WO2014041537A1

WO2014041537A1 - Protein kinase c (pkc) alpha inhibitors for treatment and prevention of toxicities caused by radiation

Info

Publication number: WO2014041537A1
Application number: PCT/IL2013/050766
Authority: WO
Inventors: Liora Braiman-Wiksman; Yuval Sagiv; Ephraim Brener; Liat Hammer; Revital MANDIL LEVIN; Olga DRATVIMAN-STOROBINSKY; Moshe BEN HAMOU; Ruth Alon
Original assignee: Healor Ltd.
Priority date: 2012-09-13
Filing date: 2013-09-11
Publication date: 2014-03-20

Abstract

The present disclosure provides methods of preventing and treating damages, in particular skin and GI damages, caused by exposure to radiation or by bio-therapy or chemotherapy against cancer, comprising administering a PKC-alpha inhibitory peptide.

Description

PROTEIN KINASE C (PKC) ALPHA INHIBITORS FOR TREATMENT AND PREVENTION OF TOXICITIES CAUSED BY RADIATION

FIELD OF THE INVENTION

The present invention relates to methods of treatment and prevention of radiation injuries. The invention further relates to prevention and treatment of damages to skin and of gastrointestinal toxicities caused by radio-, biological or chemo-therapy of cancer, comprising administration of protein kinase C alpha (PKCa) inhibitors to the patient in need of such prevention or treatment.

BACKGROUND OF THE INVENTION

Cancer therapies have led to remarkable results due to improved toxicity profiles and effects on survival. However, an unexpected constellation of toxicities has emerged. Most notably, dermatologic adverse events have gained considerable attention, due to their high frequency, visibility, and impact on physical and psychosocial health of the patient, all of which affect dose intensity and possibly clinical outcome (Balagula et al., J Am Acad Dermatol. 2011, 65, 3, 624-35). Although skin reactions are common, side effects of various anti-cancer treatments, amongst which are radiotherapy and biological therapeutics (such as epidermal growth factor receptor (EGFR) inhibitors), are considered to be responsible for the most severe skin toxicities.

Radiation-associated skin toxicities

Ionizing radiation is a widely accepted form of treatment to various types of cancer. Although in most cases the skin is not the primary target of ionizing radiation, its exposure is inevitable and radiation skin injury remains a significant problem. During radiation therapy for cancer, the skin is exposed to significant doses of radiation in order to achieve the maximal efficacy. The injury, often referred to as radiation dermatitis, can negatively affect patients' physical functioning and quality of life, cause pain and discomfort, limit activities, and may cause interruption in or cessation of treatment (Feight et al., 2011, Clin J Oncol Nurs., 15, 5, 481-92). The most radio-sensitive organ systems are bone marrow, reproductive system, gastrointestinal track, skin, muscle and the brain. In developed countries, at least 50% of patients diagnosed with cancer will receive radiation therapy during their illness. Up to an estimated 95% of patients receiving radiation therapy will experience some degree of skin reaction, which may include erythema, dry desquamation, and moist desquamation. The degree of radiation injury and the incidence of severe reactions are dependent on the total radiation dose, the dose per fraction, the overall treatment time, beam type and energy and the surface area of the skin that is exposed to radiation.

Irradiation of the skin causes immediate damage to basal keratinocytes and hair follicles stem cells, followed by a burst of free radicals, irreversible double- stranded breaks in nuclear and mtDNA, and inflammation. Radiation skin injury can be categorized as acute or late (chronic). Acute injury occurs within hours to weeks after radiation exposure, whereas late injury presents itself months to years after radiation exposure.

Radiation dermatitis generally manifests within a few weeks after the start of radiotherapy. As the cumulative dose of radiation increases, the transient erythema occurring during the first weeks of radiotherapy may evolve into the more persistent erythema to dry or even moist desquamation that reflects the damage to the basal cell layer and the sweat and sebaceous glands. Cumulative daily dose of radiation to the treatment field, including doses deposited to the skin, prevent normal skin cells from repopulating immediately, which weakens skin integrity in the irradiated area. Irradiation of the skin leads to a complex pattern of direct tissue injury and inflammatory cell recruitment, involving damage to epidermal basal cells, endothelial cells and vascular components and a reduction in skin Langerhans cells (Hymes et al., 2006, J Am Acad Dermatol., 54, 1, 28-46). In severe radiation dermatitis, there is a massive neutrophils infiltration into the epidermis and profound apoptosis. With successive doses of radiation, the opportunity for tissue healing due to cellular repopulation is reduced, even over weekend interruption of daily fractionated radiotherapy, thereby compounding the radiation insult. Chronic radiation-induced changes in the skin are characterized by the disappearance of follicular structures, an increase in collagen and damage to elastic fiber in the dermis, and a fragility of epidermal covering.

Ionizing radiation is not only a concern for cancer patients, but also a public health concern because of the potential for and reality of a nuclear and/or radiological event. It is recognized that any skin injury in the setting of radiation poising greatly increases the risk of death (Ryan., 2012, J Invest Dermatol., 132, 985-93). Although the kinetics of the clinical signs of radiation exposure may differ between cancer radiotherapy and attack/industrial radiological event, the symptoms and syndromes in exposed organ systems are similar (Williams & McBride., 2011, Int J Radiat Biol, 87, 8, 851 -68).

A wide variety of topical, oral and intravenous agents are in use to treat and prevent radiation-induced skin reactions and injuries including topical corticosteroids, amifostine (Ethyol by Meimmune) and oral enzymes, biafine cream (Ortho-McNeil Pharmaceuticals), Aloe vera creams, Hyaluronidase-based creams (Xclair by Align Pharmaceuticals), topical colony stimulating factor (CSF), moisture-maintaining hydrogels, hydrocolloid and silver dressings and others. So far, evidence has been insufficient to support the use of a particular agent for the prevention and management of acute radiation-induced skin reactions which lead to the absence of evidence-based guidelines. The clinical attempts to develop methods to treat and prevent radiation-induced skin damage are focused on attempts to treat and prevent symptoms without resolving the underlying pathophysiology caused by the radiation (Salvo et al., 2010, Curr Oncol., 17, 4, 94-112). Currently, there is no effective treatment to prevent or mitigate radiation skin injury.

