WO2011009977A2 - Use of a composition containing an extract of a gramineous plant for the prevention of cutaneous lesions caused by ultraviolet radiation - Google Patents

Abstract

The invention relates to a topically-applied pharmacological composition containing an extract of a gramineous plant from the Deschampsia, Ballica or Festuca family, to be used for the prevention or treatment of cutaneous lesions, particularly non-melanoma skin cancer, caused by exposure to UV radiation.

Description

 USE OF A COMPOSITION CONTAINING A PLANT EXTRACT

GRAMINEA FOR THE PREVENTION OF SKIN INJURIES ORIGINATED BY THE ULTRAVIOLET RADIATION TECHNICAL FIELD

 The present invention relates to the use of an extract of a plant belonging to the family of grasses from the Antarctic continent, or from areas with cold climates similar to those of the Antarctic, for the prevention of skin lesions of human skin generated by the harmful effects of exposure to UV radiation.

BACKGROUND OF THE TECHNIQUE

 It is well known that the skin, subjected to solar radiation, suffers certain damages. The main component of solar radiation, which has the greatest potential to cause damage to the skin, is ultraviolet (UV) radiation, which can be of three types: UVA (315-400 nm), UVB (290-315 nm) and UVC (240-290 nm). UVA radiation has a greater penetration, up to the reticular dermis, and causes premature photosensitivity and photoaging, also has an important role together with UVB in the carcinogenesis. The harmful effects of UVB radiation occur in the short term and mainly affect the epidermis, being the main cause of skin burns and, by itself, causes skin cancer. Finally, UVC is the most harmful, but it does not reach the surface of the earth when it is retained by the ozone layer. However, the progressive decrease of this layer is causing a notable increase in skin lesions associated with excesses in sun exposure and that are closely related to skin cancer.

One of the most common skin lesions in recent years is nonmelanoma skin cancer (CCNM) (see: "UV-induced skin damage" Toxicology 2003, 189, 21-39 by M. Ichihashi et al; "Management of nonmelanoma skin cancel "Nature Clin. Prac. 2007, 4, 462-469 by JA Neville et al.). This type of skin cancer is the most frequent and constitutes 20% of all those diagnosed in man. The most common epithelial tumors are basal cell carcinomas (CBCs; they represent 75% of all CCNM) and squamous cell carcinomas (CCEs) ₁ whose incidence in the Caucasian population is high and increases every year due to sun exposure . Actinic keratosis (QA), also known as solar keratosis, is the premalignant lesion that is most frequently detected in adult skin chronically exposed to UVA / UVB radiation. As an example, in Australia, 40-50% of the population With ages over 40 years old, he presents actinic keratosis. Between 5% and 20% of these lesions can degrade in superficial squamous cell carcinomas. Many of the CBCs and CCEs that are discovered early are treated successfully with conventional invasive intervention or pharmacological therapies: excision surgery, radiotherapy, Mohs micrographic surgery and, in recent years, also the photodynamic therapy ("Management of nonmelanoma" skin cancer "Nature Clin. Prac. 2007, 4, 462-469 by JA Neville et al .;" Current modalities and new advances in the treatment of basal cell carcinoma "Int. J. Dermatol. 2006, 45, 489-498 de RI Ceilley et al .; "Actinic keratosis" Int. J. Dermatol. 2007, 46, 895-904 by R. Rossi et al.).

 In order to protect the skin against these harmful effects of radiation, numerous sunscreens of various chemical natures and of varied physicochemical properties have been described. These compounds ensure primary protection. However, the absorption of UV radiation by the filters is never total. There is, therefore, a residual amount of energy radiations that reach the skin. Thus, sunscreens mitigate the damage caused to the skin without suppressing them completely.

 The notable increase in skin lesions that is occurring in recent years requires the need to develop new compounds that protect from sun exposure, both in the short and long term, in order to prevent skin lesions.

 The requesting firm has discovered that the topical application of an extract of a plant belonging to the grass family (Antarctic Deschampsia) on the skin prevents skin lesions, among which is nonmelanoma skin cancer.

 The photoprotective activity of the extract of Antarctic Deschampsia is already known, since patent No. 2009/000050 describes a new skin photoprotection agent against UVA (I and II) and UVB radiation containing said extract. However, the use of this extract for the purpose of preventing skin lesions such as non-melanoma skin cancer caused by UV radiation has not been described so far.

