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Title:
COMPOSITION FOR A MEDICAL DEVICE OR FOR A COSMETIC OR PHARMACEUTICAL PREPARATION COMPRISING A DECAPEPTIDE DERIVED FROM DEINOCOCCUS RADIODURANS
Document Type and Number:
WIPO Patent Application WO/2013/098743
Kind Code:
A1
Abstract:
The present invention relates to a composition comprising a decapeptide of bacterial origin, which can also produced via chemical synthesis, and the use of said composition in the therapeutic or cosmetic field or in a medical device.

Inventors:
EMANUELE ENZO (IT)
BENEDETTI RUFFO (IT)
Application Number:
PCT/IB2012/057621
Publication Date:
July 04, 2013
Filing Date:
December 21, 2012
Export Citation:
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Assignee:
BIODUE S P A (IT)
International Classes:
A61K35/74; A61K38/08; A61P17/00
Domestic Patent References:
WO2011139881A22011-11-10
WO2011139881A22011-11-10
Foreign References:
KR20040107942A2004-12-23
Other References:
DATABASE WPI Week 200532, Derwent World Patents Index; AN 2005-311366, XP002677958
COX, MM; BATTISTA, JR, NATURE REVIEWS. MICROBIOLOGY, vol. 3, 2005, pages 882 - 892
DALY, MJ ET AL., PLOS ONE, vol. 5, no. EL2570, 2010, pages 1 - 15
BLASIUS, M. ET AL., CRIT. REV. BIOCHEM. MOL., vol. 43, 2008, pages 221 - 228
SLADE, D.; RADMAN, M., MICROBIOL. MOL. BIOL. R., 2011, pages 133
J IND MICROBIOL BIOTECHNOL, vol. 36, 2009, pages 539 - 546
LUANPITPONG ET AL., APOPTOSIS, vol. 16, no. 8, August 2011 (2011-08-01), pages 769 - 82
PLOS ONE, vol. 7, no. 6, 2012, pages E39411
"OECD GUIDELINES FOR TESTING OF CHEMICAL 471 21", GENETIC TOXICOLOGY: BACTERIAL REVERSION MUTATION ASSAY, July 1997 (1997-07-01)
Attorney, Agent or Firm:
PONTIROLI, Alessandro et al. (Piazza San Babila 5, Milan, IT)
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Claims:
CLAIMS

1. A composition in the form of a cream, gel, ointment, lotion, eyedrop or ophthalmic spray for topical use in a medical device or in a cosmetic or pharmaceutical preparation comprising the decapeptide (I) with amino acid sequence

H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH (I) ,

or a salt thereof that is suitable for said preparation, for use in humans, and at least one excipient.

2. A medical device comprising the decapeptide (I) with amino acid sequence

H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH (I) ,

or a salt thereof that is suitable for use in a medical device, for use in humans .

3. The composition according to claim 1 or the medical device according to claim 2 comprising an extract of the bacterium

Deinococcus radiodurans .

4. The composition according to claim 1 or the medical device according to claim 2, wherein the decapeptide (I) is obtained by chemical synthesis.

5. The composition according to claim 1 or the medical device according to claim 2 wherein the concentration of decapeptide (I) is in the range 0.01-8 mM.

6. The composition according to one of claims 1 or 3-5 or the medical device according to claim 2 for use in the treatment or prevention of a disorder and/or pathology of the skin and skin appendages induced by chemotherapy, exposure to ultraviolet radiation or ionizing, or from oxidative damage induced by free radicals or by aging in a subject, wherein the disorder and/or pathology are selected from alopecia induced by chemotherapy or radiotherapy, androgenetic alopecia type male or female, skin damage induced by ionizing or ultraviolet radiation, aging or oxidative stress.

7. The composition for use according to claim 6 wherein the pathology is alopecia induced by chemotherapy or by radiotherapy.

8. The composition for use according to claim 6 wherein the pathology is androgenetic alopecia.

9. Cosmetic use of the composition according to one of the claims 1 or 3-5, to prevent or counteract the effects of cosmetic and/or degenerative skin aging.

10. Cosmetic treatment of the skin and skin appendages to prevent or counteract the aesthetic effects of skin aging, comprising applying the composition according to one of claims 1 or 3-5.

Description:
COMPOSITION FOR A MEDICAL DEVICE OR FOR A COSMETIC OR PHARMACEUTICAL PREPARATION COMPRISING A DECAPEPTIDE DERIVED FROM DEINOCOCCUS RADIODURANS

DESCRIPTION

The present invention relates to a composition comprising a decapeptide derived from Deinococcus radiodurans, which can also be produced through chemical synthesis, and its use in a medical device, as well as in the therapeutic or cosmetic fields.

The alterations caused by exposure of the skin surface, skin appendages and hair pathogenic agents may range from erythema to alopecia up to the onset of necrosis and skin tumors.

For example, ionizing or UV radiations, even in non-lethal doses, can cause temporary or permanent alterations of the directly exposed tissues (radiodermatitis) , which are among the most important side effects of radiation therapy of oncological diseases .

Following radiation therapy, body areas which have been subjected to the action of radiation may develop, based on the intensity and duration of treatment, erythema, edema, ulcers, alopecia, xerosis, skin discoloration, lacerations, atrophy of sebaceous and sweat glands up to the so-called "radionecrosis" , i.e. tissue cell death, mainly caused by the interruption of blood flow.

Also chemotherapy can cause hair follicle dystrophy and alopecia. In particular, the hair loss is a side effect with psychological consequences that may have a negative impact on social life and on compliance to chemotherapy by the patient, resulting in the extension of the disease or in inefficacy of the treatment.