Radiation induced gastrointestinal (GI) toxicity

Radiation-induced gastrointestinal syndrome (RIGS) is characterized by loss of crypt cells and villi depopulation, resulting in disruption of mucosal integrity and barrier dysfunction, bacterial invasion, inflammation and sepsis. Nausea, vomiting, diarrhea and abdominal cramping are hallmarks of the prodromal phase of radiation sickness, occurring hours to days following radiation exposure (Otterson., 2007, World J Gastroenterol., 13, 19, 2684-92).

Epidermal Growth Factor Receptor (EGFR) inhibition associated skin toxicity

One of the most distressing skin toxicities are those associated with EGFR inhibitor therapy (either tyrosine kinase inhibitors or monoclonal antibodies) such as Cetuximab (Erbitux by Imclone), Erlotinib (Tarceva by OSI Pharmaceuticals, Gefitinib (Iressa by AstraZenaca). EGFR-inhibitor therapy toxicity is expressed by skin rash, dry skin, hair growth disorders, pruritus, and nail changes. The cutaneous side effects can often lead to dose reductions, treatment discontinuation and reducing the overall anti-cancer treatment efficacy. EGFR inhibitor-induced skin toxicity is believed to be caused by an increase in keratinocyte production skin follicles that impairs their differentiation. In parallel, cytokine release such as IL-1, TNFa and others attracts neutrophils, monocytes, and lymphocytes to the area, resulting in an inflammatory reaction manifested as a papulopustular rash. It is estimated that rash occurs in 60% to 80% of patients, with the majority experiencing a mild to moderate rash. Severe symptoms necessitating dose alterations occur in up to 20% of the patients with rash. When EGFR inhibition therapy is combined with radiation the risk for high-grade dermatologic toxicities is significantly increased (Tejwani et al., 2009, Cancer, 115, 6, 1286-99). To date, no clinical trials have established the optimal treatment for EGFR inhibitor-associated rash. To prevent dryness and rash, delicate cleansing and moisturizing with thick, emollient-based creams are recommended. Topical and oral antibiotics have antiinflammatory effects on the rash in addition to reducing the risk of secondary infection, but there is currently no agreement on the length of the antibiotic course. Oral corticosteroids and topical nonsteroidal immunomodulatory agents are often used for their potential antiinflammatory effects. Although steroids may be beneficial in treating severe skin rash, their use may result in steroid-induced acne, which can complicate matters (Perez-Soler et al,. 2005, Oncologist, 10, 345-356).

The protein kinase C (PKC) family represents a group of phospholipid dependent enzymes catalyzing the covalent transfer of phosphate from ATP to serine and threonine residues of proteins. The family is currently considered as composed of 12 individual isoforms which belong to 3 distinct categories based on their activation by calcium ion and other factors. PKC isoforms are activated by a variety of extracellular signals and, in turn, modify the activities of cellular proteins including receptors, enzymes, cytoskeletal proteins, and transcription factors. Accordingly, the PKC family plays a central role in cellular signal processing, including regulation of cell proliferation, differentiation, survival and death. Considerable evidence has been accumulated, suggesting the involvement of the Protein Kinase C (PKC) family of serine-threonine kinases in the patho-physiology of skin, including migration, proliferation, differentiation, and new matrix formation.

The PKCa isoform, which is highly abundant in skin, was identified as a central intervention point in normal and pathological signaling pathways in skin cells. Being in the epidermis and mainly restricted to suprabasal layers, PKCa is involved in cell cycle withdrawal and is primarily associated with the keratin cytoskeleton and desmosomal cell- cell junctions. PKCa was also shown to be involved in keratinocyte differentiation and integrin-matrix regulation. It has been shown that PKC mediated signaling is a major pathway in regulation of α6β4 integrin and in mediation of skin attachment, detachment and differentiation properties. Absence of PKCa in the skin (PKCa knockout mice) leads to increased proliferation rate of basal keratinocytes. Over-expression of PKCa in transgenic mice has appeared to induce a striking inflammatory response, implicating PKCa in the epidermal inflammatory response (Wang and Smart, 1999, J Cell Sci 112, 3497-3506). PKCa is also discussed as being involved in macrophage activation and was shown to be involved in mast cell signaling (Cataisson et al., 2005, J Immunol 174, 1686-1692).

WO2009016629 discloses compositions comprising a delta-PKC activator, an alpha PKC inhibitor, and a carrier that is free of calcium and magnesium cations for decreasing inflammation at the site of a skin wound.

US20120190611 and WO2011083483 discloses methods for treatment of inflammatory disease and disorder in a subject by administering to the subject an inhibitor of PKC alpha, PKC epsilon, or PKC eta, or an activator of PKC delta.

WO2011083482 discloses treating psoriasis in a subject by administering an inhibitor of PKC alpha.

There is an unmet need for efficient and specific therapeutics against toxicities induced by radiation, chemotherapy and biological anti-cancer therapy. Improved control of dermatologic, gastrointestinal and other toxicities associated with radiotherapy may enable patients to tolerate higher doses of cancer therapies for longer durations, and this may lead to better control of their illness.

SUMMARY OF THE INVENTION

The present invention demonstrates, for the first time, that PKC alpha inhibitors are capable of preventing or reducing the pathophysiological processes associated with toxicities to gastrointestinal, skin and other organs and tissues, caused by radiation and those caused by other anti-cancer treatments such as biological therapy and chemotherapy. The present invention thus provides PKC alpha inhibitors for preventing, protecting and treating damage to organs and tissues, in particular, damages to the skin and the gastrointestinal track.

It is herein shown for the first time that administration of several specific peptide inhibitors of PKC alpha beneficially affects not only toxicity to skin, following ionizing radiation, but also toxicity to gastrointestinal tract and even survival. It is further shown for the first time, that not only damage caused by radiation can be prevented or treated by the PKC alpha inhibitor of the present invention, but also damage caused by biological therapy or chemotherapy against cancer. In particular it is shown that specific peptide inhibitors of PKC alpha are capable, not only to treat radiation-induced skin and GI toxicities but also to improve survival of treated animals.

