaneous regions irradiated by the probe. The average allowed to overcome in a very simple way the difficulties arising from the lack of spatial uniformity of the excitation illumination, but prevented simultaneous measurements of different areas to be performed. The skin region whose fluorescence was measured, was treated as previously described: ALA cream (5, 10 and 20%) was topically applied under occlusion on a 2 cm² skin area located in the inner part of the arm and subsequently irradiated with visible.

Three types of measurements were performed on: a) ALA treated skin region b) untreated skin region c) background measurements

All measurements were performed in a dark room. In measurements a) and b) the probe was positioned perpendicularly to the skin and in light contact with it. In measurements c) the probe was hold in the air. Clinical results

All subjects complained burning and itching sensations during the irradiative session. An erythematous response with pruritus and edema appeared just after the irradiation and lasted for about 24 hours. 48 Hours after the treatment a variable pigmentary response occurred, which lasted 1 - 4 weeks (depending on the skin type of the volunteer) (Fig. 1 ). Colorimetric data Colorimetric response confirmed the clinical changes.

As colorimetric slopes were similar in all subjects, in Figures 2 and 3 the a* values and the L* values of a single subject are reported.

The erythema changes (a*) and the pigmentation ones (L*) are evidenced. In vivo fluorescence

The results of the spectral measurements for the three studied cases are shown in Fig. 4. In the figure is reported the fluorescence intensity, measured as the number of counts as a function of the wavelength (expressed in μm) for the ALA-treated skin region, for the control skin region and for the background. It can be seen that, for all the three examined cases:

1) the background spectrum is approximately independent of the wavelength;

2) the spectrum of the untreated skin region shows a smooth dependence on the wavelength in the studied spectral range, with the exception of a strong drop of fluorescence intensity at 590 μm wavelength;

3) the spectrum of ALA-treated skin region show a well defined and symmetric peak at the wavelength of 635 μm, superimposed to the control spectrum.

Histopathology In Fig. 5 the control biopsy is shown. Only a slight increment in melanin pigmentation was observed in the control biopsies performed 3 days after treatment.

On the contrary, in bioptic samples performed after 7 days, a considerable increase in the size of the single melanocytes was observed, with most of the nuclei enlarged, often with an irregular profile. Moreover, also the melanocyte number was greater (Fig. 6).

Conversely, no appreciable difference in the degree of melanin pigmentation was found in the 7-days biopsies.

The immunohistochemical staining for S-100 protein further underlighted these findings (Figs. 7, 8).

In the biopsies performed 7 days after ALA exposure, a considerable positivity of melanocytes for HMB-45 protein was found, which was absent in the biopsy performed 3 days after treatment. This activation takes place only some days after the stimulation (Figs 9 and 10). Transmission Electron Microscopy (TEM

Biopsies specimens from control sites showed no melanocyte alterations. Treated areas show that melanocytes contain both simple and composite melanosomes. Some melanophages containing composite melanosomes are observed: they are heterolysomes containing melanin granules (Fig. 11). Some melanocytes contain granular electron-dense melanosomes which represent a more advanced melanization process. Discussion

In this study a pigmentary response has been obtained on normal human skin after a single ALA-PDT treatment.

As it has been already been shown (25) in our study spectral measurements of fluorescence intensity confirm the production of PpIX in ALA-treated skin areas, contrarily to what happens in the untreated areas. The spectral measurements were performed 4 hours after the topical application of ALA, which is the delay corresponding to the production peak of Pp IX (30-31). Despite the low number of studied cases, spectral analysis shows unambiguously that the excitation at the wavelength of the absorption peak of Pp IX, produces a fluorescence emission peak.

This result can not be imputed to experimental inaccuracies, but it proves that: a) Pp IX is present in tissues at least at the depths where the radiation at 500 nm can penetrate; b) the "in vivo" excitation wavelength of Pp IX is not very different from the one used in previous papers (30-31) and c) the wavelength of the emission spectrum peak of Pp IX in vivo is coincident with that found in previous studies (30). The clinical effect obtained consisting in erythema and edema after irradiation, followed by a pigmentary response, is characteristic of a classic cutaneous phototoxicity reaction.

The clinical picture is similar to the cutaneous signs of protoporphyria and is apparently directly related to the action of PpIX which generates reactive oxygen species; these in turn induce lipid peroxidation and cell membrane alterations. Other mediators and enzymes can contribute to the inflammatory response.

The hyperpigmentation that is been clinically and colorimetrically detected during the two weeks following the treatment is a hyperpigmentation arising from an increased number of active melanocytes, without increased melanin production as shown by the histologic features. This event is, on the other hand, characteristic of solar UV tanning and some other cutaneous hyperpigmentations, such as tan induced by psoralens plus UVA. Nevertheless in these last hyperpigmentations histologic examination shows an increased number of active melanocytes normal in size and cellular details.

After ALA-PDT, on the opposite, we found a considerable increase in melanocytes with most nuclei enlarged and irregular profile.

The immunohistochemical staining for S-100 and HMB-45 proteins further support these findings. Moreover, the considerable positivity for HMB-45 protein supports the hypothesis of a strong biological activation of melanocytes after the ALA-PDT.

S-100 protein is expressed by virtually all malignant melanomas and melanocytic nevi but also by other tumors. (32-33).

HMB 45, on the contrary, is a monoclonal antibody that recognizes a melanosome-associated cytoplasmic antigen. The HMB-45 Ab stains malignant melanomas and in some cases Spitz nevi, dysplastic nevi and compound nevi. The antigen is not present in vitiligo, soft tissue sarcomas, malignant

Iymphomas, intradermal nevi, Sutton nevi and melanocytic hyperplasia of the nail bed (34).

All these observations demonstrate that ALA-PDT performed on normal skin causes a cutaneous damage with erythema and edema just after irradiation and hyperpigmentation that lasts for almost 2 weeks, due to a considerable activation of melanocytes with an increase in cell number characterized by abnormal, irregular nuclei.

The positivity of melanocytes for S-100 and HMB-45 proteins supports these observations.

REFERENCES

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Claims

1. The use of δ-aminolevulinic acid for the preparation of a medicament for use in the photodynamic therapy of hypopigmentation pathologies.

2. The use as claimed in claim 1, wherein said pathologies are vitiligo; post-inflammatory, post-burns or post-cutaneous infection hypopigmentations.

3. The use of δ-aminolevulinic acid as a cosmetic.

4. The use as claimed in claim 3, wherein said cosmetic is a tanning agent.

5. The use as claimed in any one of the above claims, wherein the medicament or cosmetic is in the form of cream, ointment, emulsion, paste.

6. The use as claimed in claim 5, wherein said pharmaceutical/cosmetic are formed by a water-in-oil emulsified base.