Alopecia can also have androgenetic causes, that are related to the action of enzymes, such as 5-alpha-reductase type II, which converts testosterone to dihydrotestosterone . Aleopecia can be due to idiopathic, genetic predisposition or endocrinopathies such as the lack of Growth Hormone (GH) , virilizing tumors, hypothyroidism, menopause, polycystic ovary syndrome, or treatment with androgens. Androgenetic alopecia can affect both female and male subjects, although the latter are the most commonly affected.

In addition, skin damage can be caused by oxidative damages induced by free radicals, as in the natural aging process which may be accelerated by exposure to environmental factors that vary from air pollution to sunlight exposure.

The bacterium named Deinococcus radiodurans was isolated for the first time in 1956 as part of a program for the study of the biological effects of nuclear radiation. Deinococcus radiodurans has been the subject of several studies to determine the mechanisms for its excellent resistance to radiation (Cox, MM, Battista, JR Nature reviews. Microbiology 2005, 3, 882-892) .

However, the available studies have not clarified whether this resistance is due to individual compounds or to a synergy of factors. For example, Daly et al . (Daly, MJ et al . PLos One, 2010, 5, el2570 1-15), Blasius et al (Blasius, M. et al. Crit. Rev. Biochem. Mol. 2008, 43, 221-228) and Slade and Radman (Slade, D., Radman, M. Microbiol. Mol. Biol. R. 2011, 133) attribute a predominant role in the radioresistance of D. radiodurans to its catabolite peptide complexes with metal ions and, in particular, with manganese, which is present at high levels in the bacterium.

WO2011/139881 relates to a method for producing vaccines directed against microorganisms (viruses and bacteria) , wherein the method comprises culturing, harvesting and/or suspending the microorganism in the presence of a composition comprising a protein-free Deinococcus radiodurans extract and irradiating the microganism with a dose of radiations sufficient to render the obliterate the microorganism DNA, wherein the microorganism proteins, but not the nucleic acids, are protected against radiations by the Deinococcus radiodurans extract. In addition, WO2011/139881 mentions the administration of a extracts of Deinococcus radiodurans comprising one or more nucleosides and one or more antioxidants for preventing the side effects of radiotherapy .

KR20040107942 discloses an extract of unidentified hydrophobic substances in the cell membrane of Deinococcus radiodurans for skin protection from UV rays.

One object of the present invention is to provide effective and easy to apply compositions - that are usable in medical devices or drugs- for use in the treatment or prevention of disorders and/or diseases induced by chemotherapy, by exposure to ultraviolet or to ionizing radiations, by oxidative damage induced by free radicals or by aging, .

A task of the present invention is to provide a medical device for disorders and/or diseases induced by chemotherapy, by exposure to ultraviolet or to ionizing radiation, or by oxidative damage induced by free radicals or by aging.

Another task of the present invention is to provide a pharmaceutical composition for use in the treatment of disorders and/or of diseases induced by chemotherapy, by exposure to ultraviolet or ionizing radiation, or by oxidative damage induced by free radicals or by aging, which is free, or practically devoid, of side effects so as to be tolerable for a large number of patients.

Another task of the present invention is to provide a cosmetic method for skin and skin appendages aimed to prevent or to counteract the effects of skin aging.

In accordance with the present invention, these objects, and others which are disclosed hereinafter, are achieved by a composition in the form of a cream, gel, ointment, lotion, eyedrop or ophthalmic spray in a medical device or in a cosmetic or pharmaceutical preparation comprising a decapeptide (I) with amino acid sequence

H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH,

or an salt thereof that is suitable for said preparation, for use in humans, and at least one excipient. The objects of the invention have also been achieved by said composition for use in the treatment and/or prevention of a disorder and/or pathology of the skin and skin appendages induced by chemotherapy, exposure to ultraviolet radiation or ionizing, from damage oxidative or induced by free radicals or by aging in a subject, wherein the disorders and/or pathologies are selected from alopecia induced by chemotherapy or radiotherapy, androgenetic-type male or female alopecia, skin damage induced by ionizing or ultraviolet radiation or by oxidative stress.

These objects have also been achieved through the use of said cosmetic composition comprising the decapeptide (I) to prevent or counteract the effects of skin aging.

The objects of the invention have also been achieved through a cosmetic treatment of the skin and skin appendages to prevent or counteract the aesthetic effects of skin aging, comprising applying said composition comprising the decapeptide (I) .

The objects of the invention have also been achieved through a medical device that includes the decapeptide (I), or a salt thereof that is suitable for use in a medical device, for use in humans .

Figure 1 shows the relative cell viability of HFDPC cells treated with cisplatin (100 μΜ) for 24 h in the presence or absence of D. radiodurans extract.

Figure 2 shows the significant inhibition of cisplatin-caused apoptosis in HFDPCs cells that was achieved via pretreatment with D. radiodurans extract.

Figure 3 is a Western blot analysis showing the marked reduction of caspase 9 in cell lysates which was obtained via pretreatment with D. radiodurans extract before exposure to cisplatin.

Figure 4 shows the relative cisplatin-induced proapoptotic Bax and anti-apoptotic Bcl-2 expressions in HFDPCs cells lysates with and without pretreatment with D. radiodurans extract. Figures 5A shows the hyperexpression of COX-2 in UVC-exposed skin and 5B shows the complete inhibition of said hyperexpression following pretreatment with Deinococcus radiodurans extract (1%) . Figure 6 shows the classification category of experimentally obtained acute toxicity range estimate.

