ITMI20010685A1 - DELTA-AMINOLEVULINIC ACID FOR MEDICAL AND COSMETIC USE - Google Patents

Description

Description of the industrial invention entitled:

"DELTA-AMINOLEVULINIC ACID FOR MEDICAL AND COSMETIC USE"

The present invention relates to the use of δ-aminolevulinic acid in the treatment of skin pigmentation pathologies, or as a cosmetic.

For about a decade, δ-aminolevulinic acid (ALA) has been used for photodynamic therapy (PDT). The latter is a technique based on the administration or local application of a photosensitizing agent which, activated with light of a certain wavelength, gives rise to a photochemical reaction with consequent tissue destruction. In the past, numerous substances with photosensitizing action have been used, administered mainly by injection, for example the hematoporphyrin derivatives.

ALA, actively transported through the piasmatic membranes, is metabolized by the cells and transformed into protoporphine IX which is the true photosensitizer. Protoporfin IX absorbs light in the visible spectrum and gives rise to a photodynamic reaction which leads to the destruction of the tissue in which the reaction itself takes place.

Systemically, ALA is mainly used in the treatment of gastrointestinal, bronchopulmonary and brain tumors.

In dermatology it is used exclusively topically. In particular, it has found wide use in the PDT of numerous tumor forms: basal cell epitheliomas, squamous cell epitheliomas, actinic keratoses, Bowen's and Kaposi's diseases. Furthermore, in recent years, photodynamic therapy with ALA has also been used for the treatment of inflammatory or infectious dermatoses. ALA-PDT has been tested with encouraging results in patients suffering from psoriasis, warts, plantar warts. Although the mechanism of action in the various dermatoses has not been completely clarified, it is a common belief among experts in the field that where there is an alteration of the keratin layer or an accelerated epidermal turnover, photodynamic therapy with ALA can provide good results.

It has now been found that the topical application of ALA induces the formation of PpIX even on skin not affected by pathological phenomena that involve an alteration of the keratin layer or an accelerated epidermal turnover. In particular, it has been seen that the application of ALA on healthy skin by means of an appropriate "occlusive" dressing, that is, such as to allow greater transcutaneous penetration of the compound, and the subsequent irradiation with visible light, at lower doses than those generally used for treatment of tumors, they produce a melanic pigmentation in the treated area. This pigmentation can persist for a variable time between two weeks and two months.

The histological and histochemical data collected show that following the treatment there is an activation of epidermal melanocytes; clinically the colorimetric values are modified on all treated areas, demonstrating the changes in skin color. The control areas, treated with base cream not containing ALA, with ALA without subsequent irradiation or with visible light without previous application of ALA, did not show any type of reaction.

ALA, if applied to healthy skin with occlusive dressing, is able to penetrate the epidermis and the superficial dermis, but in such quantities as not to cause tissue necrosis following irradiation. It is likely that ALA exerts a specific action at the melanocyte level, as demonstrated in particular by the immunohistochemical stains that show a marked activation of these cells. The skin pigmentation effect observed after treatment with ALA makes it possible to predict its use in the treatment of hypopigmentation pathologies or for cosmetic purposes.

According to a first aspect, the invention is therefore directed to the use of ALA for the preparation of a drug that can be used in the treatment of hypopigmentation pathologies, following photoactivation. By "hypopigmentation pathologies" we mean a series of congenital or acquired disorders consisting of an alteration in the synthesis of melanin. Examples of such pathologies are vitiligo or post-inflammatory, post-burn or post-skin infection hypopigmentation. According to a preferred embodiment, the invention relates to the treatment of vitiligo, a dermatological disease characterized by well-defined and achromic areas with low content or free of melanocytes.

According to another aspect, the invention is aimed at the use of ALA as a cosmetic, in particular as a bronzer.