Without wishing to be bound by any specific theory, it is suggested that the peptide inhibitors of PKCa according to the present invention, even when applied topically, have not only local effect on the skin cells, but also a systemic effect, leading to prevention or alleviation of damage to the gastrointestinal tract.

Accordingly, the present invention provides in one aspect, a method of preventing, ameliorating or treating damage to organ or tissue of a subject, caused by radiation, comprising administering to a subject in need thereof a pharmaceutical composition comprising at least one PKC alpha inhibitor, thereby preventing or treating the damage to the organ or tissue.

According to some embodiments, the subject suffers from a disease or disorder which is treated by irradiation. According to other embodiments, the subject suffers from exposure to radiation through other means, accidental or otherwise.

According to some embodiments, the subject suffers from cancer.

According to other embodiments, the subject was exposed to accidental ionizing radiation.

According to some embodiments, the damage is to at least one tissue or organ selected from the group consisting of: skin, gastrointestinal track, bone marrow, reproductive system, muscle and brain. Each possibility represents a separate embodiment of the present invention. According to some embodiments the damage includes skin toxicity which is prevented, alleviated or treated.

According to some specific embodiments, the skin toxicity comprises radiation dermatitis.

According to other embodiments, the damage includes toxicity to the gastrointestinal tract which is prevented, alleviated or treated.

According to yet other embodiments, the damage includes injury to hair follicles which is prevented or treated by the PKC alpha peptide inhibitors.

According to some embodiments of the present invention, the PKC alpha inhibitor is a peptide of 6-24 amino acids comprising a sequence selected from the group consisting of:

Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser (SEQ ID NO: 1); and

Phe-Ala-Arg-Lys-Gly-Ala (SEQ ID NO: 2), or an analog, salt or derivative thereof.

According to some embodiments, the PKC alpha inhibitor is a peptide of 8-15 amino acids comprising the sequence Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser (SEQ ID NO: 1), or an analog, salt or derivative thereof.

According to other embodiments, the PKC alpha inhibitor is a peptide of 6-12 amino acids comprising a sequence selected from the group consisting of:

Phe-Ala-Arg-Lys-Gly-Ala (SEQ ID NO: 2);

Phe-Ala-Arg-Lys-Gly-Ala-Leu (SEQ ID NO: 3);

Phe- Ala- Arg-Lys-Gly- Ala-Leu- Arg (SEQ ID NO: 4);

Phe-Ala-Arg-Lys-Gly- Ala-Leu- Arg-Gln (SEQ ID NO: 5);

Phe-Ala-Arg-Lys-Gly-Ala-Arg-Gln (SEQ ID NO: 6); or an analog, salt or derivative thereof.

According to some embodiments, the PKC alpha inhibitor is selected from the group consisting of: H-Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser-OH (SEQ ID NO: 1); H-Phe-Ala-Arg-Lys-Gly-Ala-Leu-OH (SEQ ID NO: 3); and

H-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH (SEQ ID NO: 5); or an analog, salt or derivative thereof.

According to some embodiments, the PKC alpha inhibitor comprises a permeability moiety conjugated to the peptide sequence.

According to some embodiments, the permeability moiety is connected, by a covalent bond, to the N-terminus of the peptide.

According to some embodiments the permeability moiety attached to the PKC alpha inhibitor is selected from the group consisting of: i. hydrophobic moiety such as fatty acid, steroid and bulky aromatic or aliphatic compound; ii. moiety which may have cell- membrane receptors or carriers, such as steroid, vitamin and sugar; and iii. transporter peptide or amino acid.

According to some embodiments, the fatty acid comprises an aliphatic tail of 3-12 carbons. According to some particular embodiments, the fatty acid is selected from the group consisting of: myristic acid, palmitic acid and cholesterol.

According to some particular embodiments, the PKC alpha inhibitor, comprising a permeability moiety is selected from the group consisting of:

Myristoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH (SEQ ID NO: 7, also denoted MPDY-1);

Palmitoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH (SEQ ID NO: 8); and

Myristoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-OH (SEQ ID NO: 9); or an analog, derivative or salt thereof.

Within the scope of the present invention are also peptides analogs, comprising at least one amino acid or peptide-terminal modification. According to some embodiments, the invention provides a peptide analog of a PKC alpha inhibitor, comprising at least one peptide terminal modification selected from the group consisting of: modified amino-terminus and modified carboxy-terminus.

According to some specific embodiments, an analog of a PKC alpha inhibitory peptide of any one of SEQ ID NOs: 1-9 is provided, comprising at least one pep tide-terminal modification selected from the group consisting of: amino-terminus modification and carboxy-terminus modification. According to some particular embodiments, the modification is selected from the group consisting of: N-terminus acylation, C-terminus amidation and modification of the C-terminal acid to an alcohol.

A pharmaceutical composition, comprising at least one PKC alpha according to the invention, and a carrier, diluent or excipient, for preventing, ameliorating or treating damage to organ or tissue, caused by radiation or by treatment with a chemotherapy or biological therapy against cancer, is also included within the scope of the present invention.

According to some embodiments, the pharmaceutical composition is free of calcium (Ca²⁺) and magnesium (Mg²⁺) cations. According to yet other embodiment, a pharmaceutical composition comprise calcium (Ca²⁺) and/or magnesium (Mg²⁺) cations. Agents that chelate calcium (Ca²⁺) and/or magnesium (Mg²⁺) cations may also be present.

The pharmaceutical composition may comprise, according to some embodiments of the present invention, an aqueous solution or buffer. According to some embodiments, the aqueous solution is phosphate buffer. According to other embodiments, the aqueous solution is Dulbecco's Phosphate Buffered Saline (DPBS^{~ ~}, which comprises potassium chloride, potassium dihydrogen phosphate and sodium chloride and does not comprise calcium or magnesium cations).

According to some specific embodiments, the aqueous solution is DPBS^{~ ~}. According to other embodiments, the aqueous solution is acetate buffer. According to some embodiments, the aqueous solution does not contain calcium and magnesium cations. Each possibility represents a preferred embodiment of the present invention.