Figures 7 and 8 show the variations in animal weight as a result of toxicity tests in rats.

Figures 9-12 show the Raw data of Bacterial reverse mutation assays (I and II replicate) .

As used herein, the term "extract of Deinococcus radiodurans bacterium" means a solid composition or in a liquid form obtained from a culture of Deinococcus radiodurans via any type of biochemical and/or biophysical procedure known to those skilled in the art, for example after homogenization and isolation of water-soluble components, by ultracentrifugation and/or ultrafiltration.

As used herein, the term "chemotherapy" refers to a antineoplastic or antibiotic therapy which comprises the administration of chemicals with capacity to destroy, in the most possibly targeted and selective manner, pathological manifestations .

As used herein, the term "radiotherapy" defines a medical treatment comprising exposure to ionizing, corpuscular or not, or ultraviolet radiation, generally but not exclusively used for the treatment of oncological diseases.

As used herein, the term "ionizing radiation" indicates a radiation with a frequency greater than 3xl0 15 Hertz equipped with sufficient energy to ionize atoms (or molecules), and UV radiation means electromagnetic radiation with wavelength in the range 10-400 nm, usually further divided into UV-A (400-315 nm) , UV-B (315-280 nm) and UV-C (280-100 nm) .

As used herein, the terms "chemoinduced alopecia" and "radioinduced alopecia" mean the circumscribed or diffuse loss or thinning of hair and other body hair, as a result of a treatment comprising chemotherapy and/or radiotherapy.

In the context of the present invention, the term "androgenetic alopecia" refers to the loss or thinning of hair for either primary or secondary causes, with endogenous origin and due to the activity of the enzyme 5-alpha-reductase type II.

As used herein, the term "medical device" includes an apparatus, a system or another product, used alone or in combination, intended to be used in man or in an animal for the purpose of prevention, control, or alleviation of a disorder, illness or a disease, whose main action in or on the body is not achieved by pharmacological, immunological or metabolic means, but which may be assisted in its function by such means.

As used herein, the term "medical device" includes a preparation with predominant physical and/or mechanical effect. Non- limitative examples of medical device in the present invention are creams, gels, lotions including liquids for ophtalmological use such as sprays and eyedrops .

The term "excipient" means a substance commonly used in the formulation of pharmaceutical preparations or cosmetics or medical devices.

Unless otherwise indicated, all percentages are in weight of a component/overall weight of the composition.

In one aspect, the present invention relates to a composition in the form of a cream, gel, ointment, lotion, eyedrop or ophthalmic spray in a medical device or in a cosmetic or pharmaceutical preparation comprising a decapeptide (I) with amino acid sequence H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH,

or its salts, and at least one excipient.

It has surprisingly been found that topical application of a composition comprising the decapeptide (I) is able to prevent and to counteract the alterations induced by chemotherapeutic agents and radiation in the skin, hair and skin appendages in a subject undergoing chemotherapy and/or radiotherapy. The composition according to the invention can be comprised in a medical device for the prevention and the contrast of the effects of the process of aging and/or of oxidative damage induced by free radicals, irrespective of the causing agent.

In one aspect, the present invention relates to a medical device comprising the decapeptide (I) with amino acid sequence

H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH,

or a salt thereof.

In particular, the decapeptide (I) with amino acid sequence

H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH

and 1261 Da molecular weight can be synthetically produced or extracted from Deinococcus radiodurans .

Preferably, the composition or the medical device according to the present invention comprises synthetically produced decapeptide (I) or an extract of the bacterium Deinococcus radiodurans comprising the decapeptide (I) .

It has been found that the extract of the Deinococcus radiodurans bacterium comprising the decapeptide (I) or the decapeptide (I) produced via synthetic route may exert a protective role in humans with respect to damage induced by inflammative and/or DNA- damaging agents (e.g. chemoterapic drugs), by UV radiation and by various genotoxic radical species.

The effects on skin and skin appendages in the context of the present invention may be aesthetic and pathological.

The composition according to the invention may be in any form commonly used for topical application and known to the expert in the art as a component of a medical device or a pharmaceutical or cosmetic preparation, for example in the form of lotion, cream, gel, ophthalmic spray, eyedrop, ointment, paste, suspension, spray, film, gel lipophilic or hydrophilic.

Preferably, the composition or the medical device according to the invention is in the form of a cream, a gel, a lotion or an ophthalmic spray. The content of peptide (I) in the extract of Deinococcus radiodurans comprised in the formulation of the invention may be between 1 and 10 mM.

Preferably, the amount of the extract of Deinococcus radiodurans extract in the composition of the present invention is between 0.1 and 5%, more preferably between 0.5 and 2%, most preferably between 1 and 2% in weight/total weight of the composition.

It was found that the presence of peptide (I), even in low amount, is particularly advantageous for the protection of the subject from the effects of inflammative and/or DNA-damaging agents (e.g. chemoterapic drugs), of UV radiation and of various genotoxic radical species.

Preferably, the content of peptide (I) in the formulation of the invention is between 0.01 and 8 mM, more preferably between 0.02 and 1 mM, most preferably between 0.02 and 0.10 mM.

Examples of possible ingredients and excipients of the composition for topical use of the invention comprise all substances commonly used in the pharmaceutical and cosmetic industry, for example, among others, hydrophilic or lipophilic gelling agents, preservatives, antioxidants, solvents, perfumes, bulking agents, photoprotectant filters, pigments, odor absorbers, coloring agents, liposomes. The formulation for the lotion can also comprise water, volatile silicones and other water-soluble film-forming substances, antioxidants, preservatives, pH-adjusters and, optionally, fragrance.