For both therapeutic and cosmetic applications, ALA will be appropriately formulated in such a way as to ensure effective release of the active ingredient. Suitable administration forms include creams, ointments, ointments, emulsions, but other transcutaneous delivery systems can also be used. A full discussion of these pharmaceutical forms and related excipients can be found in Remington's Pharmaceutical Sciences Handbook, Mack Pub. Co., NY, USA, 17th Ed. Most preferred are creams containing a "fat", "absorption" or "emulsion" type base, capable of providing a gradual occlusive effect. Fat-type bases ensure optimal penetration of the active ingredient, but their application on the skin causes a rather unpleasant sensation and are also difficult to remove. The "absorption" bases are formed by a hydrophobic fatty base in which a water-in-oil type emulsifier is incorporated to increase Γ hydrophilicity, while the "emulsion" bases, compared to the former, additionally contain water. The latter are more suitable for cosmetic uses as they are easily spread on the skin and can be removed just as easily. The topical preparations of the invention will have an active ingredient content ranging from 0.1 to 30% of the total weight of the preparation itself.

Photodynamic therapy with ALA for the treatment of altered skin pigmentation states is generally conducted under controlled conditions, using tungsten lamps or coherent light sources with emission at 400-700 nm as a visible light source, a wavelength that is selectively absorbed by PpIX.

Controlled conditions are not required to have a cosmetic effect of tanning stimulation, as periodic application of the ALA-based formulation and subsequent exposure of the skin surface to sunlight are sufficient.

The following examples illustrate the invention more clearly.

Experimental part

Materials and methods

Patients

5 healthy volunteers (4 men and 1 woman) aged between 38 and 49 years, of phototype III and IV according to Fitzpatrick, gave their informed consent to the study.

The subjects were not taking drugs either systemically or topically and were in good physical condition.

Materials

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

A mobile projector equipped with a 150 W tungsten lamp whose emission spectrum was between 400 and 700 nm with a peak at 630 nm was used as a visible light source, Γ irradiance was 35 mW / cm2 at a distance from the 20 cm lamp.

An X-Rite 968 spectrocolorimeter was used to obtain chromometric data from the areas treated with ALA-PDT.

The spectrocolorimeter perceives reflected light in the visible spectrum (between 400 and 700 nm) but also works as a chromometer by recording the colors in a three-dimensional space.

In the system called L * a * b *, L * expresses the relative brightness of the color that goes from black to white; a * and b * represent the tint that goes from red to green and from blue to yellow respectively. Real skin color is a combination of all of the above values.

In this study, both a * was considered because it is the value that allows a real evaluation of changes in erythema, and L *, because it shows pigmentation changes.

Optical microscopy

Tissues were formalin-fixed, paraffin-embedded and cut into 5-micron thick sections.

A section of each biopsy stained with hematoxylin-eosin was examined using a Lietz Leborlux K microscope (Leica Imaging System, Ine. Cambrì dge-England) to evaluate its histological features Immunohistochemistry

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

Incubation with S-100 antibodies (S-100 DAKO dii. 1: 1000) and with anti HMB-45 (DAKO MO 634 dii. 1: 100) was performed at night at 4 ° C in a humid chamber.

The conventional ABC procedure (avidin - biotin - complex) was then applied.

Diaminobenzidine was used as a dye and hematoxylinacosine was used to highlight the nuclei.

Electron microscopy

Biopsy specimens are fixed with 2.5% glutaraldehyde.

The tissue was rinsed with 0.1 M sodium cacodylate (pH 7.4) and then fixed with 2% osmium tetroxide.

The tissues were dehydrated in increasing solutions of ethanol and placed in Epon.

Sections were obtained using a UM-4 LKB ultramicrotome; they were contrasted with uranyl acetate and lead citrate.

A ZEISS EM 109 was used as the electron microscope.

Treatment

In each subject, 5 square areas called A-B-C-D-E (2x2 cm.) Were delimited on the upper third of the arms.

Two areas acted as controls (area A: application of 20% ALA without visible irradiation and area B: visible irradiation after the application of only the vehicle without ALA).

On areas C, D, E ALA was applied at a concentration of 5.10% and 20%, and 4 hours later they were irradiated with a dose of 40 J / cm2 of visible.

ALA was applied occlusively using plastic cells (Finn chamber test) which are usually used for skin patch tests.

