阅读说明：本技术 萝卜硫素-褪黑激素类化合物 (Sulforaphane-melatonin compounds ) 是由 J·托池 J·霍芬克 于 2019-08-27 设计创作，主要内容包括：公开了一种组合物,其包含化合物并且该化合物包含6-羟基褪黑激素和6-甲基亚磺酰基己基异硫氰酸酯的偶联物或由其组成,以及该组合物用作药物的用途。此外,公开了该组合物在治疗和/或预防上皮组织疾病和/或病症的方法中的应用。(A composition comprising a compound and the compound comprising or consisting of a conjugate of 6-hydroxytyrosol and 6-methylsulfinylhexylisothiocyanate is disclosed, as well as the use of the composition as a medicament. Further, the use of the composition in a method of treating and/or preventing diseases and/or disorders of epithelial tissue is disclosed.)

1. A composition comprising a compound comprising or consisting of a conjugate of 6-hydroxytyrosol and 6-methylsulfinylhexylisothiocyanate.

2. A composition according to claim 1 for use as a medicament.

3. A composition comprising a compound and the compound comprising or consisting of a conjugate of 6-hydroxytmelatonin and 6-methylsulfinylhexylisothiocyanate for use in a method of treating and/or preventing a disease and/or disorder of epithelial tissue.

4. A composition according to claim 3 for use according to claim 3, wherein the use is in a method of treating dermatitis and/or mucositis.

5. The composition of claim 4, for use of claim 4, wherein the use is in a method of treating radiation-induced dermatitis and/or radiation-induced mucositis.

6. The composition for use of any one of claims 3-5, wherein the use is in a method of treating side effects of radiotherapy and/or chemotherapy.

7. The composition for use according to any one of claims 3-6, wherein the use comprises topical application of the composition.

8. The composition for use of claim 7, wherein the topical administration comprises applying the composition on the skin and/or mucosa.

9. The composition for use according to any one of claims 3-8, wherein the use comprises oral, mucosal, subcutaneous, intramuscular and/or parenteral administration of the composition.

10. The composition for use according to any one of claims 3-9, wherein the composition further comprises a solubilizing agent, a skin penetration enhancer, a preservative, a humectant, a gelling agent, a buffering agent, a surfactant, an emulsifier, an emollient, a thickener, a stabilizer, a humectant, a dispersing agent and/or any combination thereof.

11. The composition for use according to any one of claims 3-10, wherein the composition comprises carriers, diluents and/or excipients to facilitate the uptake of the composition in vivo or on the body surface.

12. The composition for use of claim 11, wherein the carrier and/or the excipient comprises an encapsulating material for at least temporarily encapsulating the compound.

13. The composition for use of any one of claims 11-12, wherein the carrier and/or the excipient comprises a liposome.

14. The composition for use according to any one of claims 3-13, wherein the composition further comprises an antibiotic, an antibacterial and/or an antifungal agent.

Technical Field

The present invention relates to the field of medicine, more specifically to the field of pharmaceutical compositions for the treatment of skin and/or mucosal disorders. More particularly, the present invention relates to compositions for use in methods of treating and/or preventing dermatitis and/or mucositis, to related compositions, and to related compositions for use as medicaments.

Background

Dermatitis and/or mucositis may be caused by oxidative stress, for example due to exposure to ionizing radiation. For example, radiation therapy or Radiotherapy (RT) is a treatment modality in which ionizing radiation is applied to control and/or kill malignant cells, for example in or as part of a cancer treatment strategy. Humans may also be exposed to large doses of naturally occurring ionizing radiation, such as may be encountered during space travel, or may be exposed to large doses of ionizing radiation accidentally, such as in the case of a nuclear facility failure or an inability to shield radioactive sources, such as in a hospital, non-destructive testing equipment, or radioisotope thermoelectric generators.

In radiotherapy, ionizing radiation can be used to kill cancer cells and/or shrink tumors in almost all known types of solid tumors, including brain, breast, cervical, laryngeal, lung, pancreatic, prostate, skin, spinal, gastric, uterine and soft tissue sarcomas. Radiation can also be used to treat leukemia and lymphoma. Without a cure, radiation therapy can be used as a palliative treatment for local control of tumors or symptom relief, or as a treatment that extends the life of the patient.

However, the application of radiation therapy is not necessarily limited to cancer treatment, and may be used, for example, for the treatment of trigeminal neuralgia, arteriovenous malformations, thyroid eye diseases, pterygium, and the prevention of keloid growth or ectopic ossification. Whole body irradiation may also be performed prior to bone marrow transplantation. In addition, hyperthermia or deep tissue heating is often used in combination with radiation to increase the responsiveness of large or advanced tumors to treatment. For example, advanced and/or large tumors in the body can be treated by a combination of thermal therapy and radiation therapy. By exposing the deep tissue to temperatures in the range of 43 ℃ to 50 ℃, cancer tissue that is quite deep in the body can be destroyed, which can lead to skin burns.

Unfortunately, the use of radiation therapy can have serious side effects due to the induced toxicity of healthy cells, particularly epithelial cell populations (including stem cells) within the hair follicle, epidermis and mucosa. Radiation therapy destroys cells in a target tissue by destroying their DNA, altering signal transduction pathways, and inducing apoptosis. The cytotoxic effects of radiation therapy are associated with an increase in the electron energy levels that can cause ionization of DNA and the production of Reactive Oxygen Species (ROS), including superoxide anion radicals, hydrogen peroxide and hydroxyl radicals, which can damage cells, proteins and DNA. The response of cells to radiation depends, inter alia, on the type of radiation, the energy and dose, and the tissue-specific sensitivity. However, due to physical and geometric constraints, a significant portion of the delivered dose will also be deposited in healthy tissue.

Radiation therapy often entails short-term side effects, including erythema, irritation and inflammation of the skin, as well as intermediate-and long-term side effects, such as edema, pain, fibrosis and superficial vasodilation (telangiectasia). Radiotherapy is used to treat the chest wall after mastectomy, head and neck tumors and skin tumors that may cause acute reactions and severely damage the skin and mucosa. The skin reactions may vary from acute erythema to desquamation and necrosis. Similarly, the mucosa of the mouth, throat, esophagus, trachea, intestines, bladder and rectum may also be damaged. Mouth sores and ulcers are common symptoms in patients following ionizing radiation therapy. Side effects also include xerostomia (dry mouth), xerophthalmia (dry eye) and dryness of the vaginal and anal mucosa, due to the acute effects of radiation sensed in the salivary or mucous producing accessory glands.

Long-term complications may typically occur at higher doses of radiation (e.g., over 35 Gy). Later side effects that may occur over the course of months or years include tissue scarring (e.g. due to an increase in connective tissue), secondary cancers such as breast cancer, gastric cancer, lung cancer and melanoma (which may occur in areas of the body near the irradiated area), and thyroid disease.

Radiation-induced dermatitis or radiodermatitis is a well-known and painful side effect known to occur in most radiation-treated patients. In addition, heat therapy may also induce dermatitis, and the combination of radiation therapy and heat therapy may greatly increase the risk and severity of dermatitis.

The severity of radiodermatitis may vary depending on the treatment and factors inherent to the patient. Most acute radiodermatitis reactions disappear after several weeks, but some reactions persist and may lead to complications. Delayed radiodermatitis is characterized by the formation of telangiectasias on atrophic and fragile skin, which can cause inflammation of bare tissues and severely affect epidermal barrier function.

Mucositis is another important and costly side effect of cancer therapy. As an inflammation of mucosal surfaces, mucositis is a common, potentially serious complication of chemotherapy and/or radiotherapy. It can manifest as erythema, desquamation, ulcer formation, bleeding and exudate. Mucositis may occur throughout the gastrointestinal and genitourinary tracts, from the mouth to the intestines and rectum.

It is generally believed that both radiodermatitis and mucositis result from the direct inhibition of DNA replication and epidermal and mucosal stem cell proliferation by chemotherapy or radiotherapy. These events occur in connection with the increase of the electron energy level causing the ionization of DNA and the production of Reactive Oxygen Species (ROS), including superoxide anion radicals, hydrogen peroxide and hydroxyl radicals, which destroy cells, proteins and DNA, leading to a decrease in the regenerative capacity of the basal epithelium, atrophy of the epidermis and mucosa, collagen breakdown and ulceration. The secondary effect is infection by many pathogens after the protective epidermal and mucosal barriers have been breached.

Both radiodermatitis and mucositis can have a significant adverse effect on concomitant and subsequent treatment regimens, particularly on the quality of life of the patient, as it can lead to nutritional abnormalities, increased systemic infection, pain relief with anesthetics, and delayed cancer treatment. Thus, in terms of quality-adjusted life years (QALY), such conditions may also have an adverse effect on post-treatment prognosis.

Currently, the treatment options for these pathologies are very limited. However, while there is insufficient evidence to develop an authoritative proposition to prevent and/or alleviate radiodermatitis and mucositis, some progress has been made. For example, it appears beneficial to use Low Level Laser Therapy (LLLT) or vascular laser to control the symptoms of radiodermatitis. Recent preclinical and clinical studies have shown that LLLT has biostimulating properties that allow for faster tissue regeneration and healing, reduce inflammation and prevent fibrosis. Furthermore, in delayed radiodermatitis, pulsed dye laser therapy has been shown to be beneficial in clearing radiation-induced telangiectasia.

Current treatments for mucositis include the application of hygienic fundamentals as well as local anesthetics and/or systemic analgesics to relieve pain in an effort to minimize symptoms. However, these methods do not address the underlying cause of radiodermatitis and/or mucositis.

Successful administration of protective therapies that promote normal cell growth and proliferation can enable higher dose, more aggressive cancer treatments after or during radiation therapy and/or in the presence of chemotherapeutic agents, whether or not effective treatment of dermatitis and/or mucositis is required as a side effect of cancer treatment. Thus, these protective therapies may not only address the side effects of cancer and its treatment, but may even improve the therapeutic efficacy of current cancer therapies.

WO 2012/142511 discloses anti-inflammatory and extracellular matrix-stable pro-molecular compositions and pharmaceutical formulations thereof. These compositions include a variety of phytochemically active nutritional compounds including sulforaphane and melatonin. Prophylactic and therapeutic applications are disclosed, for example, for treating acute or long-term inflammation-mediated disorders and stabilizing mammalian extracellular matrix.

WO 2012/122295 describes compositions comprising a variety of nutritional and non-chemotherapeutic drug components, including sulforaphane and melatonin, and related methods for treating pancreatic cancer.

WO 2009/051739 describes methods and compositions relating to the topical use of Nrf2 inducers (e.g. sulforaphane) for protecting the skin and mucosa from the adverse side effects of radiotherapy.

US 2014/243384 relates to the use of a composition comprising melatonin or a derivative thereof in a ratio of 2.5% to 5% w/v for the manufacture of a pharmaceutical composition for the treatment and/or prevention of (e.g. oral) mucositis (e.g. caused by radiotherapy and/or chemotherapy).

DUNAWAY SPENCER et al in "natural antioxidants: various mechanisms for photoprotection of Natural plant products, including sulforaphane and melatonin, are described IN the various mechanisms for protecting skin from solar radiation (Natural antioxidants to protective skin from radiation) ", leading edge of PHARMACOLOGY (frontier IN PHARMACOLOGY), 2018, Vol. 9,392, pp. 1-14.