The formulation for cream, optionally in the form of a microemulsion, may comprise water, emulsifiers, xanthan gum, glycerin, nicotinamide, preservatives, pH adjusting and, optionally, fragrance.

The gel formulation also comprises water, gelling agents, thickeners, nicotinamide, preservatives, pH-adjusting agents and, optionally, fragrance.

The composition according to the invention may also comprise at least another active principle for pharmaceutical or cosmetic addition to the peptide (I), for example a substance with antiinflammatory, anti-oxidant and/or analgesic properties. The composition according to the invention can be applied for the prevention and/or treatment of various disorders or diseases resulting from radiation therapy, chemotherapy, exposure to radiation or to oxidative stress, including erythema, dermatitis, edema, ulceration, alopecia, xerosis, skin discoloration, lacerations, atrophy of the skin and skin appendages to tissue necrosis .

In one aspect, the present invention relates to said composition comprising the decapeptide (I) for use in the treatment or prevention of a disorder and/or of a pathology of the skin and of skin appendages induced by chemotherapy, exposure to ultraviolet or ionizing radiation, or from oxidative damage induced by free radicals or by aging in a subject, wherein the disorders and/or pathologies are selected from alopecia induced by chemotherapy or by radiotherapy, androgenetic-type male or female alopecia, skin damage induced by ionizing or ultraviolet radiation or by oxidative stress.

Preferably, the composition according to the invention is for use in the treatment or prevention of alopecia induced by chemotherapy or by radiotherapy. More preferably, the composition according to the invention is for use in the treatment of alopecia induced by chemotherapy.

Preferably, the composition according to the invention is for use in the treatment or in the prevention of androgenetic alopecia. In another aspect, the present invention relates to the cosmetic use of said composition comprising the peptide of formula (I) to prevent or counteract the effects of cosmetic and/or degenerative skin aging.

In one aspect, the present invention relates to the cosmetic treatment of the skin and skin appendages to prevent or counteract the aesthetic effects of skin aging, comprising the topical application of said composition comprising the peptide of formula ( I ) .

The decapeptide (I) with amino acid sequence

H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH

can be obtained by chemical synthesis or by direct extraction from the bacterium Deinococcus radiodurans, according to the protocols described by Daly et al . (Daly, MJ et al . PLos One, 2010, 5, el2570 1-15) or by Yi He ( J Ind Microbiol Biotechnol, 2009, 36: 539-546) .

The effectiveness of the device and of the composition according to the present invention can be tested by assays of viability (trypan blue staining) of cell cultures of human keratinocytes (HaCaT cells) after exposure for two days to genotoxic chemotherapy (chlorambucil) in the presence and absence of preparations containing the decapeptide.

Efficacy studies of the device for the prevention of chemo- or radiotherapy-induced alopecia may be carried out on populations of cancer patients undergoing chemotherapy with known alopecia- causing agents (cyclophosphamide, etoposide, taxanes, organoplatins etc.) and/or radiotherapy. The preparation may be tested both in relation to aspects of molecular (analysis of genotoxic damage at the hair follicle) and of clinical (prevention and/ or reduction of alopecia) response.

Efficacy studies of the device for the prevention of skin damage induced by ionizing or ultraviolet radiation may be carried out in relation to aspects of molecular response (analysis of the genotoxic damage at the level of DNA extracted from skin biopsies) .

The following examples illustrates an embodiment of the invention, with no intent to limit its scope.

1. EXPERIMENTS CONDUCTED WITH DEINOCOCCUS RADIODURANS EXTRACT (1% w/w) IN HUMAN HAIR FOLLICLE DERMAL PAPILLA CELLS EXPOSED TO CISPLATIN AIM. This experiment was undertaken to measure the in vitro effects of pretreatment with Deinococcus radiodurans extract (1% w/w with content of decapeptide (I) between 2 and 8 mM, corresponding to a concentration of decapeptide I of 0.02 to 0.08 mM) on human hair follicle dermal papilla cells (HFDPCs) exposed to a common chemotherapic agent (cisplatin; 100 μΜ) with known hair growth inhibitory properties.

BACKGROUND. HFDPCs are specialized fibroblasts that form at the base of the hair follicle. HFDPCs are essential not only in the control of hair growth, formation and cycling, but also in the pathogenesis of chemotherapy-induced alopecia. For example, it is known that pretreatment of HFDPCs with cisplatin induces an increase in caspase activation and apoptotic cell death (Luanpitpong et al . , Apoptosis 2011 Aug;l 6 (8) : 769-82 ) , a mechanism involving an altered balance between the anti-apoptotic protein Bcl-2 and the pro-apoptotic molecule Bax . Indeed, cisplatin induces in the hair follicle the downregulation of the anti-apoptotic Bcl-2 protein and the up-regulation of the pro- apoptotic Bax protein, ultimately resulting in apoptotic cell death (with alopecia as the clinical sequence) .

METHODS. Human HFDPCs were obtained from PromoCell (Heidelberg, Germany) . The cells were cultured in an appropriate cell growth medium (PromoCell) containing 100 units/ml of penicillin and 100 g/ml of streptomycin (Gibco, Gaithersburg, MA, USA) in a 5% C0 2 environment at 37°C. Cis-diamminedichloroplatinum II (cisplatin, CDDP) was purchased from Sigma (St. Louis, MO, USA) . Deinococcus radiodurans extract (1%) was obtained by appropriate colture and lysis according to Daly et al . (Daly, MJ et al . PLos One, 2010, 5, el2570 1-15) or to Yi He ( J Ind Microbiol Biotechnol, 2009, 36: 539-546) ..