A pharmaceutical composition comprising a PKC alpha inhibitor according to the present invention may be administered by any suitable route of administration, including topically or systemically. Modes of administration include but are not limited to topical and transdermal routes as well as parenteral routes such as intravenous and intramuscular injections, as well as via nasal or oral ingestion. Each possibility represents a preferred embodiment of the present invention.

According to some embodiments, the PKC alpha inhibitor is administered topically. According to other embodiments, the PKC alpha inhibitor is administered systemically. According to yet other embodiments, the PKC alpha inhibitor is administered orally.

The PKC alpha inhibitor may be administered before, during or after exposure to radiation or to a chemotherapy or biological therapy (such as EGFR inhibition therapy), or as part of a treatment regimen, for example, treatment of cancer.

According to some embodiments, the PKC alpha inhibitor is administered prior to exposure to radiation or to a chemotherapy or biological therapy.

According to other embodiments, the PKC alpha inhibitor is administered after exposure to radiation or to a chemotherapy or biological therapy.

A pharmaceutical composition comprising a PKC alpha inhibitor according to the present invention may be administered alone or in conjunction with additional therapeutic or non-therapeutic agents for the conditions to be treated.

According to some embodiments, the pharmaceutical composition comprising a PKC alpha inhibitor according to the present invention is administered in conjunction with chemotherapy.

According to some embodiments the compositions are unexpectedly effective when applied topically as a sole active ingredient.

In various aspects, the present disclosure provides a kit for carrying out the method of the disclosure. In one embodiment, the kit includes a peptide inhibitor of PKCa as well as instructions for administering the peptide inhibitor.

According to other embodiments, the PKC alpha inhibitor is a peptide multimer of 12-60 amino acids, comprising at least two, identical or different, sequences wherein at least one of the sequences is set forth above. Each possibility represents a separate embodiment of the present invention. According to some embodiments the peptide multimer comprises a permeability moiety. According to particular embodiments, the permeability moiety is a fatty acid. According to some specific embodiments the peptide multimer is N-myristoylated. Each possibility represents a separate embodiment of the present invention.

Accordingly, the present invention provides methods of preventing, alleviating or treating a damage to an organ or tissue comprising administering to a subject in need thereof, a pharmaceutical composition comprising at least one PKC alpha peptide inhibitor defined above, or a peptide multimer comprising at least two, identical or different sequences.

The present invention also provides methods of preventing, alleviating or treating a damage, caused by anti-cancer therapy, to an organ or tissue comprising administering to a subject in need thereof, a pharmaceutical composition comprising a PKC alpha peptide inhibitor defined above.

According to some embodiments, the anti-cancer therapy comprises a treatment selected from the group consisting of: irradiation, chemotherapy and biological therapy.

According to some embodiments, the biological therapy comprises administration of an epidermal growth factor receptor (EGFR) inhibitor.

The present invention provides in another aspect, a method of improving the survival of a subject exposed to radiation or treated with a chemotherapy or biological therapy against cancer, comprising administering a pharmaceutical composition comprising at least one PKCa inhibitor, thereby improving the survival of the subject.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Survival following irradiation: Eleven weeks old BALB/C mice were subjected to cumulative radiation of 30Gy. Irradiated mice were treated daily, for 14 days, with PKCa inhibitory peptide Myristoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH (SEQ ID NO: 7, also denoted MPDY-1) ^g/ml, or lC^g/ml or acetate buffer as control. Mortality rate (% survival) was observed on a daily basis for a whole period of the treatment. Results are presented as a percentage of survived mice per group at the end of the experiment.

Figure 2: Prevention of radiation-induced hyperplasia: Eleven week old BALB/C mice were subjected to local 20Gy dose of ionizing radiation (X-rays, Kimtron Polaris 320kVp 10mA), to the right hind leg. Following irradiation, mice were treated daily with 1 μg/ml or lC^g/ml PKCa inhibitor MPDY-1 (SEQ ID NO: 7) or with vehicle alone (acetate buffer) as control, for 21 days. Skin biopsies of the irradiated areas were removed and submitted to paraffin embedding, followed by skin section preparation. Hematoxylin and eosin (H&E) staining of the sections was performed and assessed. Representative pictures from each group are presented.

Figure 3: Epidermal hyperplacticity: Eleven week old BALB/C mice were subjected to local 20Gy dose of ionizing radiation (X-rays, Kimtron Polaris 320kVp 10mA), to the right hind legs. Following irradiation, mice were treated daily with 1 μg/ml or 10μg/ml PKCa inhibitor MPDY-1 or with vehicle alone (acetate buffer) as control, for 21 days. Skin biopsies of the irradiated areas were removed and submitted to paraffin embedding, followed by skin section preparation. H&E staining of the sections was performed and assessed. Epidermal hyperplacticity was assessed and summarized for each group and is presented in percentage values.

Figure 4: Prevention of radiation-induces collagen degradation: Eleven week old BALB/C mice were subjected to local 20Gy dose of ionizing radiation (X-rays, Kimtron Polaris 320kVp 10mA), to the right hind legs. Following irradiation, mice were treated daily with 1 μg/ml or 10μg/ml PKCa inhibitor MPDY-1 or with vehicle alone (acetate buffer) as control, for 21 days. Skin biopsies of the irradiated areas were removed and submitted to paraffin embedding, followed by skin section preparation and staining with Masson Trichrome. Collagen deposition was assessed and summarized for each group and is presented in percentage values. Figure 5: Protection of epidermal stem cells from radiation-induced death: Eleven week old BALB/C mice were subjected to local 20Gy dose of ionizing radiation (X-rays, Kimtron Polaris 320kVp 10mA), to the right hind legs. Following irradiation, mice were treated daily with 1 μg/ml or 10μg/ml PKCa inhibitor MPDY-1 or with vehicle alone (acetate buffer) as control, for 21 days. Skin biopsies of the irradiated areas were removed and submitted to paraffin embedding, followed by skin section preparation and staining for K15 (an epidermal stem-cells marker).