TALALAY P et al disclose THE UV radiation protection effect OF topical application OF a Sulforaphane-rich extract from broccoli sprouts in "Sulforaphane mobilizes cell defense and protects skin from UV radiation (Sulforaphane cells with protected skin against UV radiation)" (PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE United STATES OF America OF THE NATIONAL ACADEMY OF SCIENCES OF THE same OF American tissue OF Industrial OF America, NATIONARY OF academic OF SCIENCES, US), volume 104,44, page 17500 and 17505).

WO 97/06779 discloses cosmetic topical compositions for use in the sun protection of skin. The composition comprises melatonin or an analog thereof.

Disclosure of Invention

It is an object of embodiments of the present invention to provide a good, efficient, effective and/or affordable medicament for the treatment of disorders of epithelial tissue, such as disorders affecting epithelial cells, such as those lining the oral cavity, gastrointestinal tract and/or urogenital tract, and/or epithelial cells of the skin, including hair follicles and epidermis.

It is an advantage of embodiments of the present invention that a higher effectiveness in treating such epithelial tissue diseases may be achieved and ease of administration may be achieved relative to existing methods known in the art.

It is an advantage of embodiments of the present invention that side effects of radiation therapy and/or chemotherapy may be removed, reduced and/or prevented.

An advantage of embodiments of the present invention is that the composition may advantageously (and synergistically) enhance the radiosensitivity of tumor cells as well as protect healthy epithelial cells from (or help recover from) oxidative stress caused by radiation.

It is an advantage of embodiments of the present invention to provide safe and effective medicaments and methods for the prevention and treatment of diseases affecting epithelial cells, which may reduce side effects of cancer treatments, such as radiodermatitis and mucositis.

An advantage of embodiments of the present invention is that potent and non-toxic compounds that induce NF-E2-related factor 2(Nrf2) can be administered to high risk non-neoplastic tissue, for example by topical, oral or other methods of administration, and thus can reduce, or even neutralize, harmful oxygen free radical formation, for example during or after radiation therapy.

An advantage of an embodiment is that the composition may be considered a topically acting formulation, for example when applied topically.

An advantage of an embodiment is that a composition is provided that has significant antioxidant activity.

It is an advantage of an embodiment to provide a composition that normalizes redox processes in irradiated tissue, retards the development of peroxidic oxidation processes of lipids in cell membranes and their breakdown, prevents the development of cellular structural changes in epidermal and subcellular structures, decreases capillary permeability, exhibits detoxification, promotes normalization of tissue metabolism and/or stimulates regenerative processes.

An advantage of an embodiment is that it provides a composition with low toxicity and/or no mutagenic, embryotoxic and/or carcinogenic properties.

An advantage of an embodiment is that a composition is provided that does not provide radioprotection to tumor tissue (e.g., may be particularly advantageous when combined with radiation therapy and/or chemotherapy, e.g., for treating side effects thereof).

It is an advantage of an embodiment to provide a composition that is rapidly metabolized to an oxidation state and cleared from an organism.

The above objects are achieved by the compositions used and the compositions according to the invention.

In a first aspect, the present invention relates to a composition comprising a compound of sulforaphane or a sulforaphane analog and melatonin or a melatonin analog for use in a method of treating and/or preventing a disease and/or disorder of epithelial tissue, such as a method of treating and/or preventing dermatitis and/or mucositis. In other words, the compound may be a conjugate (conjugate) of sulforaphane (or a sulforaphane analog) and melatonin (or a melatonin analog), i.e., a compound formed by linking at least sulforaphane (or a sulforaphane analog) and melatonin (or a melatonin analog) together. The compound comprises or consists of a conjugate of 6-hydroxytmelatonin and 6-methylsulfinylhexylisothiocyanate, and may for example comprise or consist of a compound formed by linking together at least 6-hydroxytmelatonin and 6-methylsulfinylhexylisothiocyanate.

In the composition for use according to an embodiment of the first aspect of the present invention, the use may be in a method of treating radiation-induced dermatitis and/or radiation-induced mucositis.

In the composition for use according to an embodiment of the first aspect of the present invention, the use may be in a method of treating side effects of radiotherapy and/or chemotherapy.

In the composition for use according to an embodiment of the first aspect of the present invention, the use may include topical administration of the composition.

In the composition for use according to an embodiment of the first aspect of the present invention, such topical application may comprise applying the composition on the skin and/or mucosa.

In the composition for use according to an embodiment of the first aspect of the present invention, the use may include oral, mucosal, subcutaneous, intramuscular and/or parenteral administration of the composition.

In the composition for use according to an embodiment of the first aspect of the present invention, the composition may further comprise a solubilizing agent, a skin penetration enhancer, a preservative, a humectant, a gelling agent, a buffering agent, a surfactant, an emulsifier, an emollient, a thickener, a stabilizer, a humectant, a dispersing agent and/or any combination thereof.

In the composition for use according to an embodiment of the first aspect of the present invention, the composition may further comprise a carrier and/or an excipient to facilitate the uptake of the composition in vivo or on the body surface.

In the composition for use according to an embodiment of the first aspect of the invention, the carrier and/or excipient may comprise a non-toxic filler material, i.e. a filler material that is substantially non-toxic to human organisms.

In the composition for use according to an embodiment of the first aspect of the present invention, the carrier and/or excipient may comprise a diluent.

In the composition for use according to an embodiment of the first aspect of the present invention, the carrier and/or excipient may comprise an encapsulating material for at least temporarily encapsulating the compound.

In the composition for use according to an embodiment of the first aspect of the present invention, the carrier and/or excipient may comprise a liposome.

In the composition for use according to an embodiment of the first aspect of the present invention, the composition may further comprise an antibiotic, an antibacterial agent and/or an antifungal agent.

In a second aspect, the present invention relates to a composition comprising a compound, wherein the compound comprises or consists of a conjugate of 6-hydroxytyrosol and 6-methylsulfinylhexylisothiocyanate.

In a third aspect, the present invention relates to the use of a composition according to an embodiment of the second aspect of the invention as a medicament.

Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and features of other dependent claims as appropriate and not merely as explicitly set out in the claims.

These and other aspects of the invention are apparent from and will be elucidated with reference to the embodiments described hereinafter.

Brief description of the drawings

Fig. 1 shows a radiation lesion with focused gamma radiation applied to the back skin of a miniature pig in an example illustrating an embodiment of the invention.

Fig. 2 shows a schematic view of the example of fig. 1 for illustrating an embodiment of the present invention.

Fig. 3 shows the results of the examples of fig. 1 and 2, showing pictures of the skin appearance of pigs treated with vehicle and with a composition according to an embodiment of the invention before irradiation, 7, 21 and 35 days after irradiation.

Fig. 4 shows the change in clinical score of pigs over time after irradiation in the examples of fig. 1 and 2 illustrating embodiments of the invention.

Figure 5 shows histological skin changes 35 days after irradiation in pigs treated with vehicle and with a composition according to an embodiment of the invention. Stained with hematoxylin and eosin and images were taken at 400x magnification.

Figure 6 shows the change in basal cell density over time in the skin of a miniature pig treated with a vehicle and with a composition according to an embodiment of the invention. (. p <0.05 and. p <0.01 vs. vehicle-treated irradiated animals)

Figure 7 shows the change in epidermal thickness over time of skin of a mini-pig treated with a vehicle and with a composition according to an embodiment of the invention after irradiation. (. p <0.05 and. p <0.01 vs. vehicle-treated irradiated animals)

Figures 8 to 14 show the reduction in the expression of Cyclooxygenase (COX) -2 in the skin after irradiation of pigs treated with a composition according to an embodiment of the invention. Counterstaining with hematoxylin and images were taken at 400x magnification.

Figure 8 shows COX-2 expression in skin prior to radiation exposure.

Figure 9, figure 10 and figure 11 show COX-2 expression in the skin of vehicle-treated piglets at 7, 21 and 35 days post-irradiation, respectively.

Figure 12, figure 13 and figure 14 show COX-2 expression in skin of piglets treated with compositions according to embodiments of the invention at 7, 21 and 35 days post-irradiation, respectively.

Fig. 15 to 21 show that the expression of Nuclear Factor (NF) - κ B is reduced in skin after irradiation of pigs treated with a composition according to an embodiment of the invention. Counterstaining with hematoxylin and images were taken at 400x magnification.

FIG. 15 shows NF- κ B expression in skin before radiation exposure.

Figure 16, figure 17 and figure 18 show NF- κ B expression in the skin of vehicle-treated mini-pigs at 7, 21 and 35 days post-irradiation, respectively.

Figure 19, figure 20 and figure 21 show NF- κ B expression in skin of a mini-pig treated with a composition according to an embodiment of the invention at 7, 21 and 35 days post-irradiation, respectively.

Figures 22 to 27 show the peripheral blood counts of the mini-pigs of the above examples 3, 7, 21 and 35 days after irradiation before focused radiation, for the case of treatment with vehicle and the case of treatment with a composition according to an embodiment of the invention, respectively. Fig. 22 shows the number of blood cells, fig. 23 shows the counts of neutrophils, fig. 24 shows the counts of eosinophils, fig. 25 shows the counts of lymphocytes, fig. 26 shows the counts of erythrocytes, and fig. 27 shows the counts of platelets. Mean ± standard error of the mean is shown.

Fig. 28 shows wound healing of biopsy lesions stimulated by a composition according to an embodiment of the invention three days after irradiation in the above example. From left to right, the appearance of the unirradiated biopsy lesions 32 days after biopsy, 35 days after irradiation combined with vehicle treatment, and 35 days after irradiation combined with treatment with a composition according to an embodiment of the invention are shown, respectively.

Figure 29 shows the change over time (scored on a clinical scale) of biopsy wounds in skin following irradiation in pigs treated with vehicle and pigs treated with compositions according to embodiments of the invention. 6OHM-6-HITC administration can affect the formation of biopsy wounds in the skin. Mean ± standard error of the mean is shown.

Fig. 30 shows the change in body weight of a mouse with time after X-ray irradiation in a second example for illustrating an embodiment of the present invention.

Fig. 31 shows a lifetime process after X-ray irradiation in a second example for explaining the embodiment of the present invention.

Fig. 32 shows the mean ± SEM clinical score (n ═ 10) for oral mucositis in a second example used to illustrate an embodiment of the invention. The tongue of the mice was irradiated with 20Gy on day 0. P <0.05, clearly different from the control values (Steel-Dwass test).

Fig. 33 shows mean ± SEM values of total oral mucositis scores between day 0 and day 12 for each group in a second example used to illustrate an embodiment of the invention. P <0.05, clearly different from the control values (Steel-Dwass test).

Fig. 34 shows a histological photograph of a tongue specimen after X-ray irradiation in a second example for illustrating an embodiment of the present invention. Tongue specimens were fixed with 10% buffered formalin and embedded in paraffin. Sections (3mm) were stained with hematoxylin and eosin (x 200). a: complete, b: control (oral mucositis), c: 6OHM-6-HITC 30mg/kg, d: 6OHM-6-HITC 300 mg/kg.