Results

i. Pretreatment with D. radiodurans extract increases HFDPCs viability after exposure to CDDP. HFDPC cells were treated with cisplatin (100 μΜ) for 24 h in the presence or absence of D. radiodurans extract. Cytotoxicity in HFDPCs was determined by MTT colorimetric assay. After specific treatments (CDDP either with or without D. radiodurans extract exposure), cells were incubated in 96-well plates with 500 g/ml of MTT for 4 h at 37 °C. The intensity of the formazan product was measured at 550 nm using a microplate reader. Relative cell viability was calculated by dividing the absorbance of treated cells by that of the non-treated cells in each experiment. (Figure 1)

Compared to vehicle-treated cells (mean viability: 0.95 ± 0.05%), cisplatin exposure a significant reduction of cell viability (0.45 ± 0.05%), which was significantly inhibited by the pretreatment with D. radiodurans extract (0.77 ± 0.06%, P < 0.001) . Plots are means and standard deviations of four independent replicates.

ii . Pretreatment with D. radiodurans extract inhibits HFDPCs apoptosis and caspase 9-expression after exposure to CDDP

As expected, the exposure to cisplatin (100 μΜ) for 24 h led to a marked increase of apoptosis in HFDPCs cells (from 2.2 ± 1.2% to

40.7 ± 7.6%) according to the results of the Hoechst 33342 assay.

However, such a marked increase was significantly inhibited by the pretreatment with D. radiodurans extract (apoptotic rate:

19.2 ± 5.6%, P < 0.001) (Figure 2)

This phenomenon was accompanied by a marked reduction of caspase

9 in cell lysates by Western blot analysis (Figure 3) .

iii . Pretreatment with D. radiodurans extract inhibits CDDP- induced antiapoptotic Bcl-2 downregulation and proapoptotic Bax upregulation

As expected, the exposure to cisplatin (100 μΜ) for 24 h led to a marked decrease of antiapoptotic Bcl-2 expression in HFDPCs cell lysates (mean relative expression compared to control cells: 0.37) . However, such a marked decrease was significantly inhibited by the pretreatment with the D. radiodurans extract (mean relative expression compared to control cells: 0.74, P < 0.001) . Similarly, the exposure to cisplatin (100 μΜ) for 24 h led to a marked increase of proapoptotic Bax expression in HFDPCs cell lysates (mean relative expression compared to control cells: 2.46) . However, such a marked increase was significantly inhibited by the pretreatment with D. radiodurans extract (mean relative expression compared to control cells: 1.40, P < 0.001) ( Figure 4 ) .

2. EXPERIMENTS CONDUCTED TO ASSESS CYCLOOXYGENASE (COX) -2

IMMUNOHISTOCHEMICAL EXPRESSION IN HUMAN SKIN IN VIVO AFTER EXPOSURE TO 5-FOLD MED OF UVC RADIATION WITH AND WITHOUT PRETREATMENT WITH DEINOCOCCUS RADIODURANS EXTRACT (1%)

AIM. This experiment was undertaken to assess whether in vivo pretreatment with Deinococcus radiodurans extract (1% w/w with content of decapeptide (I) between 2 and 8 mM, corresponding to a concentration of decapeptide I of 0.02 to 0.08 mM) on human skin can prevent the previously reported hyperexpression of cyclooxygenase (COX) -2 (PLoS One 2012; 7(6): e39411) after exposure to 5-fold MED of UVC radiation.

BACKGROUND. COX-2 is one of the rate-limiting enzymes in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Growing evidence indicates that COX-2 protein expression is enhanced in skin epidermal cancer cells and that COX-2 plays a pivotal role in regulating cell growth. In addition, expression of COX-2 at 6 h correlates to the intensity of heat-induced erythema 24 h after UVC irradiation.

METHODS. Skin biopsies taken 24 h post UVC irradiation of a 1 cm 2 skin area in the lateral site of the left or right buttock were fixed in 10% neutral buffered formalin, processed routinely, and embedded in paraffin. Localization of COX-2 protein expression was investigated using the monoclonal mouse anti-COX-2 IgG antibody (Transduction Laboratories, Lexington, KY, USA) . Slides were deparaffinized in xylene, were hydrated through a graded series of ethanol and were immersed in 3% H 2 0 2 for 5 min to block endogenous peroxidase activity. After microwave pretreatment in citrate buffer (pH 6.0) for antigen retrieval, the sections were blocked with normal bovine serum for 5 min, and were then incubated overnight at 4 °C with the COX-2 antibody at a dilution of 1:100. Non-immune mouse immunoglobulin G was used as the negative control at equivalent conditions in place of the primary antibody. The bound primary antibody was visualized using the enhanced labelled-polymer system and amino-ethylcarbazole as the color-developing reagent. Slides were counterstained with Mayer's hematoxylin .

Results

As expected, the expression of COX-2 was markedly higher in UVC- exposed skin (Figure 5A) , but such hyperxpression was entirely abrogated with pretreatment with Deinococcus radiodurans extract (1%) (Figure 5B) .

3. TOXICITY STUDY

A toxicology study was performed on the D. Radiodurans extract comprising peptide (I) according to the invention in order to obtain the necessary data to evaluate its toxic effects after dermal exposure.

The following test was performed:

acute dermal toxicity according to OECD M° 402

During the assay of acute dermal toxicity the test product (neat extract of Deinococcus radiodurans comprising decapeptide (I) in concentration comprised between 2 and 8 mM) was administered with a dose of 2000 mg/kg by topical application to a group of 10 rats (5 males and 5 females) .