Figure 6: Prevention of hair follicles damage in PKCa depleted mice: Eleven weeks old BALB/C normal or PKCa depleted mice were subjected to an array of irradiation dosages ranging from 30Gy to 40Gy. Irradiated mice were left untreated for 14 days following irradiation. Skin biopsies of the affected areas were removed and paraffin embedded for histological analysis. H&E staining of the slides was performed and assessed. Representative pictures from control vs PKCa depleted groups are shown.

Figure 7: IL-6 and IL-1 alpha levels in response to LPS stimulation: Keratinocytes were derived from newborn C57BL/6J mice skin. The cells were grown for 5 days in 24 wells plates. Cells were then treated with Dulbecco's Phosphate Buffered Saline^"7" (DPBS^"/_), LPS or LPS+PKCa inhibitor (HO/02/02, MPDY-1 SEQ ID NO: 7). Medium containing secreted cytokines was collected following 48 hr incubation and analyzed using a Luminex system.

Figures 8: IL-6 and TNF alpha levels in response to LPS stimulation: Experimental design as in Figure 7.

Figure 9: IL-1 alpha secretion in TNF alpha-stimulated keratinocytes: Keratinocytes were derived from newborn C57BL/6J mice skin. The cells were incubated for 5 days in 24 wells plates. Cells were then treated with DPBS^_/", TNFa or TNFa +PKCa inhibitors (SEQ ID NO: 7 and SEQ ID NO: 9). Medium containing secreted cytokines was collected following 48 hr incubation and analyzed using a Luminex system.

Figures 10-12: Cytokine levels in response to IL-17a stimulation. Keratinocytes were derived from newborn C57BL/6J mice skin. The cells were incubated for 5 days in 24 wells plates. Cells were then treated with DPBS ^{~ ~} (control), IL-17a or IL-17a +PKCa inhibitors (SEQ ID NOs: 7, 9 and 1). Medium containing secreted cytokines was collected after 48 hr and analyzed using a Luminex system. Figure 10 - IL-6 levels after addition of 0.1, 1 and 10 μ§/ηι1 PKC alpha inhibitors (SEQ ID NOs: 7 and 9); Figure 11 - IL-1 alpha levels after addition of 1 μ§/πύ PKC alpha inhibitor (SEQ ID NOs: 7 and 9); Figure 12 TNF alpha levels after addition of 0.1 , 1 and 10 μ^ιηΐ PKC alpha inhibitor (SEQ ID NOs: 7, 9 and 1).

Figure 13: IL-1 alpha secretion in IL-17 alpha - stimulated keratinocytes: Keratinocytes were derived from 129 mice tail's skin or PKCa depletion mice tail's skin. The cells were incubated for 5 days in 24 wells plates. Cells were then treated with DPBS ^{~ ~} (control) or IL- 17a. Medium containing secreted cytokines was collected following 48 hr incubation and analyzed using a Luminex system.

Figure 14: IL-6 secretion in LPS - stimulated splenocytes: Splenocytes were derived from 129 mice spleen or PKCa depletion mice spleen. Cells were incubated for 6 days in 24 wells plates, and then were treated with DPBS ^{~ ~} (control) or LPS. Medium containing secreted cytokines was collected after 48 hr and analyzed using a Luminex system.

Figures 15-16: Inhibition of PKC alpha reduces cytokine secretion. Bone marrow cells were derived from B6 mice. Macrophages were incubated for 6 days in 24 wells plates, and then were treated with DPBS^_/" (control), LPS or LPS+ PKCa inhibitor MPDY-1 (SEQ ID NO: 7). Medium containing secreted cytokines was collected after 48 hr and analyzed using a Luminex system. Figure 15 - IL-1 beta levels, Figure 16 - IL-1 alpha and TNF alpha levels.

Figures 17: The reduction of TNF alpha- induced Granulocyte colony- stimulating factor (G- CSF) secretion in response to MPDY-1 is not interrupted by Erbitux. HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY-1 (1 μg/ml) and Erbitux (Erb - 1 μg/ml) for two hours, followed by activation with TNFa (10 ng/ml). 48 hours post activation, medium was collected from plates and cytokines were measured using Multiplex ELISA Arrays assay.

Figure 18: The reduction of TNF alpha- induced MG-CSF secretion in response to MPDY-1 is not interrupted by Erbitux. HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY-1 (1 μg/ml) and Erbitux (Erb - 1 μg/ml) for two hours, followed by activation with TNFa (10 ng/ml). 48 hours post activation, medium was collected from plates and cytokines were measured using Multiplex ELISA Arrays assay. Figure 19: The reduction of TNF alpha- induced G-CSF secretion in response to MPDY-1 is not interrupted by Erbitux (an epidermal growth factor receptor (EGFR) inhibitor used for the treatment of cancers). HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY-1 (1 μg/mϊ) and Erbitux (Erb - 1 μg/mϊ) for two hours, followed by activation with LPS (10 μg/ml) . 48 hours post activation, medium was collected from plates and cytokines were measured using Multiplex ELISA Arrays assay.

Figure 20: The reduction of LPS-induced TNFa secretion in response to MPDY-1 is not interrupted by Erbitux (an epidermal growth factor receptor (EGFR) inhibitor used for the treatment of cancers). HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY-1 (1 μg/ml) and Erbitux (Erb - 1 μg/ml) for two hours, followed by activation with LPS (10 μg/ml). 48 hours post activation, medium was collected from plates and cytokines were measured using Multiplex ELISA Arrays assay.

Figure 21: The reduction of LPS-induced IL-6 secretion in response to MPDY-1 is not interrupted by the chemotherapy drug Epirubicin. HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY-1 (1 μg/ml) and Epirubicin (Epi -100 μΜ) for two hours, followed by activation with LPS (10 μg/ml). 48 hours post activation, medium was collected from plates and cytokines were measured using Multiplex ELISA Arrays assay.

Figure 22: The reduction of LPS-induced IL-lb secretion in response to MPDY-1 is not interrupted by the chemotherapy drug Epirubicin. HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY-1 (1 μg/ml) and Epirubicin (Epi -100 μΜ) for two hours, followed by activation with LPS (10 μg/ml). 48 hours post activation, medium was collected from plates and cytokines were measured using Multiplex ELISA Arrays assay.