Fig. 35 shows a histological photograph of a tongue specimen after X-ray irradiation in a second example for illustrating an embodiment of the present invention. Tongue specimens were fixed with 10% buffered formalin and embedded in paraffin. Apoptotic cells were assessed by TUNEL staining (x 400). a: complete, b: control (oral mucositis), c: 6OHM-6-HITC 30mg/kg, d: 6OHM-6-HITC 300 mg/kg.

Figure 36 shows the percentage of apoptosis observed after TUNEL staining in a second example used to illustrate an embodiment of the invention. P <0.05, clearly different from the control values (Steel-Dwass test).

Fig. 37 shows the effect of a composition according to an embodiment of the invention on Myeloperoxidase (MPO) activity (in units) in the tongue of mice with oral mucositis, in a second example to illustrate an embodiment of the invention. P <0.05, clearly different from the control values (Steel-Dwass test).

Figure 38 shows the effect of a composition according to an embodiment of the invention on the activity (in units) of 2-thiobarbituric acid-reactive substance (TBARS) in the tongue of mice determined to have oral mucositis in a second example used to illustrate an embodiment of the invention. P <0.05, clearly different from the control values (Steel-Dwass test).

The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

Any reference signs in the claims shall not be construed as limiting the scope of the invention.

Detailed Description

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. The dimensions and relative dimensions do not correspond to actual reductions to practice of the invention. It is to be noticed that the term 'comprising', used in the claims, should not be interpreted as being limitative to the parts listed thereafter, but does not exclude other elements or steps. It is therefore to be understood that the presence of the stated features, integers, steps or components is indicated but does not preclude the presence or addition of one or more other features, integers, steps or components or groups thereof. Reference in the specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments as would be apparent to one of ordinary skill in the art. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various aspects of the invention. However, the methods in this disclosure should not be understood to reflect the intent: the claimed invention requires more features than are expressly recited in each claim. Moreover, as the following claims reflect, inventive aspects may lie in less than all features of a single foregoing disclosed embodiment. Thus, the following claims are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention. Furthermore, when some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are intended to be included within the scope of the invention and form different embodiments, as would be understood by those skilled in the art. For example, in the appended claims, any of the claimed embodiments may be used in any combination. Numerous specific details are set forth in the description herein. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been described in detail so as not to obscure the understanding of this description.

In a first aspect, the present invention relates to a composition comprising a compound of sulforaphane (sulforaphane) or a sulforaphane analog (e.g. a synthetic sulforaphane analog) and melatonin or a melatonin analog (e.g. a synthetic melatonin analog) (e.g. fused or conjugated), for use in a method of treatment and/or prevention of diseases and/or conditions of epithelial tissue, such as for use in a method of treatment and/or prevention of dermatitis and/or mucositis. In other words, the compound may be a conjugate of sulforaphane (or a sulforaphane analog) and melatonin (or a melatonin analog), i.e., a compound formed by linking at least sulforaphane (or a sulforaphane analog) and melatonin (or a melatonin analog) together. The compound comprises or consists of a conjugate of 6-hydroxytmelatonin and 6-methylsulfinylhexylisothiocyanate, and may for example comprise or consist of a compound formed by linking together at least 6-hydroxytmelatonin and 6-methylsulfinylhexylisothiocyanate.

Sulforaphane is an aglycone breakdown product of the glucosinolate sulforaphane, also known as Sulforaphane Glucosinolate (SGS). The molecular formula of the sulforaphane is C₆H₁₁NOS₂And has a molecular weight of 177.29 daltons. Sulforaphane is also known as 4-methylsulfinylbutyl isothiocyanate and (-) -1-isothiocyanato-4 (R) - (methylsulfinyl) butane. The sulforaphane has a structural formula as follows:

reference to a sulforaphane analog may generally refer to an isothiocyanate, such as 6-methylsulfinylhexylisothiocyanate. Sulforaphane analogs can refer to any metabolite of the isothiocyanate family, or synthetic derivatives thereof. The sulforaphane analogs can be obtained from an extract of at least one isothiocyanate-containing plant or vegetable, or can be synthetic.

Melatonin (N-acetyl-5-methoxytryptamine) is a modified tryptophan that can be synthesized by acetylation and methylation of 5-hydroxytryptamine. The structural formula of melatonin is:

in a particular example, reference to a melatonin analog may refer to 6-hydroxy melatonin. Melatonin analogs may refer to any metabolite of melatonin or synthetic analogs thereof.

The compositions used according to embodiments may reduce Reactive Oxygen Species (ROS) and inflammation by activating NF-E2-related factor 2(Nrf2) phase II enzymes. In particular, the compounds can specifically target the neutralization of free radicals by activating the Nrf2/Keap1/ARE pathway. The compounds may have pleiotropic effects, e.g., multiple genes and/or related pathways may be targeted, thereby achieving surprisingly effective effects through synergistic effects of melatonin (class) and sulforaphane (class) properties.

Use in a method for treating dermatitis and/or mucositis may refer to treatment of an animal, for example treatment of a mammal, for example a human. The treatment is not necessarily a treatment after diagnosis of dermatitis and/or mucositis, but may also refer to a prophylactic treatment in consideration of anticipation of future development of dermatitis and/or mucositis. For example, the compositions may be used to treat a subject having a past condition or to a subject predisposed to a skin or mucosal disease or condition. For example, the composition may be used to alleviate radiation therapy symptoms in a patient, or as a prophylactic measure in such a patient.

The composition advantageously has a curative effect on dermatitis and mucositis caused by radiation-related factors (but not necessarily limited thereto), has an advantageously long duration of action, and can be considered safe, e.g., without toxic side effects. Furthermore, the treatment can be advantageously short and can be effected rapidly, and can be widely used for clinical treatment of skin injury and inflammation, as well as radiation therapy-induced dermatitis and mucositis. The compositions comprising the pharmaceutical compounds may advantageously protect the skin and mucous membranes of subjects undergoing treatment with ionizing radiation.

Dermatitis and/or mucositis may refer to damage to or disease of the skin or mucosa, as will be apparent to those skilled in the art, and may include any abnormality of the skin and mucosa in which oxidative stress is involved as a causative or substantive causative factor. Radiation may be associated with the cause of such damage or disease. Examples of lesions or diseases that the composition may be used to treat include, but are not limited to, acute erythema, skin irritation, inflammation, edema, desquamation, skin necrosis, mouth soreness and ulceration, pain, fibrosis, telangiectasia, dry mouth, dry eye, dryness of vaginal mucosa, breast cancer, gastric cancer, lung cancer, melanoma and thyroid disease.

In the composition for use according to an embodiment of the first aspect of the present invention, the use may be in a method of treating radiation-induced dermatitis and/or radiation-induced mucositis. In the composition for use according to an embodiment of the first aspect of the present invention, the use may be in a method of treating side effects of radiotherapy and/or chemotherapy. The composition for use according to an embodiment of the present invention may be a pharmaceutical composition for preventing and/or treating dermatitis and/or mucositis caused by radiotherapy.

The pharmaceutical composition can be used for preventing and/or treating skin radiation burn lesions, such as skin radiation burn lesions caused by ionizing and/or non-ionizing radiation. For example, the compositions may be used for topical protection of the skin and mucosa of a patient from the toxic side effects of radiotherapy, and/or for prophylaxis. When applied topically (but not necessarily limited to topical application) to a body region and/or surrounding area of a patient exposed to ionizing radiation, the composition can protect the skin and mucosa of a patient receiving ionizing radiation treatment by inducing Nrf2 phase II enzymes.

For example, a subject to be treated for use according to an embodiment of the invention may be subjected to short-term or long-term effects of ionizing radiation therapy. The subject may be affected by: acute erythema, skin irritation, inflammation, edema, desquamation, skin necrosis, mouth sores and ulcers, pain, fibrosis, telangiectasia, dry mouth, dry eye, dryness and irritation of vaginal or rectal mucosa, melanoma, breast cancer, gastric cancer, lung cancer or thyroid disease.

Thus, embodiments of the present invention may specifically relate to compositions according to embodiments of the present invention for use in the treatment of any of the conditions and/or diseases mentioned above and/or for use in the treatment of any combination thereof.

However, the subject to be treated may also have little or no symptoms, e.g., may be treated with a composition for use according to an embodiment of the present invention to prevent radiation burn lesions of the skin. The composition may advantageously have low toxicity and may advantageously provide high efficacy in protecting skin from ionizing and non-ionizing radiation. This may also enable higher radiation doses to be used in radiation therapy due to increased tolerance to higher doses, and/or may enable more aggressive staging schemes to be employed, for example, where the same or similar dose is delivered in fewer stages and/or the delay between delivery of successive dose stages is shorter.

However, embodiments of the invention are not necessarily limited to use in connection with side effects of radiation therapy, e.g., may be equally useful in the treatment of dermatitis and/or mucositis caused by non-radiation induced oxidative stress. Although radiation therapy can cause high levels of acute oxidative stress, a variety of other sources of oxidative stress are known that may cause dermatitis and/or mucositis. For example, oxidative stress may play a role in many skin diseases as well as skin aging. For example, atopic eczema or Atopic Dermatitis (AD) is a chronic recurrent inflammatory skin disease in which oxidative stress may be involved, accompanied by factors such as genetic susceptibility to immune disorders and hypersensitivity reactions. In another example, dermatitis or mucositis may be caused by (or promoted by) hyperthermia.

Oxidative stress refers to the excessive formation of oxidants in cells, i.e. in the skin or mucosa in the context of the present disclosure, (acutely or chronically) relative to the antioxidant defense capacity of these cells. Oxidants such as free radicals, Reactive Oxygen Species (ROS) and Nitric Oxide Species (NOS) are produced during normal metabolic activity, and cellular defense mechanisms for these oxidants, such as enzyme-based systems (e.g., redox proteins) and non-enzyme-based systems (e.g., possibly dependent on specific vitamins, glutathione, coenzyme Q10 and other antioxidants), may become overloaded. If improperly controlled, the oxidizing agent may react with many macromolecules in the cell and may even initiate a chain reaction, resulting in severe cell damage and/or apoptosis.

In the compositions for use according to embodiments of the first aspect of the present invention, the compound may comprise or consist of a conjugate of 6-hydroxytmelatonin and 6-methylsulfinylhexylisothiocyanate, e.g. may comprise or consist of a compound formed by linking together at least 6-hydroxytmelatonin and 6-methylsulfinylhexylisothiocyanate. Such compounds can be prepared by coupling 6-hydroxytyrosol (6OHM) (melatonin analog) and 6-methylsulfinylhexylisothiocyanate (6-HITC) (sulforaphane analog). The structural formula of 6-hydroxytmelatonin (6OHM) is:

6-Methylsulfinyl hexyl isothiocyanate (6-HITC) has the structural formula:

thus, the hybrid 6-HITC-60HM of two molecules can be represented by the following structural formula:

thus, in a preferred embodiment, the compound that may be considered to be an inducer of the Nrf2 phase II enzyme may be a compound resulting from the fusion of a synthetic sulforaphane analog consisting of 6-methylsulfinylhexylisothiocyanate with a synthetic melatonin analog consisting of 6-hydroxytmelatonin (6 OHM). Thus in the present disclosure, the compound may be referred to as 6-HITC-6OHM, i.e. the composition for use according to embodiments of the invention may comprise a 6-HITC-6OHM conjugate.