During the study, the animals have been daily observed for 14 days (5 days a week) to detect possible toxic symptoms.

Findings included:

-mortality;

-assessment of major organic functions;

-assessment of the status of integumentary apparatus;

-assessment of the status of mucosae; -assessment of somatomotory activity and of the sensory status.

The body weight has been weekly registered.

At the end of the study a post-mortem observation was carried out on all animals.

The results can be summarized as follows:

Mortality: during the study no mortality was detected.

Clinical symptomatology; during the study neither symptoms nor signs of toxicity were recorded.

Weight increase: treated animals' weight complied with the standard of species and race in first and in the second week of the study.

Post-mortem observations: no abnormalities were recorded at the autopsy .

BIBLIOGRAPHY

OECD, Guidelines for Testing of Chemicals n° 402 of February 24 lh 1987 - Acute dermal toxicity-

Official Journal of European Communities, REGULATION (EC) No 1272/2008 of the European Parliament and of the council of 16 December 2008 on classification, labeling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006

METHOD

Characterisation

Specie: Rat

Breed: SD

No: 10 (5 males and 5 females)

Weight range: 170-212 g at the beginning of the assay

Sex: Males and females

Supplier: Charles River - Calco (LC) Italy

Food: VRF1 Batch: 9099

Bedding: Lignocell Batch: 03018120702

Justification of the assay system

To perform the study, rats were used which belonged to the choice specie quoted in OECD. Quarantine

Purchased animals, before being used for this study, were put in quarantine for 5 days under daily observation.

At the end of the period of quarantine they were carefully examined to verify their qualification to the study.

Animal selection

Animals used in the study were haphazardly selected among the eligible ones that were available at the time of the study.

Animal identification

Animals selected for the study were marked with indelible colouring in different parts of the body, as follows:

No sign (B) 1

Head ( ) 2

Tail (C) 3

Head tail (TC) 4

Leg ( Z ) 5

Cages were identified via a tag.

Housing

Animals were put in cages of transparent polycarbonate with sizes of cm 42.5x26.6x18, separated by sex so that 5 animals of the same sex were in each cage.

Stabling rooms were lighted with fluorescent lamps and kept with cycles of 12 hours of light and 12 hours of darkness.

Temperature and humidity, controlled by air conditioning system, have been continuously registered.

Cleaning and disinfection

Cages and stabling room were cleaned before housing animals and periodically disinfected.

Feeding

Animals were fed with complete pelletized diet provided by Harlan,

Italy.

Watering

Animals were watered with purified water available ad libitum.

ASSAY SAMPLE PREPARATION The test substance, comprising peptide (I) in 5% weight/total weight of the composition, was used neat.

EXPERIMENTAL DESIGN

10 rats were used, 5 males and 5 females.

TREATMENT

Skin preparation

24 hours before the beginning of the test the back and the sides of the animals were shaved on an area of about 20 cm 2 . Appl i ca tion

The test substance was given, at a ratio of 2000 mg/kg, with a square of gauze directly on animals skin

and fixed with a skin patch in a area of about 10% of the total body surface.

The square was put on the back of animals and further fixed with hypo-allergenic occlusive adhesive tape.

The whole trunk of the animal has been protected with an elastic bandage. Bandage has been left in situ for 24 hours, during this period animals were kept in single cages.

Removal

24 hours after application bandage and adhesive tape were removed .

Skin was cleaned from sample excess by a tampon soaked in physiological solution.

OBSERVATIONS

The general status of all animals has been daily monitored after 1-3-5 hours from the start of treatment and daily (5 days per week) for 14 days.

All activities related to the study, as well as remarks and exams were daily registered in dated and signed forms.

During the study the following observations were done:

Mortali ty

Animals were observed in the morning of each working day.

Clinical symptomatology Every clinical symptom, including possible variations of somatomotory activity, was daily registered for each individual animal .

Clinical observations included a general objective exam (G.O.E.) with regards to:

assessment of major organic functions;

assessment of the status of integumentary apparatus;

assessment of the status of mucosae;

assessment of somatomotory activity and of the sensory status. Body weight

Animals were weighted before experiment, after 7 days and at the end of the experiment.

Pos tmortem

At the end of the observation period, animals were sacrificed and a post-mortem examination was carried out.

RESULTS INTERPRETATION

Results were interpreted according to Official Journal of the European Union 1272/2008 (CLP) dated December 16 th , 2008 (Figure 6) RESULTS

Mortality

During the study no mortality was detected.

Clinical symptomatology

During the study, neither symptoms nor signs of toxicity were recorded .

Weight increase

Treated animals' weight complied with the standard of species and race in first and in the second week of the study.

Figure 7 shows weight increases of individual treated animals.

Body weight of treated animals were detected at the beginning, after

7 and 14 days of treatment as reported in Figure 8.

Post-mortem observations

At the autopsy, no abnormalities have been observed in all the treated animals.

DEVIATION No deviation has been recorded from study program

CONCLUSIONS

On the basis of obtained results, according to Official Journal of (he European Union 1272/2008 (CLP) dated December 16" 1 , 2008 and OECD n° 402 of February 24 lh 1987, the test substance comprising 2- 8mM of the peptide of formula (I) has a LD 50 > 2000 mg/Kg bw and can be considered NOT TOXIC - NOT HARMFUL.

4. GENOTOXICITY STUDY

A genotoxicological study was performed on the D. Radiodurans extract comprising peptide (I) according to the invention in order to obtain the necessary data to evaluate its mutagenic effects after dermal exposure.