Thus, the present invention has as its object the use of a composition for the prevention of skin lesions caused by UV radiation comprising an extract of a grass from the Antarctic continent, such as Antarctic Deschampsia, or grasses that can be grown in climates Colds similar to those of the Antarctic continent, such as the Baltic and Festuca grasses or others of the Deschampsia family; distilled water and a conventional excipient or additive used to obtain a formulation in the form of cream, gel, or liquid such as lotions, oils, suspensions or ointments for topical application on the skin.

 DESCRIPTION OF THE INVENTION

 The present invention is based on the use of preventive properties and skin lesions of an extract of a plant belonging to the family of grasses, such as Deschampsia, Baltic and Festuca, for application in the prevention or treatment of skin lesions both in humans and animals, especially nonmelanoma skin cancer.

 From the extract of the grass plant a pharmaceutical composition for topical use is prepared. Optionally, this composition may have other components such as antioxidants, physical filters, chemical filters, tanning agents, humectants, etc.

An extract of Antarctic Deschampsia (DA), which is presented in brown powder, is kept in the dark, in an airtight container with silica to prevent hydration and at a temperature of 4 ⁰ C until use.

 From the extract, a topical application pharmacological composition is prepared, the concentration of which varies between 150 mg / ml and 500 mg / ml, preferably 300 mg / ml.

 A composition is prepared, which we will now call EADA

(aqueous DA extract), with a concentration of 300 mg / ml of the DA extract. For this, said extract is dissolved in distilled water and subsequently in the inert gel Carbopol in a 3: 7 ratio.

 Once said composition was prepared, different tests were performed in mice to verify its preventive effect of skin lesions caused by exposure to UV radiation.

 Effect of EADA on the skin of mice (SKH-D when applying UV radiation

The mouse model chosen "hairless Mouse" (SHK-1) is the most frequently used for studies of UV-induced carcinogenesis (see: "Dependence of skin cancer induction by ultraviolet irradiation of albino hairless mice." Cancer Res. 1993 , 53, 53-60 of FR of Gruijl et al. And "UV-induces skin cancer in a hairless Mouse model." Bioessays. 1993, 17, 651-660 of FR of Gruijl; PD Forbes et al.). In this mouse model, UV radiation causes lesions similar to actinic keratosis after several weeks of irradiation and subsequently carcinomas of squamous cells The time required for the development of said lesions varies depending on the spectrum of the source of irradiation, the frequency of exposure and the dose used. In general, histological studies show that small papillomatous lesions (less than 4 mm) are actinic keratoses, while larger ones are squamous cell carcinomas. UV radiation does not generate melanomas or basal cell carcinomas in this animal model, a fact that the cause is unknown.

 In order to carry out the study, 72 female SHK-1 mice, about 25-30 g of body weight and about 6-7 weeks of age were used at the beginning of the trial.

The animals were distributed in groups of 6 animals per cage and were kept in a room whose temperature was 22 ⁰ C and their relative humidity between 50% and 75%, values that were controlled at all times and with light cycles / darkness of 12 hours. The animals received food and water ad libitum during the time of experimentation.

Each mouse received 25 μl of the EADA solution for 1.5 cm ² of the dorsal surface, or only of 50% diluted Carbopol in distilled water (v: v) (vehicle) by direct application on the skin.

 The following groups of mice were made, each consisting of 12 animals, as indicated below and in Table 1:

 - Group 1: Control untreated (mice untreated with EADA and without subsequent irradiation with UV light)

 - Group 2: UV control (mice exposed only to UV light)

 - Group 3: EADA + UV (subject to topical application of EADA and subsequent irradiation with UV light)

 - Group 4: vehicle without the DA + UV extract (subject to topical application of the vehicle the DA extract and subsequent irradiation with UV light)

 - Group 5: EADA control (subject to topical application of EADA, without subsequent irradiation with UV light)

 - Group 6: vehicle control without EADA (subject to topical application of the vehicle without EADA and without subsequent irradiation with UV light)

 Table 1. Distribution of animals in the different experimental groups. * EADA was prepared in the vehicle at a concentration of 300 mg / ml. ** Carbopol inert gel.

 The mice were irradiated with a set of 6 UV lamps according to the standardized protocol for skin damage caused by chronic exposure to UV radiation (see: "Chronic Photodamage in Skin of Mast Cell-deficient Mice." Photochem. Photodamage. 1999, 70, 246-253 of S. González et al.).