6OHM can be considered as the main active metabolite of melatonin, i.e. melatonin is oxidatively converted in vivo to 6-hydroxy melatonin by cytochrome P450 isoenzymes. Melatonin is a 3-alkylindole organic compound, i.e. having an indole moiety with an alkyl chain in the-3 position, whereas 6-hydroxymelatonin is advantageously a hydroxyindole organic compound, i.e. having an indole moiety with a hydroxyl group. Furthermore, 6OHM is advantageously stable, bioavailable and has good free radical scavenging activity, e.g. higher stability, better bioavailability and better free radical scavenging activity compared to melatonin.

6-methylsulfinylhexylisothiocyanate may be obtained from horseradish, but the embodiments of the present invention are not limited thereto. Sulforaphane can be synthesized by oxidation of 4-methylthiohexyl isothiocyanate, in which the oxygen group in the isothiocyanate group is replaced with sulfur. On the other hand, 6-methylsulfinylhexylisothiocyanate can be synthesized by oxidation of 6-methylthiohexylisothiocyanate, in which the oxygen group in the isothiocyanate group is also substituted with sulfur. Although the two isothiocyanates have the corresponding structures, -N ═ C ═ S, their alkyl chain lengths differ. As shown in vitro, 6-methylthiohexyl isothiocyanate is advantageously potent (e.g., more potent than sulforaphane) in inducing the Nrf2 pathway.

When melatonin is transformed in vivo by cytochrome P450 isozymes, these isozymes may also activate genotoxic agents, which is undesirable, for example, for preventing and/or treating radiodermatitis or mucositis in cancer patients. However, sulforaphane may inhibit cytochrome P450 isozymes, so that melatonin is not metabolized (or metabolized to a lesser extent). Thus, 6-HITC-6OHM compounds may be particularly suitable for stimulating the Nrf2 pathway, as the main metabolite of melatonin is provided without relying on the action of cytochrome P450 isozymes, which may even be inhibited to avoid or reduce genotoxicity.

In the composition for use according to an embodiment of the first aspect of the present invention, the use may include topical administration of the composition. In the composition for use according to an embodiment of the first aspect of the present invention, such topical application may comprise applying the composition on the skin and/or mucosa. The local administration may relate to an area of the body of the subject that has been, is being or will be exposed to ionizing radiation, or to such an area including surrounding areas. Thus, the composition may be applied directly to the skin of the relevant part of the subject's body to prevent or minimize short-term and/or long-term side effects caused by radiotherapy or another condition, such as hyperthermia.

A therapeutically effective amount of a compound (which may be considered to be an Nrf2 phase II enzyme inducer) may be administered to a subject. Compositions for topical administration may be provided in the form of, but are not necessarily limited to, wound dressings, sprays, ointments, creams, emulsions, lotions, gels, or sun blocks. Such excipients are well known in the art. Topical administration may, for example, include application to the skin or mucous membranes, including surfaces of the lungs, stomach, vagina, mouth, and/or eye.

In the composition for use according to an embodiment of the first aspect of the present invention, the use may include oral, mucosal, subcutaneous, intramuscular and/or parenteral administration of the composition. Thus, the composition may comprise a variety of suitable carriers and/or excipients.

In the compositions according to embodiments, for example, in these compositions for topical application, various additives known to those skilled in the art may be included. For example, in the composition for use according to an embodiment of the first aspect of the present invention, the composition may further comprise a solubilizing agent, a skin penetration enhancer, a preservative (e.g., an antioxidant), a humectant, a gelling agent, a buffering agent, a surfactant, an emulsifier, an emollient, a thickener, a stabilizer, a humectant, a dispersing agent, a pharmaceutical carrier and/or any combination thereof.

In the composition for use according to an embodiment of the first aspect of the present invention, the composition may further comprise a carrier and/or an excipient to facilitate the uptake of the composition in vivo or on the body surface. In the composition for use according to an embodiment of the first aspect of the present invention, the composition may further comprise an antibiotic, an antibacterial agent and/or an antifungal agent. Examples of such agents include, but are not limited to, parabens, chlorobutanol, phenol sorbic acid, and the like. An advantage of an embodiment is that prevention and/or treatment of infection may be achieved. This is synergistic with the advantage of treating dermatitis and/or mucositis, as dermatitis and/or mucositis can significantly increase the risk of infection, for example, due to a compromised skin barrier.

Suitable skin penetration enhancers are well known in the art and may include: lower alkanols, such as methanol, ethanol and 2-propanol; alkyl methyl sulfoxides such as dimethyl sulfoxide (DMSO), decyl methyl sulfoxide (C10 MSO), and tetradecyl methyl sulfoxide; a pyrrolidone; urea; n, N-diethyl-m-toluidine; C2-C6 alkanediol; dimethylformamide (DMF); n, N-Dimethylacetamide (DMA) and/or tetrahydrofurfuryl alcohol.

Examples of solubilizing agents include, but are not limited to, hydrophilic ethers such as diethylene glycol monoethyl ether (ethoxydiglycol, available asCommercially available) and diethylene glycol monoethyl ether oleate (available asCommercially available); polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil, polyethylene glycol (PEG), especially low molecular weight PEG (e.g., PEG 300 and PEG 400), and polyethylene glycol derivatives, such as PEG-8 caprylic/capric glycerides (available as PEG-C)Commercially available); alkyl methyl sulfoxides such as DMSO; pyrrolidone, DMA and mixtures thereof.

In the composition for use according to an embodiment of the first aspect of the invention, the carrier and/or excipient may comprise a non-toxic filler material, i.e. a filler material that is substantially non-toxic to human organisms. These filler materials may be solid, semi-solid or liquid. In the composition for use according to an embodiment of the first aspect of the present invention, the carrier and/or excipient may comprise a diluent.

Suitable pharmaceutical carriers may include any such materials known in the art, for example, any liquids, gels, solvents, liquid diluents, solubilizers, polymers, and the like, which are non-toxic (to human organisms) and do not interact significantly with the other components of the composition or the skin in a deleterious manner. In the composition for use according to an embodiment of the first aspect of the present invention, the carrier and/or excipient may comprise an encapsulating material for at least temporarily encapsulating the compound. In the composition for use according to an embodiment of the first aspect of the present invention, the carrier and/or excipient may comprise a liposome.

Prevention and/or treatment of infection can be achieved by including in the compositions of the present invention antibiotics as well as various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like.

The composition for parenteral injection for use according to an embodiment may be in (e.g., may consist of): sterile aqueous or non-aqueous solutions, dispersions, suspensions and/or emulsions, and/or sterile powders which are reconstituted into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

The compositions for use according to embodiments of the present invention may further comprise adjuvants such as, but not limited to, preservatives, wetting agents, emulsifying agents and dispersing agents. Compositions for use according to embodiments of the invention may include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form of the composition may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Where prolonged effectiveness of the composition is desired by slowing the absorption of the composition upon subcutaneous or intramuscular injection, the composition may comprise a liquid suspension of a crystalline or amorphous material that is poorly water soluble. The rate of absorption of the drug may then depend on its rate of dissolution, which in turn may depend on the crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered drug form may be achieved by dissolving or suspending the drug in an oil carrier.

Injectable depot forms can be prepared by forming a microencapsulated matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of release of the drug can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

The compositions for use according to embodiments of the present invention may be provided in solid dosage forms for oral administration, such as, but not limited to, capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one of the following: excipients or carriers, for example sodium citrate or dicalcium phosphate, and/or a) fillers or extenders, for example starches, lactose, sucrose, glucose, mannitol and silicic acid, b) binders, for example carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, c) humectants, for example glycerol, d) disintegrants, for example agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) dissolution retarders, for example paraffin, f) absorption promoters, for example quaternary ammonium compounds, g) wetting agents, for example acetyl alcohol and glycerol monostearate, h) absorbents, for example kaolin and bentonite, and i) lubricants, for example talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and capsids, e.g., enteric coatings or other coatings such as extended release, sustained release, delayed release and immediate release coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) in a delayed manner only, or preferentially, in a certain part of the intestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. The active compounds can also be in microencapsulated form, if appropriate with one or more of the abovementioned excipients.

Liquid dosage forms for oral administration may include, but are not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. In addition to inert diluents, these oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

One of ordinary skill in the art will appreciate that effective amounts of the agents in the compositions used in the methods of the present invention can be readily determined empirically. It will be appreciated that when administered to a human patient, the total daily amount of a composition according to the invention can be determined by the attending physician within the scope of sound medical judgment. The therapeutically effective dose level for a particular patient may depend upon a variety of factors, such as the type and extent of the response to be achieved, the activity of the particular composition employed, the age, weight, general health, sex, and diet of the patient, the duration of the treatment, the drug used in combination or concomitantly with the use of embodiments of the invention, and/or other factors known in the medical arts. A pharmaceutically or therapeutically effective amount refers to an amount effective to elicit a clinically significant cellular response. For example, in uses according to embodiments of the invention, the amount of the compound in the composition (e.g., for topical administration to a subject) may range from about 0.005% to about 1% by weight.

The composition may be provided by dissolving the compound as the Active Pharmaceutical Ingredient (API) in an ointment or similar product for topical application, such that the API concentration reaches 0.005% to 1% (by weight). Such compositions may have radioprotective and therapeutic properties. The composition may advantageously partially penetrate the skin to deposit the API therein. The composition can improve the radiation resistance of the skin when applied to the skin prior to irradiation, and can prevent or minimize erythema, wet and dry scaling that can lead to radiodermatitis. In the case of radiodermatitis, the composition can reduce or eliminate edema, hyperemia, itching, pain and burning irritation of the skin, accelerate healing and promote rapid normalization of tissue and cellular structures.

In a second aspect, the present invention relates to a composition comprising a compound, wherein the compound comprises or consists of a conjugate of 6-hydroxytyrosol and 6-methylsulfinylhexylisothiocyanate. For details of the composition according to embodiments of the second aspect of the invention, reference is made to the above description relating to embodiments of the first aspect of the invention, which is not necessarily limited to the medical use described in relation to embodiments of the first aspect of the invention.

In a third aspect, the present invention relates to the use of a composition according to an embodiment of the second aspect of the invention as a medicament.

In a fourth aspect, the present invention relates to a method of synthesizing a composition according to an embodiment of the second aspect of the present invention. The method comprises providing a compound and may comprise adding a further product to the compound to form a composition, for example a further product as described in relation to embodiments of the first aspect of the invention. The process may comprise adding a solution of N, N' -thiocarbonyldiimidazole (e.g. 0.26 mmol, 46.8mg) in THF (e.g. 2mL) to a solution of 2- (6-hydroxy-5-methoxy-1H-indol-3-yl) -acetamide (e.g. 0.26 mmol, 50mg) in dry tetrahydrofuran (e.g. 3 mL). For example, the mixed solution may be maintained at 0 ℃ for 10 minutes. The resultant may be allowed to warm to room temperature and may be stirred, for example for 3 hours, until completion. Thereafter, the solvent can be removed under reduced pressure and purified by flash chromatography on silica gel (e.g., using hexane: CH)₂Cl₂0-60%) to give the product as a pale yellow oil (e.g., 54.4mg, 90% yield).