The following test was performed:

Bacterial reverse mutation assay (Ames test)

The Bacterial reverse mutation assay was performed on five mutant strains of Salmonella typhimurium

{TA1535, TA1537, TA98, TA100, TA102) .

The presumed mutagenic activity of the test substance was determined by comparing number of reverting colonies in treated cultures with the number of the reverting organisms in the control cultures. The direct incorporation method in a plate was used both in the presence of, and without, an enzymatic system for metabolic activation.

The test substance was prepared as solution in water with a concentration equivalent to 50 mg/ml and 4 different concentrations of semi-log intervals between them were prepared. The assay was performed in two replicates.

BIBLIOGRAPHY

- OECD GUIDELINES FOR TESTING OF CHEMICAL 471 21*, July 1997. Genetic Toxicology: Bacterial Reversion Mutation Assay.

PRINCIPLE OF THE TEST

The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid, for this reason an appropriate minimal glucose agar and an overlay agar (Top agar) containing Histidine and biotin in very small quantitative, to allow for a few cell divisions, is used, sufficient to permit the survival of the microorganisms and show a mutation when this happened. Many of the test strains have several features that make them more sensitive for the detection of mutations, including responsive DNA sequences at the reversion sites, increased cell permeability to large molecules and elimination of DNA repair systems or enhancement of error-prone DNA repair processes. The revertants bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.

EXPERIMENTAL PROCEDURES ASSAY SYSTEM

Microorganisms

Identification ofthe strains

Mutant Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 were used.

Preparation of the test suspensions

One day before use, the strains were transferred into 100 mi of Cultural Medium (Tryptic soy Broth, TSB) , and were incubated at 37°C ± 1°C for 15-16 hours to obtain fresh cultures in exponential growth so to expose at mutagenic effects an high number of cells. During the test the bacterial strains were kept in an ice bath to avoid their vitality reduction.

Genetic characteristics

Established procedures for stock culture preparation, marker verification and Storage were used. The phenotypic characteristics checked, were: Histidine requirement, presence or absence of R-factor plasmids where appropriate (i.e. Ampicillin resistance in strains TA98, TA100 and Ampicillin + tetracycline resistance in strain TA102); the presence of characteristic mutations as UVrB and rfa mutation. The ranges of number of spontaneous revertants frequency were validating.

CULTURE MEDIA AND REAGENTS Ready to use

- Tryptone Soya Broth (TSB) Moltox

Minimal Glucose Agar (bottom agar plates ) Moltox

Top agar (supplemented with Biotin and Histidine) Moltox S9 Moltox

Regensys AMoltox

- Regensys BMoltox

PBS Lonza

Histidine/Biotin Master plates Moltox

Ampicillin Master plates Moltox

Ampicilln/Tetracycline Master plates Moltox

- Tryptone Soya Agar (TSA) OXOID

Crystal violet discs Moitox

DMSO (Dimethyl sulfoxide) SIGMA

Water for injection use Eurospital

Sodium Azide (NaN3) Moltox

- 9-Aminoacridina (9-AAc) Moltox

2-Nitrofluorene (2NF) Moltox

Mitomycin C Moltox

2-Aminoantracene (2AAn) Moltox

Ciclophosfamide (CP) Moltox

ENZYMATIC SYSTEM FOR METABOLISM ACTIVATION

The enzymatic System for metabolism activation (S9 mix) was prepared adding to S9 (an hepatic homogenate obtained from the liver of adult male rats which had previously been induced with "aroclor 1254" soybean oil solution) to Regensys A and to Regensys B containing respectively phosphate-buffered salt solution and Glucose -6-phosphate and 153 mg NADP for the activation. S9 mix was subjected to a sterility control to exclude any possible contamination. The product toxicity in the presence of S9 Mix was also tested. EQUIPMENT

Standard microbioiogy laboratory were used.

EXPERIMENTAL DESIGN

Preparation of the assav sample

The solvent should not be suspected of chemical reaction with the test substance and should be compatible with the survival of the bacteria and the S9 activity.

The test substance, comprising peptide (I) in 5% weight/total weight of the composition, was used neat as starting material. A solution in water of the test substance at equivalent concentration of 50 mg/ml and 4 subsequent dilutions of semi-log intervals between them were prepared, at a concentration equivalent to 15 mg/ml, 4.5 mg/ml, 1.35 mg/ml, 0.41 mg/ml.

Preliminary tests

These tests were performed for each replicate.

Microbiological controls

Aliquota of maximum concentration of the assay samples, solvent, PBS, S9 mix and Top Agar, were directly piaced in TSA plates.

Determination of solubility

The solubility was verified for all test concentrations during the test.

Determination of toxicity

The sample toxicity assay for the test strains was determining any reduction in culture growth and number

of spontaneously reverting organisms caused by the assay sample. Positive controls

Positive controls were prepared at these concentrations, and were use for the subsequent strains:

Negative controls

Negative controls were performed in the presence and in the absence of solvent used for the preparation of test substance dilutions .

Verification of the genette characteristic

The following genetic characteristics of the strains were verified :

rfa: sensitivity to crystal violet;

His: need for Histidine as a growth factor;

UvrB: sensitivity to ultraviolet radiation;

R. Amp: resistance to Ampicillin;

R. Tetra: resistance to Tetracycline.

ASSAY PROCEDURE

The whole assay was performed in triplicate.