The emission spectrum of the lamp assembly is shown in Fig. 1 The integrated power of the lamp group (radiance: 3.1 mW / cm ² ) was measured by the company Iberlélite SA using a specific spectrograph and source (Shamrck SR- 163; EMCCD Newton; Power Meter UP19K).

The distance of the lamps to the mouse skin was approximately 15 cm and the daily dose that each mouse received under UV was about 160 mJ / cm ² (Total spectrum energy), applied for 3-4 days per week.

In order to prepare the animals for the performance of the test, the mice of the corresponding experimental groups (groups 3-6) received topically EADA, or only the vehicle once a day (10:00 am, morning application ) during the first week of experimentation. Subsequently, the animals received the morning application of EADA or the vehicle 3-4 times per week and six hours later they were exposed to the source of irradiation of the UV light (16:00 h, evening UV application). The mice were subjected to UV light until most of those in group 2 (UV control) showed papillomatous lesions or carcinomas equal to or greater than 0.15-0.2 cm in diameter. This took place at 21 weeks of exposure to UV light, after each animal had accumulated a total UV dose of approximately 11,760 mJ / cm ² .

Once the mice developed the tumors, the UV exposure sessions were suspended and the mice were kept for 4 more weeks, without treatment, until slaughter (25 weeks after starting the experiment). All animals were sacrificed by CO ₂ .

The evaluation of the skin damage generated in the mice was evaluated by carrying out a photographic study in order to show the effects of EADA, in relation to exposure with UV light and through the analysis of the clinical characteristics of the skin. In the photographic study, the appearance of the skin, the moment of the appearance of the tumors and the number and size thereof were mainly observed. For the estimation of the number and size of the tumors, only those located on the back of the animal, in the treated regions, have been taken into account, while that those that appeared in other positions (head, neck, lateral-ventral regions of the trunk) have not been counted. Biopsies of the dorsal skin, from the middle and lower areas, were removed to each animal, separating the tumors.

 On the other hand, statistical studies were carried out using the SPSS package, selecting different types of tests according to the variables studied (type of treatment and number and size of lesions) and the size of the sample. The selected tests: kruskal-Wallis and U Mann-Whitney for the analysis and the size and number of the lesions and Pearson's chi-square to determine the possible relationship with the type of treatment (Control, vehicle, UV, EADA + UV, vehicle + UV).

Results

 Table 2 shows the results obtained from the evaluations carried out on the skin of the mice of all the groups at specific times (5, 10, 18 and 21) weeks after starting the test.

 Table 2. Clinical analysis of the general state of the skin of the experimental groups and evolution of lesions induced by UV light in the different experimental groups. At each time, the evaluations were carried out by three different people.

Table 3 also shows the results at the end of the experimentation, that is, 25 weeks after the start of the experiment. In this table we can see the total number of papillomatous lesions with a diameter greater than 0.15 cm in each experimental group at the time of the evaluation, as well as the number of mice with tumors in relation to the total number of mice, indicated in brackets.

 Table 3. Total number of papillomatous lesions larger than 0.15 cm in diameter present in each experimental group at the time of the evaluation (10, 18, 21 and 25). In parentheses the number of mice with tumors is collected in relation to the total number of mice. Animals (1 or 2 respectively) that have been sacrificed 21 weeks after starting the trial because they had tumors larger than 0.5 cm in diameter

 The mice of the control groups that were not exposed to UV light (groups 1, 5 and 6) presented an excellent cutaneous condition both during the experiment, where their skin was flushed, and at the end of it (Table 3 ), Which indicated that neither the EADA nor the vehicle (Carbopol + water) caused visible changes in the skin of the animals. It is noteworthy, however, the presence of a solid tumor located on the right side of the animal and in caudal and ventral position.

 However, skin alterations were observed in animals subjected to chronic UV exposure (Group 2). After 24 hours of exposure to UV light, all mice showed an erythematous reaction, which disappeared between 3 and 5 days after irradiation. Likewise, most of the mice presented wounds and dry skin during the first weeks of exposure to UV light. The first papillomatous lesions began to appear around week 18 of irradiation. Similar results were observed in Group 4, corresponding to animals treated with vehicle and subsequently exposed to UV light. It is noteworthy that in this case a mouse with inguinal axillary tumors was found, as well as two mice with ocular lesions. The animals of Group 3 presented a mild erythema after irradiation and, in general, showed a good cutaneous appearance with few wounds and few papillomatous lesions.