The following theoretical considerations are provided to assist the skilled person in understanding the various aspects of the invention and to simplify the practice of the invention. However, the present invention and embodiments of the present specification should not be construed as limited by the accuracy and/or completeness of this theoretical framework.

Upon exposure to ionizing radiation, free radicals may form in the exposed cells, thereby initiating subsequent damage to DNA and organelles. Such damage can cause a variety of reactions by the immune system. A few minutes after exposure may initiate an immune response to radiotherapy-induced oxidative stress (but not necessarily limited to radiotherapy-induced oxidative stress, even ionizing radiation-induced oxidative stress), and this immune response may even last years after irradiation. These effects may depend on the radiation dose and the organ being irradiated. Furthermore, different immune cells may respond differently to ionizing radiation. The cells and molecules of the immune system are divided into two parts, the innate immune system and the acquired immune system. Most immune responses are mediated by soluble molecules, including cytokines and chemokines. The innate and acquired immune systems respond differently to DNA damage and cell death caused by ionizing radiation. Several types of cell death can occur following exposure to radiation and can stimulate different pathways in the immune system. Mechanisms of cell death that occur following irradiation include mitotic dysfunction, necrosis, apoptosis, autophagy, and senescence. The response of immune cells to these cell death pathways results in the production of cytokines that stimulate various signaling pathways in normal tissues. Immunogenic cell death pathways include necrosis and necrotic death (necroprotic). In contrast, apoptotic death induced by cellular oxidative stress and oxidative DNA damage is anti-immunogenic. The balance between these pathways determines the cytokine profile secreted by immune cells, as well as the immunogenicity or tolerability of radiation. Apoptotic cells, in conjunction with macrophages, stimulate macrophages to synthesize and release tolerogenic cytokines such as TGF- β, IL-10, platelet activating factor and PGE2, thereby inhibiting inflammatory responses. Following cell death, secretion of the damage-associated molecular pattern (DAMP), such as high mobility group box 1 protein (HMGB1) and oxidized DNA, results in the production of inflammatory cytokines such as IL-1, IL-2, IL-6, TNF- α and IFN- γ. Both the immunogenic and tolerogenic responses of immune system cells to ionizing radiation involve several early and late effects associated with radiation therapy.

Macrophages and T lymphocytes are important for the release of cytokines and chemokines in response to immune challenges. The response of lymphocytes to ionizing radiation can be mediated by subgroups of T helper 1(Th1) and T helper 2(Th 2). Secreted cytokines with these subgroups have different effects on cells. Long-term follow-up data from nuclear disaster survivors (e.g. in chernobiles and japan) and patients receiving cancer radiotherapy show that the balance between the Th1/Th2 cytokine profiles changes. Exposure to ionizing radiation was associated with a decrease in Th1 and an increase in Th 2. These results indicate that ionizing radiation inhibits cell-mediated immunity and stimulates humoral immunity. The Th1 cytokine is involved in inflammatory reactions including the activation of macrophages and T cells, while the Th2 cytokine stimulates humoral and allergic reactions of the immune system. This imbalance between Th1 and Th2 cytokine production is associated with long-term side effects following radiation exposure.

The response of macrophages and T cells to high doses of radiation, such as that seen in radiotherapy, results in changes in the cytokine profile of irradiated tissue, including blood as well as non-irradiated tissue. Key molecules involved in radiation-induced immune responses and non-irradiated tissue damage include transcription factors (e.g., NF-. kappa.B), protein kinases (e.g., MAPK), cytokines (e.g., IL-1. beta., IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, IL-33, and IFN-. gamma.), TNF-. alpha.and growth factors (e.g., TGF-. beta., bFGF, IGF-1, and PDGF). Inflammatory cytokines and growth factors (e.g., TGF- β and IGF-1) stimulate production of prostaglandins, ROS and NO by macrophages, T cells, neutrophils and non-immune cells. These immune responses lead to inflammation, redness, pain, and also to oxidation of DNA, lipids, and proteins, as well as increased risk of carcinogenic and non-cancerous diseases (e.g., heart disease). In addition, long-term upregulation of inflammatory cytokines and growth factors (e.g., TGF- β, bFGF and IGF-1) upon exposure to high doses of radiation can lead to remodeling of the extracellular matrix (ECM), resulting in serious effects such as atrophy, vascular damage and fibrosis that can affect normal functioning of tissues.

Modulation of the immune response in radiation therapy is an interesting goal to increase the effectiveness of the tumor response and to manage the side effects of normal tissues. For these goals, it seems most important to manage the immune response to radiation therapy. The interesting properties of melatonin in tumor and normal tissues can help to achieve proper management of normal tissues and cancer response to radiation therapy.

Compositions according to embodiments of the invention, for example when topically applied to skin and mucosal and/or surrounding areas exposed to ionizing radiation, may be considered to be Nrf2 phase II enzyme inducers. This may significantly improve the mechanical elasticity of the skin and mucosa and may prevent or reduce skin and mucosal damage in mammals, especially humans exposed to heat and/or ionizing radiation, for example in radiotherapy, hyperthermia, space environments or nuclear accidents. In particular, topical administration of pharmaceutically effective amounts of the pharmaceutical compounds before, during or after exposure to radiation can provide effective immunomodulatory protection against short-term and long-term damage to the skin and mucosa.

The compounds of the compositions according to embodiments of the invention may advantageously induce the transcription factor NF-E2-related factor 2(Nrf2) to prevent or treat damage to or diseases of the skin or mucosa caused by oxidative stress, e.g. due to radiotherapy or other radiation exposure and/or due to hyperthermia. According to the pleiotropic profile of sulforaphane and melatonin, complementary cytoprotective effects can be achieved by combining these compounds (or their analogs) in a single molecule. The compound according to the embodiments may react with cysteine present in Keap1 to release Nrf2, Nrf2 then acting as a drug to couple with intracellular GSH to produce an effective melatonin-based antioxidant compound, i.e. a prodrug of the conjugate. This drug-prodrug mechanism may lead to a good pharmacological profile in therapeutic applications, e.g. as an adjuvant to radiotherapy.

The transcription factor NF-E2 related factor 2(Nrf2) belongs to the CNC (Cap-N-Collar) transcription factor family and has a highly conserved basic region-leucine zipper (bZip) structure. Nrf2 plays a key role in the constitutive and inducible expression of antioxidant and detoxifying genes (commonly referred to as phase II genes) that encode defense enzymes, including drug metabolizing enzymes such as glutathione S-transferase, nadp (h): quinone oxidoreductase and UDP-glucuronic acid glycosyltransferase, as well as antioxidant enzymes such as heme oxygenase-1 (HO-1) and glutamylcysteine synthetase (GCS), in response to oxidative and exogenous stress. These enzymes ARE regulated by promoters known as Antioxidant Response Elements (ARE) or electrophilic response elements (EpRE). Phase II genes are responsible for cellular defense mechanisms including the scavenging of reactive oxygen or nitrogen species (ROS or RNS), the detoxification of electrophiles and the maintenance of intracellular reduction.

Nrf2 actin-binding regulatory protein, commonly referred to as Keap1, sequesters in the cytoplasm of the cell. When cells are exposed to oxidative or electrophilic stress, the Keap1-Nrf2 complex undergoes a conformational change, and Nrf2 is released from the complex and into the nucleus. Active Nrf2 dimerizes with small Maf proteins, binds to ARE and activates phase II gene transcription.

The Keap1-Nrf2 system has antioxidant and detoxifying functions and is involved in the steady state regulation. Compounds that activate this system are considered therapeutic agents for a variety of diseases.

Nine classes of phase II enzyme inducers are known: 1) bisphenols, phenylenediamine and quinones; 2) michael (Michael) acceptors; 3) an isothiocyanate; 4) hydroperoxides and hydrogen peroxide; 5)1, 2-dithiol-3-thiones (1, 2-dithiole-3-thiones); 6) dithiols; 7) trivalent arsenic; 8) a divalent heavy metal; and 9) carotenoids, curcumin and related polyenes. These phase II enzyme inducers are considered to be very effective antioxidants because, unlike direct antioxidants, they are not consumed in stoichiometric amounts during the redox reaction, have a longer duration of action, support the function of direct antioxidants (such as tocopherol and coenzyme Q), and enhance the synthesis of the strong antioxidants glutathione.

Ethacrynic Acid (EA) diuretic, electrophilic michael receptors, oltipraz (oltipraz) and the isothiocyanate sulforaphane have been shown to inhibit Lipopolysaccharide (LPS) -induced secretion of high mobility group protein 1(HMGB1), HMGB1 is a pro-inflammatory protein from immune-stimulated macrophages that is involved in the pathogenesis of inflammatory diseases. Oltipraz prevents liver and bladder carcinogenesis by enhancing the detoxification of carcinogens. The cytoprotective effect of Keratinocyte Growth Factor (KGF) on oxidative stress in wounded and inflamed tissues, including wounded skin, has been associated with KGF stimulating Nrf2 during skin wound repair.

Isothiocyanates are mainly derived from cruciferous vegetables, are potent antioxidants, and are effective agents in chemoprevention of tumors through activation of phase II enzymes, inhibition of carcinogen-activated phase I enzymes, and induction of apoptosis. In plants, isothiocyanates are formed by hydrolysis of glucosinolates, which are β -glucosinolate-N-hydroxy sulfates, which when vegetables are macerated by predators, food preparation or chewing destroys cells, activating and releasing myrosinase. The resulting aglycones undergo non-enzymatic intramolecular rearrangement to give isothiocyanates, nitriles and epithionitriles.

Sulforaphane, a potent phase II enzyme inducer in isolated murine hepatoma cells, has been identified in broccoli. Sulforaphane prevented the formation of mammary tumors in SD (Sprague-Dawley) rats, promoted skin tumorigenesis in mice, and increased heme oxygenase-1 (HO-1) expression in human liver cancer HepG2 cells. Sulforaphane also inhibits activation of the Ultraviolet (UV) light-induced activator protein-1 (AP-1), a promoter of skin carcinogenesis, in human keratinocytes. Topical application of sulforaphane extract may increase the phase II enzyme nad (p) H: quinone oxidoreductase 1(NQO1), glutathione S-transferase A1 and heme oxygenase 1 levels.

In addition, sulforaphane can protect human epidermal keratinocytes from sulfur mustard, potent cytotoxic agents as well as powerful mutagens and carcinogens, and inhibit cell growth, activate apoptosis, inhibit Histone Deacetylase (HDAC) activity and reduce the expression of estrogen receptor- α, epidermal growth factor receptor and human epidermal growth factor receptor-2, which are key proteins involved in breast cancer proliferation in human breast cancer cells. In addition, sulforaphane has been shown to eradicate H.pylori from human gastric xenografts.

In addition to the inhibitory effect of sulforaphane on repair of radiation-induced DNA DSB through damage to NHEJ and HRR pathways, sulforaphane significantly enhanced the radiosensitivity of human tumor cells in vitro and in vivo. This repair inhibition appears to be due, at least in part, to the enhanced apoptosis induced by the combination therapy.

Melatonin is primarily secreted by the pineal gland, but may also be synthesized, for example, by the bone marrow, immune cells, brain, and intestinal tract. Melatonin has two important metabolites, N1-acetyl-N2-formyl-5-methoxy kynurenine (AFMK) and N1-acetyl-5-methoxy kynurenine (AMK). Among them, AFMK is the most abundant.