Figure 23: The reduction of TNF alpha- induced IL-6 secretion in response to MPDY-1 is not interrupted by the chemotherapy drug Cyclophosphamide (CPA). HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY-1 (1 μg/ml) and Cyclophosphamide (CPA-100 μΜ) for two hours, followed by activation with TNFa (10 ng/ml). 48 hours post activation, medium was collected from plates and cytokines were measured using Multiplex ELISA Arrays assay. Figure 24: The reduction of LPS-induced IL-6 secretion in response to MPDY-1 is not interrupted by the chemotherapy drug Cyclophosphamide. HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY-1 (1 μg/ml) and Cyclophosphamide (CPA-100 μΜ) for two hours, followed by activation with LPS (10 μg/ml). 48 hours post activation, medium was collected from plates and cytokines were measured using Multiplex ELISA Arrays assay.

DETAILED DESCRIPTION OF THE DISCLOSURE

Radiation-induced changes in the skin are characterized by the disappearance of follicular structures, and damage to elastic fiber in the dermis. Following ionizing radiation, irradiated skin exhibited abnormal morphology expressed by reduced cellularity and marked reduction in collagen fibers as well as, an increase in fibrotic tissue. Significant loss of follicular structure is also observed, expressed by abnormal hair follicles and reduction in mature sebaceous glands. As shown herein for the first time, these toxic effects of radiation were markedly reduced and in some parameters completely prevented in animals treated with PKCa peptide inhibitors. Surprisingly, treated animals not only suffered less from skin toxicity, but also survived longer.

The efficient therapeutics of the present invention take into account the pathological process initiated by radiation and act in a specific point of intervention in order to stop the pathological process from advancing. The present invention thus provides improved control of dermatologic toxicities, that may enable patients to tolerate higher doses of cancer therapies for longer durations, and this may lead to better control of their illness.

It is to be understood, that this disclosure is not limited to particular compositions, methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, as the scope of the present disclosure will be limited only in the appended claims.

The principles and operation of the methods according to the present disclosure may be better understood with reference to the figures and accompanying descriptions. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, some preferred methods and materials are now described.

As used herein, the term "subject" refers to a mammalian subject. As such, treatment of any animal in the order mammalian is envisioned. Such animals include, but are not limited to horses, cats, dogs, rabbits, mice, goats, sheep, non-human primates and humans. Thus, the method of the present disclosure is contemplated for use in veterinary applications as well as human use.

"Treatment" of a subject herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with a tissue or organ toxicity as well as those in which it is to be prevented. Hence, the subject may have been diagnosed as having skin or GI toxicity, for example, or may be predisposed or susceptible to suffer from such toxicities.

The expression "effective amount" refers to an amount of an inhibitor of PKC alpha according to the invention that is effective for preventing, ameliorating or treating a tissue or organ toxicity such as skin or GI radiation toxicity compared to no treatment with such agents. Such an effective amount will generally result in an improvement in the signs, symptoms and/or other indicators of a tissue or organ toxicity.

"Permeability" refers to the ability of an agent or substance to penetrate, pervade, or diffuse through a barrier, membrane, particularly cells' membrane, or a skin layer. Any conjugate which succeeds in penetrating into the cells whether by a passive diffusion (e.g., lipophilic moieties that penetrate the lipid bilayer of the cells), or a passive mechanist (e.g., encapsulation or liposome uptake or the like), or by active uptake (e.g. attachment to a moiety that is transported into the cells or through the membrane), is included within the scope of the present invention.

A "permeability moiety", denoted also "a permeability enhancing moiety", according to the invention may be any moiety biological or chemical (natural, semi-synthetic or synthetic) capable of facilitating or enhancing entry, penetration, pervading or diffusion of the PKC inhibitor to which it is conjugated, through a barrier, membrane, particularly cells' membrane, or a skin layer, or into the target cells. Non-limiting examples of permeability moieties include hydrophobic moieties such as lipids, fatty acids, steroids and bulky aromatic or aliphatic compounds; moieties which may have cell-membrane receptors or carriers, such as steroids, vitamins and sugars, natural and non-natural amino acids and transporter peptides. More specific examples include cationic protein transduction domains (PTDs) such as HIV-1 TAT, Drosophila Antennapedia, poly-arginine (R7) (synthetic), PTD-5 (synthetic), amphipathic PTDs such as transportan (chimeric, galanin fragment plus mastoparan), KALA and more. Other examples are small organic molecules, notably lipophilic that are known to promote transfer across cell membranes of agents that are complexed or covalently attached to them.

Non-limiting examples for lipidic moieties which may be used according to the present invention: Lipofectamine, Transfectace, Transfectam, Cytofectin, 2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DMRIE), 2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DLRIE), (+/-)-N-(3- aminopropyl)-N,N-dimethyl-2,3-bis (dodecyloxy)-l -propanaminium bromide (GAP-DLRIE), 1 ,2-Dioleoyloxy-3-(trimethylammonium)propane (DOTAP), 1 ,2-dioleoyl-glycero-3- phosphatidyl ethanolamine (DOPE), Dioleoyl lecithin DOPC), Didecyldimethylammonium bromide (DDAB), 2,3-dioleyloxy-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-l- propanaminium trifluoro acetate (DOSPA), l-(4'-carboxyethyl)-6-diphenyl-l,3,5-hexatriene (DPH-PA), l-(4-(trimethylamino)phenyl)-6-phenylhexa-l ,3,5-triene (TMADPH), Cetrimonium bromide (CTAB), lysyl-PE, 3-(N-(N',N'- dimethylaminoethane)carbamoyl)cholesterol (DC-Cho), alanyl cholesterol; 1 ,2- dipalmitoylphosphatidylethanolamidospermine (DPPES), Chlorfenethol (DCPE), 4- dimethylaminopyridine (DMAP), 1 ,2-dimyristoylphosphatidylethanolamine (DMPE), dioleoylsuccinylglycerol (DOGS), dipentafluorophenylcarbonate (DPEPC), Pluronic, Tween, polyethylene glycol oleyl ether (BRIJ-96), plasmalogen, phosphatidylethanolamine, phosphatidylcholine, glycerol-3-ethylphosphatidylcholine, dimethyl ammonium propane, trimethyl ammonium propane, diethylammonium propane, triethylammonium propane, dimethyldioctadecylammonium bromide, a sphingolipid, sphingomyelin, a lysolipid, a glycolipid, a sulfatide, a glycosphingolipid, cholesterol, cholesterol ester, cholesterol salt, oil, N-succinyldioleoylphosphatidylethanolamine, 1 ,2-dioleoyl-sn-glycerol, 1 ,3-dipalmitoyl-2- succinylglycerol, l,2-dipalmitoyl-sn-3-succinylglycerol, l-hexadecyl-2- palmitoylglycerophosphatidylethanolamine, palmitoylhomocystiene, N,N'-Bis