Likewise, as reflected in Table 3 and in Fig. 2, there was a notable increase in the number and size of the tumors of the mice of Groups 2 and 4, while the mice treated with EADA before their exposure to the UV light source showed a lower number of tumors (papillomatous lesions and carcinomas). The average number of tumors counted for Groups 2 and 4 was 7.5 ± 3.6 and 3.0 ± 1, 3 respectively and the average size of these tumors was 0.27 ± 0.11 cm and 0.21 ± 0.1 cm respectively (Table 4). However, mice treated with EADA before exposure to the UV light source showed a smaller number of tumors (papillomatous lesions and carcinomas) (1, 25 ± 1, 2) and of a smaller size (0.125 ± 0.07 cm).

Table 4. Total number of papillomatous lesions larger than 0.15 mm in diameter and their size present in each experimental group at the end of the experiment (25 weeks after starting irradiation with UV light). ^a Two mice of Group 2 and two mice of Group 4 were sacrificed at 21 weeks of initiating the experiment for presenting a tumor larger than 0.5 cm. ⁶ Very thin mouse, without tumors. ⁰ Mouse that presented a tumor in the uncounted right ear. ¹ * Mouse that presented an uncounted ventral tumor. ⁶ Mouse that presented two axillary tumors, lymph node position not counted. Statistical analyzes show that the number of papillomatous lesions induced in mice by chronic exposure to UV light is significantly larger and in addition the average size of them is also larger than observed in mice treated with EADA before being exposed to UV light. These differences have been revealed by the Mann-Whitney U test:

 - Variable "number of tumors": P = 0.002; Sig. <0.05

- Variable "average tumor size": P = 0.005; Sig. <0.05

 Likewise, significant differences have been observed in relation to the number and size of tumors among mice treated with EADA before being exposed to

The UV light, with respect to the mice treated with the vehicle (Carbopol) and then irradiated with UV light. In the latter case, both the number and size of the tumors are significantly larger, according to the Mann-Whitney U test:

- Variable "number of tumors": P = 0.012; Sig. <0.05

 - Variable "average tumor size": P = 0.002; Sig. <0.05

 The result of Pearson's chi-square test shows that the number of tumors developed depends on the type of treatment to which the mice are subjected, while the average size of tumors does not depend on the treatment applied:

 - Variable "number of tumors": P = 0.011; Sig. <0.05

 - Variable "average tumor size": P = 0.132; Sig. <0.05

 Finally, it is verified that by applying Pearson's chi-square test that the average size of the tumors is dependent on the number of tumors found in each treatment (P <0.0001, Sig <0.05).

 The results obtained throughout this study allow us to conclude that the topical application of EADA containing the extract of Antarctic Deschapsy (300 mg / ml) on the skin of the back of SHK-1 mice has a preventive effect of skin damage caused by UV light Mice treated with EADA before exposure to UV light have, in the short term, a marked decrease in skin alterations (dryness, wounds), in relation to those not treated with the extract and subjected to UV light. Likewise, treated mice develop, in the long term, a smaller number of papillomatous lesions and carcinomas compared to untreated animals.

Claims

1. Use of a topical application pharmacological composition that contains an extract of a plant belonging to the grass family from the Antarctic continent, or from areas with cold climates similar to those of the Antarctic continent, for the prevention of skin lesions caused by The ultraviolet radiation in both humans and animals. 2. The use according to claim 1 wherein the grass is selected from the Deschampsia, Baltic or Festuca family. 3. The use according to claim 1 and 2 wherein the extract is from the grass Antarctic Deschampsia. 4. The use according to claims 1, 2 and 3 wherein the composition has a concentration ranging between 150 mg / ml and 500 mg / ml, preferably 300 mg / ml, of the extract prepared by dissolving said extract in distilled water and subsequently in an inert gel or compatible vehicle. 5. The use according to all the preceding claims in which the pharmacological composition can be presented in the form of cream, gel, gel-cream, foam or any other formulation galenically accepted for topical use 6. The use according to all the preceding claims wherein the pharmacological composition contains products selected from the group comprising: antioxidants, physical filters, chemical filters, tanning agents and humectants. 7. The use according to claim 1 to 5 wherein the skin lesion is nonmelanoma skin cancer. 8. The use according to claim 7 wherein the non-melanoma cancer is a basal cell carcinoma. 9. The use according to claim 7 wherein the non-melanoma cancer is a squamous cell carcinoma. 10. The use according to all the preceding claims in which the phamacological composition is applied up to six hours before exposure to ultraviolet radiation.