Melatonin and its metabolites have potent antioxidant and radioprotective properties. It reduces ROS/NO production by different oxidation factors, including ionizing radiation. It is also consumed very rapidly during oxidative stress. This suggests that melatonin may be effective as a first line protective factor against increased ROS/NO production.

Melatonin acts as a powerful ROS/NO scavenger, interacting with free radicals and oxidative DNA damage. It can protect cells from hydroxyl radicals (the most common type of radical after irradiation), hydrogen peroxide, nitric oxide (a product of immune cells), peroxynitrite anion and singlet oxygen. Moreover, melatonin ameliorates different levels of oxidative stress, such as modulation of molecular pathways and cellular functions, as will be explained in detail below. It appears that the mechanism by which melatonin scavenges free radicals is different from most other mechanisms. Antioxidants such as vitamins C and E stimulate the redox system and may also promote the production of ROS. Some studies have suggested that melatonin interacts with ROS and RNS without stimulating the redox system. Furthermore, melatonin converts free radicals into stable products, such as N-acetyl-5-methoxykynuramine (kynuramine), N (1) -acetyl-N (2) -formyl-5-methoxykynuramine, and 6-hydroxytemetonin, which are substantially unreactive with other molecules. However, in some cases, melatonin may stimulate mitochondrial ROS production through oxidative phosphorylation.

Another important mechanism of antioxidant action of melatonin is the stimulatory genes and enzymes, such as Nrf2, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and Glutathione Reductase (GR), all of which contribute to detoxification. Since the inhibitory effect of antioxidant enzymes is an important role of ionizing radiation in irradiated and extraterrestrial organs, this property of melatonin may enhance the clearance of free radicals produced by radiation.

Immune cells (e.g., macrophages, T cells, and neutrophils) and subcellular organelles (e.g., mitochondria, endoplasmic reticulum, cell membranes, and lysosomes) play an important role in the early and late response of cells and tissues to radiation. Mitochondrial and cell membrane production of ROS and RNS and release of lysosomal lytic enzymes play an important role in radiation injury. In addition, immune cells, including macrophages, T cells and neutrophils, produce ROS and NO in response to stress conditions. A significant feature of melatonin, compared to other antioxidants and radioprotectors, is its ability to enter most organs and their subcellular organelles. Melatonin can affect different immune cells and organelles and mitigate functional changes due to ionizing radiation in these cells and organelles. These characteristics suggest that melatonin is a good candidate for protecting normal tissues during radiotoxicity in different tissues. Protection of the mitochondrial membrane, restoration of mitochondrial respiratory frequency and membrane potential against ROS/NO are unique properties of melatonin, while other antioxidants do not.

Mitochondria are the major source of ROS production in cells, and they play a key role in oxidative damage after irradiation. Maintenance of mitochondrial integrity may be important for oxidative damage and ROS production during melatonin mitigation of oxidative stress (e.g., exposure to ionizing radiation). Furthermore, there is some evidence for reduced oxidative damage and functional impairment of cell membranes and lysosomes.

Melatonin can regulate proliferation and cytokine secretion via receptors on immune cells. Administration of melatonin can improve survival and increase the number of precursor B and NK cells in the bone marrow. Ionizing radiation has a potent effect on immune cells (e.g., T and B lymphocytes). Among immune cells, these cells are most sensitive to radiation. Radiation-induced lymphopenia in the bone marrow is an important side effect of radiation therapy, which may limit the radiation dose received by a tumor. Melatonin treatment can significantly improve DNA damage and reduce the number of peripheral and bone marrow lymphocytes after irradiation. Reduce DNA damage and cell death (especially in radiosensitive cells), making melatonin a suitable radioprotectant and immunomodulator for managing the radiation immune response.

Cytokines are key mediators of normal tissue response to ionizing radiation. Exposure to ionizing radiation upregulates a variety of cytokines, including inflammatory and anti-inflammatory cytokines. Studies have shown that melatonin has mitogenic and anti-inflammatory effects, depending on the circumstances. Melatonin can up-regulate the production of IL-2, IL-12 and IFN- γ. In addition, melatonin induces increased monocyte responses to granulocyte-macrophage colony stimulating factor (GM-CSF), IL-3, IL-4, and IL-6. These effects result in increased NK cell activity and increased production of granulocytes, macrophages, neutrophils and erythrocytes following melatonin treatment. On the other hand, there is evidence that treatment with melatonin can increase IL-10 production and activate the anti-inflammatory Th2 immune response.

In response to inflammation, melatonin acts as a potent anti-inflammatory compound and reduces the over-expression of inflammation-associated Th1 cytokines (e.g., TNF- α, IL-1 β, and IFN- γ) and promotes a Th2 response. Melatonin suppresses the release of two inflammatory cytokines TNF- α and IL-8 secreted by neutrophils. These cytokines are particularly important in chronic inflammation. Therefore, melatonin can reduce acute and chronic consequences of inflammation following radiation therapy by these routes. A recent study has shown that administration of melatonin prior to pulmonary irradiation can improve upregulation of TNF- α, TGF- β and IL-6. Moreover, it showed increased SOD and catalase activity and GSH levels, and reduced oxidative damage in lung tissue compared to irradiation alone. TGF-beta has inhibitory effect on SOD and catalase gene expression. Thus, a decrease in TGF- β levels upon exposure to ionizing radiation may help mitigate oxidative damage.

Certain transcription factors (e.g., NF-. kappa.B, AP-1, c-jun, c-fos, and STAT families) and protein kinases (e.g., MAPK) play a key role in controlling the response of cells to ionizing radiation. NF-. kappa.B stimulates transcription of DNA, cell cycle progression, response to DNA damage, cytokine production (particularly inflammatory cytokines), cell growth and differentiation, and cell survival. NF-. kappa.B is present in several types of cells. Abnormal up-regulation is associated with many malignancies, such as ovarian cancer, colon cancer, leukemia and lymphoma. Upon irradiation, NF-. kappa.B stimulates the expression of inflammatory cytokines, such as IL-1. alpha., IL-1. beta., TNF-. alpha., and IL-6 mRNA.

It appears that there is crosstalk between NF-. kappa.B, AP-1 and MAPK signaling pathways. Thus, inhibition of one of these transcription factors with a selective inhibitor is not sufficient to manage the up-regulation of these cytokines upon exposure to radiation. Melatonin has inhibitory effects on NF-. kappa.B gene expression under stress conditions such as radiation. Modulation of functional retinoid-related orphan receptor-alpha (ROR-alpha) transcription factors is involved in this effect. Inhibition of this signaling pathway can reduce the production of ROS and its consequences, such as mucositis following irradiation. Furthermore, melatonin may down-regulate increases in MAPK, including oxidative stress-induced p38 and JNK. One report shows that the inhibition of c-jun, c-fos and STAT upregulation by melatonin may improve inflammatory responses. Thus, the regulatory effects of melatonin on transcription factors may constitute good evidence for the immunomodulatory properties of melatonin on radiation oncology.

It is well recognized that cyclooxygenase 2(COX-2) is one of the most important factors involved in radiation-induced inflammation. Melatonin is capable of inhibiting COX-2 production. Melatonin and its metabolites, AFMK and AMK, have previously been studied for their anti-inflammatory effects by preventing COX-2 and iNOS activation and reducing their products, including PGE2 and nitric oxide. Melatonin was found to have no effect on COX-1, but only selectively inhibits COX-2. This suggests that melatonin and its metabolites act as anti-inflammatory agents without certain side effects due to COX-1 inhibition, such as gastrointestinal disorders. This anti-inflammatory effect of melatonin is not due to its antioxidant effect, but is involved in other pathways.

The production of NO by macrophages is a bactericidal property of the immune system and is involved in inflammation and oxidative damage caused by ionizing radiation. Melatonin can inhibit the expression of iNOS in macrophages and reduce the production of NO and its consequences. This effect may be mediated by inhibition of STAT-1 signaling and by inhibition of NF-. kappa.B signaling by inhibition of nuclear translocation and DNA binding activity of the NF-. kappa. B p50 subunit. Overproduction of PGE2 and NO plays a key role in the development and persistence of inflammation and also causes its symptoms including vasodilation, pain, fever and edema. Abnormal increases in NO production can lead to oxidative stress on the nitro group, resulting in DNA damage, lipid peroxidation and protein oxidation. This damage induces several transcription factors that lead to chronic inflammation, such as NF-. kappa.B and MAPK.

COX-2, iNOS and other enzymes (such as NADPH oxidase) are important in the redox pathway. These enzymes also have other effects related to inflammation and redox systems, such as mitochondria following exposure to radiation, which amplify oxidative damage caused thereby. Both the antioxidant and anti-inflammatory effects of melatonin inhibit COX-2, iNOS, and NADPH oxidase. Melatonin scavenges ROS and NO, and attenuates the formation of peroxides and peroxynitrites, and decreases the expression of transcription factors associated with chronic inflammation.

Epigenetic regulation suppresses the effects of melatonin on COX-2 and iNOS transcriptional activation by inhibiting histone acetyltransferase activity. Furthermore, melatonin down-regulates the activation of NF-. kappa.B and MAPK, such as JNK, ERK and p38, which are activated by oxidative stress. These effects of melatonin inhibit inflammatory mediators such as TNF- α and IL-1 β, as well as COX-2, PGE2 and iNOS. These suppressive effects on the redox system enhance activity after irradiation and are one of the ways in which melatonin may exert a protective effect against toxicity caused by ionizing radiation.

Chronic changes in the immune response to ionizing radiation are important examples of delayed effects of radiation therapy. Responses such as chronic upregulation of inflammatory cytokines, chemokines, growth factors, adhesion molecules, and immune cell infiltration can lead to pathological changes in the irradiated tissue. Pathological damage caused by ionizing radiation is manifested as irreversible changes in tissue structure, resulting in impaired normal function. Such damage may occur months to years after exposure.

Long-term upregulation of NF-. kappa.B, inflammatory cytokines and chemokines, adhesion molecules (such as VCAM and ICAM-1), immune cell infiltration, etc., are associated with different pathological lesions. Skin, lung, heart, brain, liver, intestine, kidney, spleen and colon are the most important tissues affected by irreversible pathological changes. The most important pathological changes caused by ionizing radiation include pneumonia, fibrosis, necrosis, vasodilation and obstruction, and edema. This damage is followed by early reactions such as cell death and acute inflammation. These tissue changes lead to diseases such as altered barrier function of the skin, altered respiratory function, heart disease, gastrointestinal disorders, and the like. Thus, management of the immune system's response to radiation may reduce the risk of pathological changes and their consequences following radiation therapy.

Melatonin can improve pathological changes caused by ionizing radiation. Local and/or oral administration of melatonin can prevent or alleviate chronic inflammation and oxidative damage, fibrosis, necrosis, thrombosis, vascular damage, and an increase in the number of immune cells in various tissues (e.g., heart, lung, parotid and submandibular glands, kidney, spinal cord, lens, urogenital system, etc.). The use of melatonin to prevent pathological damage caused by ionizing radiation at different times before and after exposure is a promising outcome.