(dodecyaminocarbonylmethylene)-N,N'-bis((-N,N,N-trimethylammoniumethyl-ami nocarbonylmethylene)ethylenediamine tetraiodide; N,N"-

Bis(hexadecylaminocarbonylmethylene)-N,N', N"-tris((-N,N,N-trimethylammonium- ethylaminocarbonylmethylenediethylenetri amine hexaiodide; N,N'-Bis

(dodecylaminocarbonylmethylene)-N,N"-bis((-N,N,N-trimethylammonium

ethylaminocarbonylmethylene)cyclohexylene- 1 ,4-diamine tetraiodide; 1 ,7,7-tetra-((- N,N,N,N-tetramethylammoniumethylamino-carbonylmethylene)-3- hexadecylaminocarbonyl- methylene- 1 , 3, 7-triaazaheptane heptaiodide; N,N,N',N'-tetra((- N,N,N-trimethylammonium-ethylaminocarbonylmethylene)-N'-(l ,2-dioleoylglycero-3- phosphoethanolamino carbonylmethylene)diethylenetriam ine tetraiodide; dioleoylphosphatidylethanolamine, a fatty acid, a lysolipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, a sphingolipid, a glycolipid, a glucolipid, a sulfatide, a glycosphingolipid, phosphatidic acid, palmitic acid, stearic acid, arachidonic acid, oleic acid, a lipid bearing a polymer, a lipid bearing a sulfonated saccharide, cholesterol, tocopherol hemisuccinate, a lipid with an ether- linked fatty acid, a lipid with an ester-linked fatty acid, a polymerized lipid, diacetyl phosphate, stearylamine, cardiolipin, a phospholipid with a fatty acid of 6-8 carbons in length, a phospholipid with asymmetric acyl chains, 6-(5-cholesten-3b-yloxy)-l-thio-b-D- galactopyranoside, digalactosyldiglyceride, 6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxy- 1-thio-b-D-galactopyranoside, 6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxyl-l-thio-a-D- mannopyranoside, 12-(((7'-diethylamino-coumarin-3-yl)carbonyl)methylamino)-octadecanoic acid; N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methyl-amino) octadecanoyl]-2- aminopalmitic acid; cholesteryl)4'-trimethyl-ammonio)butanoate; N-succinyldioleoyl- phosphatidylethanolamine; 1 ,2-dioleoyl-sn-glycerol; l,2-dipalmitoyl-sn-3-succinyl-glycerol; l ,3-dipalmitoyl-2-succinylglycerol, l-hexadecyl-2-palmitoylglycero-phosphoethanolamine, and palmitoylhomocysteine. The PKC alpha peptide inhibitor according to the invention may be conjugated to a permeability moiety by any known means, for example via its amino, carboxy, S-S groups. The conjugation between the PKC alpha inhibitory peptide and the cell entering moiety may also involve a linker. Suitable linkers are known in the art. Preferably the linker is of the type that can be cleaved by intracellular enzymes this separating the PKC alpha inhibitory peptide from the permeability moiety.

The term "linker" denotes any chemical compound, which may be present between the permeability enhancing moiety and the peptide. Preferably, the linker may be cleaved from the peptide by chemical means, by enzymatic means, or may decompose spontaneously. The linker may be pharmacologically inert or may itself provide added beneficial pharmacological activity. The term "spacer" denoting a moiety used to allow distance between the permeability-enhancing moiety and the peptide, may also be used interchangeably as a synonym for linker.

The linker may optionally comprise a protease specific cleavable sequence. A "Protease specific cleavable sequence" denotes any peptide sequence which comprises a peptide bond cleavable by a specific protease, which is more abundant within or in proximity to the malignant cells. Non-limiting examples for protease specific cleavable sequence are described in WO 02/020715. Typically a protease specific cleavable sequence includes peptides of from about two to about fourteen amino acids comprising at least one site that is cleaved by a specific protease.

No n- limiting examples for specific biodegradable sequences that are degraded by proteases that are more abundant within or in proximity to the malignant cells are: Matrix metalloproteinases (for example collagenases, gelatinases and stromelysins); Aspartic proteases (for example cathepsin D, cathepsin E, pepsinogen A, pepsinogen C, rennin); Serine proteases (for example plasmin, tissue-type plasminogen activator (tPA), urokinase- type plasminogen activator (uPA); cysteine proteases (for example cathepsin B, cathepsin L, cathepsin S); asparaginyl proteases (for example legumain).

As disclosed in the Examples, peptide inhibitors of PKCa were capable, not only to reduce skin toxicity from radiation but also prevent GI toxicity and increase survival of treated animals. In various embodiments, the inhibitors of PKC alpha used according to the methods of the present invention are peptides. The term "peptide" is used herein to designate a series of natural, non-natural and/or chemically modified amino acid residues connected one to the other by peptide (amide) or non-peptide bonds, typically between the alpha-amino and carboxy groups of adjacent residues.

Peptides according to the present invention may are typically linear but cyclic versions of the peptides disclosed herein, are also within the scope of the present invention. Cyclization of peptides may take place by any means known in the art, for example through free amino and carboxylic groups present in the peptide sequence, or through amino acids or moieties added for cyclization. Non limiting examples of cyclization types are: side chain to side chain cyclization, C-to-N terminal cyclization, side chain to terminal cyclization, and any type of backbone cyclization incorporating at least one N^a-co-substituted amino acid residue/s as described for example in WO 95/33765.