Clinical and colorimetric evaluations were made before the application of ALA, 30 minutes after and 1, 2, 3, 7 and 14 days after the irradiation session.

During the measurements the room temperature was under control (23 ° C). Volunteers rested for 10 minutes before each measurement.

Three and seven days after irradiation, biopsies were performed using punches of 2.5 mm in diameter both from the areas treated with visible 20% ALA and from the two control areas.

Biopsies were observed with light microscopy and transmission electron microscope (TEM).

In vivo fluorescence: experimental setup

A dye laser (PTI model PL 201) was used as the light source to determine the fluorescence.

The measuring probe consists of 56 quartz fiber heads; 27 of these fibers carry the exciting radiation from the laser to the patient's skin. Twenty-eight fibers, on the other hand, whose heads are placed alternately in reference to the exciting ones, transmit the autofluorescence from the irradiated tissues to the entrance slot of the spectrograph.

The light analysis tool was a Chromex 500 IS / SM monochromator spectrograph (Creruy Tumer asymmetric configuration, focal length = 0.5 m) equipped with toroidal collimator and camera mirrors.

In our measurements, the radiation excitation was obtained using the PLD 500 dye (PTI Canada Inc.) in the dye laser which under the excitation at 337 nm produces a fluorescence emission at 500 nm (30).

Spectral measurements were made by averaging the spectrum recorded on all CCD files.

Fluorescence radiation was obtained on average from all the different skin regions irradiated by the probe.

The average made it possible to overcome in a very simple way the difficulties that arose from the lack of spatial uniformity of the lighting excitation, but prevented simultaneous measurements of different areas.

The skin region whose fluorescence was measured was treated as previously described: ALA (5.10 and 20%) applied locally in an occlusive state on an area of 2 cm <2> in the inner part of the arm and subsequent irradiation with visible.

Fluorescence measurements were made on:

a) skin treated with ALA

b) untreated skin

c) environmental measures

All measurements were made in a dark room. In measurements a) and b) the probe was positioned perpendicular to the skin in light contact with it. In measurement c) the probe was held in the air.

Clinical results

All subjects complained of a burning and itching sensation during the irradiative session.

A reaction characterized by erythema and moderate edema appeared after irradiation and lasted about 24 hours.

Forty-eight hours after the treatment, pigmentation of varying intensity appeared which lasted 1-4 weeks (depending on the phototype of the volunteer) (Fig. 1).

Colorimetric data

The colorimetric response confirmed the clinical changes.

Since the colorimetric curve was similar in all subjects, the a * values and the L * values of a single subject are shown in Figures 2 and 3.

Changes in erythema (a *) and pigmentation changes (L *) are highlighted.

Fluorescence in vivo

The results of the spectral measurements for the 3 cases studied are shown in Figure 4. The figure shows the fluorescence intensity, measured as the number of counts as a function of the wavelength (expressed in μm) for the skin region treated with ALA, for the control region and for the environment. It can be seen that for all 3 cases examined:

1. the spectrum of the environment is approximately independent of the wavelength;

2. the spectrum of the untreated skin region shows a quiet dependence on the wavelength in the spectral area studied, with the exception of a strong decrease in fluorescence intensity at a wavelength of 590 μm;

3. the spectrum of the skin region treated with ALA shows a well defined and symmetrical peak at a wavelength of 635 pm superimposed on the control spectrum.

Histopathology

Figure 5 shows the histology before treatment

Only a slight increase in pigmentation was observed in the biopsy performed 3 days after treatment.

In contrast, a considerable increase in the size of individual melanocytes was found in biopsy samples taken 7 days later, with most of the nuclei enlarged, often with an irregular profile.

Furthermore, the number of melanocytes also increased. (Fig. 6).

Furthermore, no difference in the amount of melanin (pigmentation) was found in the biopsies taken 7 days after the therapy.

Immunohistoclinical staining for the S-100 protein confirmed these data (Figs. 7 - 8).

The staining for ΗΜΒ-45 shows a considerable positivity of the melanocytes to this antigen, absent instead in the biopsies carried out 3 days after the treatment.