In addition to protecting normal tissues from ionizing radiation, several studies have reported that melatonin has an inhibitory effect on tumor growth. The possible synergistic effects of melatonin administration and radiation therapy or chemotherapy can improve survival and improve early and late side effects in cancer patients.

The anticancer effects of melatonin are produced by inhibiting the proliferation and growth of tumor cells. This property may be associated with the inhibition of the tumor cell cycle. Stimulating cell death and inhibiting tumor cell proliferation reduces the likelihood of recurrence and enhances treatment. The antiproliferative effects of melatonin may be associated with negative regulation of NF-. kappa.B. This factor has a proliferative effect by acting directly on cyclin D1. Furthermore, melatonin induces apoptosis by activating caspase-dependent apoptotic pathways, enhancing tumor necrosis factor, down-regulating Bcl-2 and survival (by inhibiting nuclear translocation of NF- κ B p 65). For example, Ramos cells (human burkitt lymphoma B cells) are very sensitive to melatonin, which is caused by dose-dependent cell cycle arrest and apoptosis in G1 phase. On the other hand, melatonin in MCF-7 cells induces a delay in cell cycle progression, which is largely mediated by the involvement of the TGF- β pathway. In addition to apoptosis, melatonin can induce other cell death pathways in cancer cells, including autophagy and aging.

Angiogenesis plays a key role in tumor growth and metastasis. Inhibition of angiogenesis is a promising approach to improve cancer response to radiation therapy. Inflammation in tumor cells in response to radiation therapy stimulates the up-regulation of different genes that cause angiogenesis and tumor growth. Melatonin may reduce angiogenesis by scavenging ROS production and inhibiting HIF-1 α, sphingosine kinase 1, COX-2, and Vascular Endothelial Growth Factor (VEGF). Furthermore, melatonin reduces the effects of growth factors on tumor cells by inhibiting insulin-like growth factor 1(IGF-1), Epidermal Growth Factor Receptor (EGFR) and endothelin 1(ET-1), which are powerful stimulators of angiogenesis in cancer cells, suggesting that melatonin inhibits angiogenesis in gastric cancer cells and tumor-bearing nude mouse models. The results indicate that the main mechanism of antiangiogenesis in gastric cancer cells is to decrease the expression of HIF-1 alpha, VEGF and the nuclear receptor RZR/ROR gamma. Using cell and mouse models, the effects of melatonin on angiogenesis and tumor size in breast cancer are promising. Tumor size determination using SPECT imaging showed that melatonin treatment reduced vascular growth and size of the implanted human breast cancer in the mouse model. Furthermore, in vitro studies, the use of melatonin can reduce the viability of breast cancer cells. In a clinical study involving 20 metastatic patients, administration of melatonin resulted in a significant decrease in VEGF plasma levels, whereas no effect was seen in patients in progress.

Natural Killer (NK) cells play an important role in inhibiting tumor growth and metastasis. NK cells can kill a variety of tumor cells, especially those derived from lymphomas and leukemias. Studies have demonstrated that melatonin has a positive effect on NK cell activity. The effect of melatonin on mouse immune cell populations has been evaluated, indicating that NK cell populations continue to rise in spleen and bone marrow for two weeks. These results indicate that melatonin enhances the anti-tumor function of NK cells.

Although the exact mechanism of the stimulating effect of melatonin on NK cells has not been fully established, it has been proposed to increase IL-2 production by stimulating T cell melatonin receptors.

Hereinafter, examples and experimental results are provided to help the skilled person understand the present invention and to simplify it to practice. However, these examples and experimental results are merely illustrative and not intended to limit the present invention.

In these examples, compound 6-HITC-60HM was synthesized as described above in relation to an embodiment of the fourth aspect of the invention. Rf 0.87 (dichloromethane, 100%); 1H NMR and 13C NMR data consistent with previously reported findings; rf 0.87(DCM, 100%); 1H NMR (300MHz, CDCl3) δ H7.91 (1H, bs, NH),7.20(1H, d, J ═ 8.6Hz, H7),7.01(1H, d, J ═ 2.4Hz, H4),6.91(1H, d, J ═ 2.4Hz, H2),6.81(1H, dd, J ═ 2.4Hz, J ═ 8.6Hz, H6),3.81(1H, s, OCH3),3.69(2H, t, J ═ 6.8Hz, OCH2CH2NCS),3.06(2H, t, J ═ 6.8Hz, OCH2CH2 NCS); 13C NMR (75MHz, CDCl3) delta C154.2,131.4,127.3,123.8,112.5,112.2,110.9, 56.1,45.7, 26.5; HRMS (ES +) mass calculation. For C12H12N2SO 232.0670; found [ (M + H) + ]233.0740, found [ (M + Na) + ] 255.0567; and (5) analyzing and calculating. For C12H12N2SO: C, 62.04; h, 5.21; n,12.06, S, 13.80. Found C, 62.26; h, 5.38; n, 11.88; s, 13.56.

Formulations according to embodiments of the invention were studied experimentally in vivo in animal models by the Association Research & Development, Belgium, Research center for Addison Research & Development, Belgium, Belgi. The results of these tests are described below.

Example 1: dermatitis treatment and prevention

Male Gentiana minipigs (average body weight 19 kg; range 18-20 kg; age 6-7 months) obtained from the institute for animal breeding and genetics, university of Gentiana, Germany, were used in these experiments. Minipigs were provided with tap water and commercial laboratory piglet feed (Purina laboratory pig feed 5085) from Purina, germany, containing crude protein, fat, fiber and ash, as well as calcium, phosphorus and moisture (14.5, 4, 5, 8, 0.55, 1 and 14% respectively). In addition, no antibiotic supplements were used. All animal experiments were performed according to the German animal welfare method.

A formulation suitable for topical administration according to an embodiment of the present invention was obtained by adding 200mg of carbomer (carbomer 934P; Lubrizol) to 2.5mL of distilled water and dissolving 200mg of 6OHM-6-HITC in 2mL of ethanol. An appropriate amount of the ethanol dispersion was transferred to the aqueous dispersion of carbomer. Methanol (1.25mL) was mixed with 1mL ethanol and added to a mixture of 6OHM-6-HITC and carbomer, which was stirred slowly and the carbomer was allowed to soak for 2 hours. Adding triethanolamine (100 mg; Sigma, USA)Aldrich (Sigma-Aldrich, USA)) to neutralize the carbomer solution and promote gel formation, and then the pH was adjusted to 6.8. The carrier cream was prepared using the same ingredients and the same method as the cream, but 6OHM-6-HITC was omitted from the mixture. The topical gel formulation produces the highest permeability of 6OHM-6-HITC without causing skin irritation or anti-inflammatory effects. For topical treatment, 6OHM-6-HITC or a carrier cream (concentration 200 mg/cm)²) The pigs were painted twice daily on irradiated skin for 35 days and the first application was made immediately after irradiation.

To observe the effect of gamma radiation on the skin of miniature pigs (3 animals per group), the skin of the back was irradiated. For all procedures, animals were anesthetized with teletamine/zolazepam (Zoletil 50; vibaac, Germany) and medetomidine (Domitol; Pfizer Animal Health Germany). Three to four days prior to irradiation, the animal's hair was cut from the area to be exposed and the location of the exposed area was marked and tattooed with indian ink. Use of⁶⁰The area was gamma irradiated with Co gamma radiation (Theratron 780; AECL canada) at a dose of 50Gy and at a dose rate of 130.1 cGy/min (area size, 5 x 2cm, rectangle; distance of radiation source to skin, 80 cm; depth, 1 cm, and filler (bolus)1 cm). As shown in figure 1, each pig was irradiated to 50Gy depending on the available lateral skin area. Fig. 2 shows the irradiation and puncture site of a sequential biopsy 21. The puncture site is indicated by a triangular mark on fig. 1.

Pigs were evaluated weekly for five weeks after irradiation and scored for their skin response using a clinical status scoring system. The presence and appearance of skin reactions and the characteristics of surgical scars were examined. Responses were assessed according to previous skin lesion models using the following scoring system: grade 1.0, normal skin; grade 1.5, minimal erythema and slight dryness of the skin; grade 2.0, moderate erythema and dry skin; grade 2.5, marked erythema and dry desquamation; grade 3.0, dry peeling and minimal dry encrustation; grade 3.5, dry peeling, dry encrustation and minimal surface incrustation; 4.0 grade, wet desquamation and moderate scab of macula shape; grade 4.5, combined wet desquamation, ulceration and large and deep scab; grade 5.0, open wound and full-thickness skin exfoliation; grade 5.5, necrosis.

A 5mm needle biopsy was performed under anesthesia to obtain skin samples from non-irradiated healthy skin and irradiated skin areas 3, 7, 21 and 35 days after irradiation. After collection, the skin biopsy samples were mounted on cork stoppers, keeping the size 5 mm. A biopsy of non-irradiated skin was obtained from each pig prior to irradiation. All biopsy samples were processed and fixed in 10% buffered formalin, then embedded in paraffin, then cut into 4 μm thick coronal sections and deparaffinized. Next, the sections were stained with hematoxylin and eosin and examined by light microscopy. The longest ridge (rete ridge) on each slide was chosen and measured from the bottom of the basal layer to the bottom of the stratum corneum, avoiding the area where the contents appeared to be tilted. An average is then calculated based on the measurements of each section on each slide. The cell density of the basal layer is determined by counting cells in the basement membrane to a depth of at least 5 mm. Results are expressed as cells per mm of basement membrane. Degenerated cells (i.e., cells exhibiting atrophy and shrinkage necrosis) were excluded from these calculations.

After incubation in normal horse serum for 60 minutes to prevent non-specific binding, the skin sections were incubated overnight at 4 ℃ in phosphate buffered saline-Tween (PBS-T) with mouse antinuclear factor (NF) - κ B (sc-109, 1: 200; Santa Cruz Biotechnology, Santa Cruz, Inc., Santa Cruz, Calif.) and mouse anti-COX-2 (18-7379, 1: 200; Zymed, USA). The sections were then incubated with biotinylated horse anti-mouse IgG (VECTASTAIN Elite ABC Kit; U.S. Vector laboratories). Immunoreactivity was assessed using avidin-biotin peroxidase complex (VECTASTAIN Elite ABC Kit; Vector laboratories). The peroxidase reaction was developed using the diaminobenzidine substrate kit (DAB substrate kit SK-4100; Vector laboratories). As a control, immunohistochemical analysis of some of the test sections in each experiment omitted the primary antibody. The sections were then counterstained with hematoxylin prior to installation.

Blood samples were collected via the auricular vein into sample tubes containing ethylenediaminetetraacetic acid at various time points (3, 7, 21 and 35 days post irradiation before irradiation). Peripheral eosinophils were automatically counted using the Hemavet System (Drew science, UK).

Data are presented as mean ± Standard Error of Mean (SEM) values. Differences between groups were assessed by one-way analysis of variance (ANOVA) followed by multiple comparisons by Student-Newman-Keuls post hoc tests. In all cases, p <0.05 was considered significant.