Plate test without metabolic activation

0.1 mL of assay sample, 0.1 mi of the bacterial cell suspensions and 0.5 mi of PBS was added to aliquotted top agar (containing Biotin and Histidine) in tube, then briefly stirred and poured into minimal glucose agar plates.

At the same time, negative controls, solvent controls and positive controls were also prepared.

The plates were then incubated at 37°C±1°C for 48 hours.

After incubation, the reverted colonies of the assay sample at the different concentrations, as well as those of the negative controls (with and without solvent) and positive controls, were counted in each plate.

Three replications were performed with the assay sample, negative and positive controls.

Plate test with metabolic activation

0.1 mi of assay sample, 0.1 mi of the bacterial celi suspensions and 0.5 mi of the enzymatic system for metabolism activation was added to aliquotted top agar (containing Biotin and Histidine) in tube, then briefly stirred and poured into minimal glucose agar plates .

At the same time, negative controls, solvent controls and positive controls were also prepared.

The plates were then incubated at 37°C±1°C for 48 hours.

After incubation, the reverted colonies of the assay sample at the different concentrations, as well as those of the negative controls (with and without solvent) and positive controls, were counted in each plate.

Three replications were performed with the assay sample, negative and positive controls.

Verification of the genetic characteristic His-and R Factor verification

Three sets of plates according to the scheme were used:

a) Minimal glucose agar plates with Biotin;

b) Minimal glucose agar plates with Histidine and Biotin;

e) Minimal glucose agar plates with Histidine, Biotin and

Ampicillin; d) Minimal glucose agar plates with Histidine, Biotin, Ampicillin and Tetracycline;

The strains were seeded on the plates using sterile loops, and incubate at 37°C+1°C for 48 h.

RFA verification

To 2.0 ml of top agar (containing Biotin and Histidine) kept liquid in sterile tubes in a thermostated bath at

45°C+2°C were added 0.1 mi of bacterial strain. The whole was stirred and then poured into TSA plates.

On the agar solidifies were place a disk of sterile filter paper, on which are deposited 10 μΐ of crystal violet 1 mg ml. The plates were incubated at 37aC+l°C for 48 hours.

UvrB verification

The test strains were seeded on TSA plates, an half of the plate were covered with opaque material and irradiation with UVB rays for about 10 seconds. Incubate the plates at 37°C+1°C for 48 hours .

ASSAY VALIDITY CRITERIA

In the microbiological controls performed on MGA and TSA plates, top-agar, solvent used, assay sample at the highest concentration tested, PBS and S-9 Mix should not be contaminated by more than two colonies per plates.

On average, for the TA1535, TA1537 and TA98 strains, the number of reverting colonies developed by the positive control should be at least 300% higher than that of the respective negative control (lower limit for positive control/negative control ratio for strains with low mutation frequency) ; for the TA100 and TA102 strains, the number of reverting colonies developed by the positive control should be at least 200% higher than that of the respective negative control (a 20% of variation is accepted at the lower limit of the positive control/negative control ratio in strains with high mutation frequency) . The number of spontaneously reverting colonies per plate in the negative controls should be included between the validated limits .

The control of genetic characteristics of strains should give the following results:

In the His- verification, the test strains with His- genetic markers should be negative for growth on minimal glucose agar plates and biotin, positive for growth on minimal glucose agar plates with biotin and Histidine.

In the R-Factor verification, the test strains with genetic markers for Ampicillin R Factor (Salmonella typhimurium TA 98, TA 100 and TA 102) should be positive for growth on minimal glucose agar plates with Biotin Histidine and Ampicillin.

Strain with genetic markers for R Factor Tetracycline (Salmonella typhimurium TA 102) should be positive for growth on minimal glucose agar plates with Biotin Histidine Ampicillin and tetracycline .

In rfa verification, the test strains with rfa factor should be show a halo of inhibition of growth around the disk of about 7 mm in diameter.

In the UvrB- verification, the test strains exposed to UVB should be positive for growth only on the side of the plate covered.

INTERPRETATION OF RESULTS

According to the reference standard OECD 471:1997, there are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertants colonies per plate in at least one strain with or without metabolic activation System; bioiogical reLevance of the results should be considered first.

Statistical methods may be used as an aid in evaluating the test results; however, statistical significance should not be the only determining factor for a positive response. For the present experimentation a validated statistical test has been used for the evaluation of the obtained results.

This test performs a statistical comparison between the number of revertants colonies counted in presence of the test substance and the number of spontaneous revertants colonies on solvent control plates .

If the observed difference can be considered not statistically significant, the test substance could be considered as non mutagenic in this test.

RESULTS (Figures 9-11)

Neither reproducible increase in the number of revertants colonies per plate in any strain with or without metabolic activation system was detected. Besides, the statistical test applied showed no significant difference between the numbers of revertants colonies for assay sample vs. negative control.

Genetic characteristics of the bacterial strains

The verification of the genetic characteristics showed that the test strains maintained the required genetic properties io both the assay repetitions.

Toxicity of the test substance

On the basis of the results obtained during the test, the test substance did not show toxic effects either in the presence or absence of the enzymatic system for metabolism activation.

Negative control and Positive controls

The number of spontaneously reverting colonies in the negative control plates did not exceed the established limits and ali the positive controls caused a significant increase of number of reverting colonies.

Microbiological controls

The microbiological control performed did not show any contamination .

DEVIATIONS

No deviation has been recorded during the study.

CONCLUSIONS On the basis of the results, interpreted according to OECD 471:1997, the D. Radiodurans extract comprising peptide (I) according to the invention proved to be NOT MUTAGENIC for all the test strains, either in the presence or absence of metabolic activation.