This activation develops only a few days after the stimulus (Figs.

9 - 10) and not in the following three days.

Transmission electron microscopy

Control site biopsy samples did not demonstrate melanocyte alterations.

From the treated areas it is evident that melanocytes contain various simple and compound melanosomes.

There are some melanophages containing compound melanosomes. These are heterolysomes containing melanin granules (Fig. 11). Some melanocytes contain electrondensed granular melanosomes which represent a more advanced stage of melanization.

Discussion

In this study, a pigment reaction was obtained on the skin after a single ALA-PDT treatment.

As has already been demonstrated (25), in our study the spectral measurements of fluorescence intensity show that PpIX is produced in the skin areas treated with ALA, as opposed to what happens in the untreated areas. The spectral measurements were performed 4 hours after the topical application of ALA, time necessary for the formation of delay of PpIX (30-31).

Despite the low number of cases studied, the spectral analysis unambiguously demonstrates that the excitation at the wavelength corresponding to the absorption peak of PpIX, produces a maximum of fluorescence emission.

This result cannot be attributed to experimental inaccuracies but demonstrates that: a) PpIX is present in tissues at least at depths where 500 nm radiation can penetrate; b) the wavelength in the excitation in "vivo" of PpIX is not very different from that used in the previous documents (30-31), c) that the wavelength of the maximum of the spectral emission of PpIX in vivo coincides with that found in previous studies (30).

The clinical effect obtained consisting of erythema and edema after irradiation performed by a pigment reaction, is characteristic of a classic phototoxic skin reaction.

The clinical picture is similar to the skin manifestations of protoporphyria and should be directly caused by the action of PpIX which induces the formation of reactive oxygen species; these produce a peroxidation of the lipids and alterations of the cell membranes.

Other mediators and enzymes can contribute to the inflammatory reaction.

The hyperpigmentation that was clinically and colorimetrically highlighted during the two weeks following the treatment is hyperpigmentation due to an increased number of active melanocytes without increased production of melanin as demonstrated by the histological characteristics.

This usually happens in normal tanning and in some other skin hyperpigmentation such as pigmentation caused by psoralens plus UVA.

However, in the latter hyperpigmentation the histological examination shows an increased number of active melanocytes which are normal in size and cytological characteristics.

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

Immunohistochemical staining for S100 and HMB-45 proteins further underscore this data.

Furthermore, the considerable positivity for the HMB-45 protein supports the hypothesis of a strong biological activation of melanocytes after ALA-PDT.

The SI 00 protein is expressed by practically all malignant melanomas and melanocytic nevi but also by other tumors. HMB-45, on the other hand, is a monoclonal antibody that recognizes a cytoplasmic antigen associated with melanosomes.

HMB-45 stains malignant melanoma and in some cases, Spitz nevi, dysplastic nevi and compound nevi.

The antigen is not present in vitiligo, soft tissue sarcomas, malignant lipomas, intradermal nevi, Sutton's nevi and melanocytic hyperplasia of the nail bed (34).

All these observations demonstrate that treatment with ALA PDT carried out on normal skin causes skin damage with erythema and edema after irradiation and stable hyperpigmentation for two weeks, due to a considerable activation of melanocytes with an increase in the number of cells characterized by nuclei. irregular and abnormal.

The positivity of melanocytes for SI 00 and HMB-45 proteins confirm these observations.

Claims (6)

CLAIMS 1. Use of δ-aminolevulinic acid for the preparation of a drug to be used following photoactivation in the treatment of hypopigmentation diseases. Use according to claim 1, wherein said pathologies are vitiligo, post-inflammatory hypopigmentation, post-burn or post-skin infection. 3. Use of δ-aminolevulinic acid as a cosmetic. 4. Use according to claim 3, wherein said cosmetic is a bronzer. Use according to any one of the preceding claims, wherein the medicament or cosmetic is in the form of a cream, ointment, emulsion, ointment. 6. Use according to claim 5, wherein said pharmaceutical / cosmetic forms are constituted by an emulsified base of the water-in-oil type.