Total changes in irradiated skin over time were observed in both vehicle-treated and 6 OHM-6-HITC-treated mini-pigs 35 days after radiation exposure. One week after irradiation, the exposed areas of the skin appeared desquamated with bright red erythema. The reaction gradually increased in severity during the first 5 weeks after irradiation, with continued wet desquamation and tissue disruption progressing to the dermis. Clinical changes were similar in piglets receiving vehicle treatment and 6OHM-6-HITC treatment when evaluated 1 week after irradiation. However, the beneficial effect of 6OHM-6-HITC treatment on dermatitis occurred 2 weeks after irradiation. As shown in fig. 3 and 4, the group treated with 6OHM-6-HITC showed reduced severity of skin reactions compared to the irradiation group treated with vehicle.

Hematoxylin and eosin stained sections were examined to assess basal cell density and epithelial depth in pig skin treated with or without 6 OHM-6-HITC. Skin sections collected from each pig before irradiation showed normal morphology. Changes in basal cell density and epithelial layer thickness paralleled the progression of the observed clinical changes, as shown in fig. 5, 6 and 7.

Radiation exposure of the skin gradually reduced the density of basal cells in the epidermis until 5 weeks after radiation. However, the decrease in basal cell counts was significantly improved 21 days and 35 days after irradiation (p <0.01 and p <0.05 relative to vehicle-treated irradiation groups, respectively; see FIG. 6).

Five weeks after irradiation, the epidermal thickness gradually decreased significantly. However, 6OHM-6-HITC treatment may preserve basal cell numbers, preventing this reduction 35 days after irradiation (p <0.01 relative to vehicle treated irradiation group). These results indicate that 6OHM-6-HITC significantly reduced skin damage in irradiated swine skin (see FIG. 7).

In normal pig skin, i.e. without irradiation and treatment, the staining of cyclooxygenase 2(COX-2) was barely visible, with some staining in the sebaceous glands and subcutaneous tissue, but no visible epidermal staining, as shown in FIG. 8. COX-2 expression was detectable in the epidermis of irradiated skin, and evaluation between 1 and 3 weeks after exposure showed COX-2 expression in the stratum granulosum and stratum corneum, as shown in fig. 9 and 10, respectively. Five weeks after irradiation, as shown in fig. 11, plaque-like stained areas were observed in all skin layers. However, COX-2 expression in irradiated skin was lower than in vehicle-treated skin in 6 OHM-6-HITC-treated skin, as shown in fig. 12, fig. 13, and fig. 14.

In normal pig skin, almost no NF-. kappa.B staining was seen, and some staining was seen in the sebaceous glands, hair follicles and epidermis. In addition, as shown in fig. 15, NF- κ B was expressed in the basal level cytoplasmic region of the epidermis, whereas no nuclear staining was detected. As shown in FIGS. 16 and 17, respectively, NF- κ B expression increased in irradiated skin between 1-2 weeks after irradiation. As shown in fig. 18, diffuse cytoplasmic staining was observed in all epidermal layers three weeks after irradiation, while nuclear staining was detected five weeks later. As can be seen in fig. 19, fig. 20 and fig. 21, respectively, NF- κ B expression was lower in irradiated skin treated with 6OHM-6-HITC than in irradiated skin treated with vehicle, and nuclear expression decreased rapidly. 7 days after exposure, focused radiation exposure temporarily reduced the white blood cell count of peripheral blood, including neutrophils and lymphocytes. Analysis of peripheral blood samples of the 6 OHM-6-HITC-treated group did not initially show the radioprotective effect of 6 OHM-6-HITC. However, 21 and 35 days after irradiation, skin inflammation caused by focused radiation increased the number of neutrophils in the peripheral blood. In contrast, neutrophiles and eosinophiles were reduced in the blood of the 6 OHM-6-HITC-treated group, but the reduction was not significant, indicating that 6OHM-6-HITC reduced radiation-induced skin inflammation, as shown in fig. 22 to 27. Healing of the biopsy wound progressed over time, with stable healing of the skin wound observed prior to irradiation. Biopsy wounds in normal skin healed within 6 to 12 days, but 3 days after irradiation, biopsy lesions did not appear to heal. Exposure of the skin to radiation can significantly delay wound healing as observed at various time points after irradiation. However, as shown in fig. 28 and 29, 6OHM-6-HITC treatment attenuated delayed healing of biopsy wounds induced by radiation 3 days after irradiation.

Example 2: treatment and prevention of mucositis

In a second example illustrating an embodiment of the invention, six week old ICR mice (30-40g) obtained from the institute for animal breeding and genetics, university of Gonggen, Germany, were used. Animals were housed in a room maintained at 22 ± 2 ℃ in a 12 hour light/dark cycle with lights on at 7:00 a.m. Mice were fed a standard rodent diet and were allowed free access to water. In addition, no antibiotic supplements were used. All animal experiments were performed according to the German animal welfare method. Mice (n ═ 20 per group) were anesthetized (sodium pentobarbital, 50mg/kg body weight, injected intraperitoneally) and then irradiated. The mouse only needs to be irradiated at the tip of the tongue, so the rest of the body is shielded by a lead device (0.5 mm thick). The tongue was fixed on the outer surface of the lead device with tape and irradiated with a single radiation dose of 20 Gy. Radiation was generated at a focal length of 350mm using a 150kV potential (20mA) X-ray source and the beam was hardened by a 1.0mm aluminum filter system (MBR-1520R-3; Hitachi, Tokyo, Japan). The dose rate was 5.1 Gy/min. The 6OHM-6-HITC compound was dissolved in a small amount of 1M NaOH solution. The pH was adjusted to 7 with 1M HCl. The concentration was adjusted to 3mg/mL or 15mg/mL in 0.9% (physiological) saline solution. Body weight was recorded and the tongue was observed daily after irradiation. 6OHM-6-HITC was injected 30 minutes prior to irradiation by the intraperitoneal route. The control group received irradiation, but no 6OHM-6-HITC compound was applied.

Oral mucositis scoring is based on an improvement of the Sonis et al method. To assess the severity of oral mucositis, mice were anesthetized with isoflurane daily. Oral mucositis scores were: 0 is normal; 1 ═ partial congestion, erythema and swelling; 2 ═ global congestion, erythema and swelling; 3 ═ epidermolysis, congestion and erythema; extensive epidermal detachment and bleeding; 5-bleeding and abscess. Myeloperoxidase (MPO) activity is a marker of neutrophils in inflamed tissue. MPO activity was measured in the mouse tongue using a modification of the Chen et al method. After killing the mice by cervical dislocation 12 days after irradiation, tongue samples (n ═ 10 per group) were removed and stored at-70 ℃ until needed for analysis. The sample was weighed and homogenized for 1 minute in 10 volumes of 50mM potassium phosphate buffer (pH 6) containing 0.5% cetyltrimethylammonium bromide (sigma-aldrich, germany). After freezing and thawing the homogenate three times, it was centrifuged at 10,000 Xg for 15 minutes at 4 ℃. The supernatant was collected and reacted with 0.167mg/mL O-dianisidine dihydrochloride (Wako biochemicals) and 0.0005% H2O2(Wako biochemicals) in 50mM phosphate buffer (pH 6). MPO activity was measured at 450nm using a Microplate Reader (NJ-2300; Tokyo Biotec, Japan). MPO activity was calculated by measuring the slope of absorbance calibrated using MPO standards (Wako Biochemical Co., Ltd.) and expressed as MPO/g tongue. TBARS occurs naturally in biological samples. They include lipid hydroperoxides and aldehydes, which increase in concentration in response to oxidative stress. After killing the mice by cervical dislocation 12 days after irradiation, tongue samples (n ═ 10 per group) were removed and stored at-70 ℃ until needed for analysis. Samples were weighed and homogenized in 0.5-1 mL Phosphate Buffered Saline (PBS) per 100mg of tongue. The samples were centrifuged at 1500 Xg for 10 min at 4 ℃. The supernatant was collected and diluted in assay buffer. TBARS levels were measured using a microplate reader (Biotec) at 540 nm. TBARS levels were calculated by measuring the absorbance slope calibrated using TBARS standards and expressed as TBARS/g tongue. In addition, after killing the mice 12 days after irradiation, the tongue was removed for histopathological analysis. The samples were fixed in 10% neutral buffered formalin, dehydrated and embedded in paraffin (Wako biochemicals). Tissue sections were obtained and stained with hematoxylin and eosin (H & E) and examined under an optical microscope (magnification 200). After killing the mice 12 days after irradiation, the tongue was removed for Immunohistochemical (IHC) analysis. Paraffin-embedded tissue sections were used for TUNEL staining (400 x magnification). Dewaxing, hydration and protein digestion are performed. Subsequently, the 3-terminal of the DNA was labeled with 100. mu.L (or 50. mu.L) of TdT reaction solution at 37 ℃ for 10 minutes. Sections were washed with PBS and labeled with POD-conjugated antibody. The color was developed with 100. mu.L of 3,3' -diaminobenzidine solution for 5 minutes at room temperature, then washed with double distilled water and counterstained. After these operations, dehydration, cleaning, mounting and inspection were performed under an optical microscope. The number of TUNEL positive cells on the tip of the tongue was counted and expressed as a percentage. Results are reported as the mean of the means and standard error or as the mean. Data were analyzed using one-way analysis of variance (ANOVA) followed by a Steel-Dwass test. P <0.05 was considered significant. Body weight after irradiation was measured daily.

The 20Gy irradiation of the mouse tongue resulted in weight loss, which reached a maximum on day 12. After day 12, body weight increased as shown in figure 30. Food and water intake is also reduced, with concomitant weight loss. FIG. 31 shows the dose dependence of 6OHM-6-HITC (30 and 300 mg/kg). The number of surviving mice decreased after day 12. No significant difference in survival was found. Even in the case of 300mg/kg of 6OHM-6-HITC, no behavioral impairment was observed in mice. Pathophysiological changes in the mouse tongue were assessed by macroscopic and histological methods. Figure 32 shows the scores for oral mucositis. No oral mucositis was observed on day 7, but appeared on day 8. Severity scores reached a maximum on day 12. Thereafter, the score decreases with time. The oral mucositis score of the group to which 6OHM-6-HITC was administered at 300mg/kg was significantly lower on days 10 and 12 than that of the control group. The total scores of 30 and 300mg/kg of 6OHM-6-HITC from day 0 to day 12 were significantly lower than those of the control group, respectively (FIG. 33). A histopathological section of the tongue sample is shown in fig. 34. In the control group, epidermal laxity of the tongue was observed. However, in the 6OHM-6-HITC group, the degree of epidermal relaxation was maintained, and infiltration of inflammatory cells was reduced. TUNEL staining was performed to confirm cell damage due to radiation. Figure 35 shows photographs of TUNEL staining used to assess apoptosis. The control group had considerable apoptosis (23.7% of TUNEL positive cells). In contrast, the percentage of TUNEL positive cells decreased to 9.9% and 8.6% after treatment with 30 and 300mg/kg6OHM-6-HITC, respectively (see also FIG. 36). MPO activity and TBARS levels were measured 12 days after irradiation. MPO activity was significantly reduced in the groups administered with 6OHM-6-HITC at 30 and 300mg/kg, as compared with the control group (see FIG. 37). The groups administered 6OHM-6-HITC at 30 and 300mg/kg, respectively, had lower TBARS levels than the control group, and 6OHM-6-HITC (300mg/kg) caused a significant decrease compared to the control group (see FIG. 38).