The peptides of the present invention are preferably synthesized using conventional synthesis techniques known in the art, e.g., by chemical synthesis techniques including peptidomimetic methodologies. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. Solid phase peptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). A skilled artisan may synthesize any of the peptides of the present invention by using an automated peptide synthesizer using standard chemistry such as, for example, t-Boc or Fmoc chemistry. Synthetic peptides can be purified by preparative high performance liquid chromatography (Creighton T. 1983, Proteins, structures and molecular principles. WH Freeman and Co. N.Y.), and the composition of which can be confirmed via amino acid sequencing. Some of the peptides of the invention, which include only natural amino acids, may further be prepared using recombinant DNA techniques known in the art. The conjugation of the peptidic and permeability moieties may be performed using any methods known in the art, either by solid phase or solution phase chemistry. Some of the preferred compounds of the present invention may conveniently be prepared using solution phase synthesis methods. Other methods known in the art to prepare compounds like those of the present invention can be used and are comprised in the scope of the present invention. The term "amino acid" is used in its broadest sense to include naturally occurring amino acids as well as non-naturally occurring amino acids including amino acid analogs. In view of this broad definition, one skilled in the art would know that reference herein to an amino acid includes, for example, naturally occurring proteogenic (L)-amino acids, (D)- amino acids, chemically modified amino acids such as amino acid analogs, naturally occurring non-proteogenic amino acids such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid. As used herein, the term "proteogenic" indicates that the amino acid can be incorporated into a protein in a cell through a metabolic pathway. The amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and sequential, divergent and convergent synthetic approaches to the peptide sequence are useful in this invention. When there is no indication, either the L or D isomers may be used.

Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions includes replacement of one amino acid with another having the same type of functional group or side chain e.g. aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, affinity to the target protein, metabolic stability, penetration into the central nervous system, targeting to specific cell populations and the like. One of skill will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Also included within the scope of the invention are salts of the peptides, analogs, and chemical derivatives of the peptides of the invention.

As used herein the term "salts" refers to both salts of carboxyl groups and to acid addition salts of amino or guanido groups of the peptide molecule. Salts of carboxyl groups may be formed by means known in the art and include inorganic salts, for example sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as salts formed for example with amines such as triethanolamine, piperidine, procaine, and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, acetic acid or oxalic acid. Salts describe here also ionic components added to the peptide solution to enhance hydrogel formation and /or mineralization of calcium minerals.

A "chemical derivative" as used herein refers to peptides containing one or more chemical moieties not normally a part of the peptide molecule such as esters and amides of free carboxy groups, acyl and alkyl derivatives of free amino groups, phospho esters and ethers of free hydroxy groups. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Preferred chemical derivatives include peptides that have been phosphorylated, C-termini amidated or N-termini acetylated.

"Functional derivatives" of the peptides of the invention as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the peptide, do not confer toxic properties on compositions containing it and do not adversely affect the antigenic properties thereof. These derivatives may, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed by reaction with acyl moieties. The term "peptide analog" indicates molecule which has the amino acid sequence according to the invention except for one or more amino acid changes or one or more modification/replacement of an amide bond. Peptide analogs include amino acid substitutions and/or additions with natural or non- natural amino acid residues, and chemical modifications which do not occur in nature. Peptide analogs include peptide mimetics. A peptide mimetic or "peptidomimetic" means that a peptide according to the invention is modified in such a way that it includes at least one non-coded residue or non-peptidic bond. Such modifications include, e.g., alkylation and more specific methylation of one or more residues, insertion of or replacement of natural amino acid by non-natural amino acids, replacement of an amide bond with other covalent bond. A peptidomimetic according to the present invention may optionally comprises at least one bond which is an amide-replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. The design of appropriate "analogs" may be computer assisted. Additional peptide analogs according to the present invention comprise a specific peptide or peptide analog sequence in a reversed order, namely, the amino acids are coupled in the peptide sequence in a reverse order to the amino acids order which appears in the native protein or in a specific peptide or analog identified as active. Whether completely or partially non-peptide, peptidomimetics according to this invention provide a spatial arrangement of chemical moieties that closely resembles the three-dimensional arrangement of groups in the peptide on which the peptidomimetic is based. As a result of this similar active-site structure, the peptidomimetic has effects on biological systems, which are similar to the biological activity of the peptide.

A modified amino acid residue is an amino acid residue in which any group or bond was modified by deletion, addition, or replacement with a different group or bond, as long as the functionality of the amino acid residue is preserved or if functionality changed (for example replacement of tyrosine with substituted phenylalanine) as long as the modification did not impair the activity of the peptide containing the modified residue.

Peptide PKC alpha inhibitors may have modified amino acid sequences or non- naturally occurring termini modifications. Modifications to the peptide sequence can include, for example, additions, deletions or substitutions of amino acids, provided the peptide produced by such modifications retains PKCa inhibitory activity. Additionally, the peptides can be present in the formulation with free termini or with amino-protected (such as N- protected) and/or carboxy-protected (such as C-protected) termini. Protecting groups include: (a) aromatic urethane-type protecting groups which include benzyloxycarbonyl, 2- chlorobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, isonicotinyloxycarbonyl and 4- methoxybenzyloxycarbonyl; (b) aliphatic urethane-type protecting groups which include t- butoxycarbonyl, t-amyloxycarbonyl, isopropyloxycarbonyl, 2-(4-biphenyl)-2- propyloxycarbonyl, allyloxycarbonyl and methylsulfonylethoxycarbonyl; (c) cycloalkyl urethane-type protecting groups which include adamantyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl and isobornyloxycarbonyl; (d) acyl protecting groups or sulfonyl protecting groups. Additional protecting groups include benzyloxycarbonyl, t-butoxycarbonyl, acetyl, 2-propylpentanoyl, 4-methylpentanoyl, t- butylacetyl, 3-cyclohexylpropionyl, n-butanesulfonyl, benzylsulfonyl, 4- methylbenzenesulfonyl, 2-naphthalenesulfonyl, 3-naphthalenesulfonyl and 1- camphorsulfonyl.

According to some embodiments the amino terminus of the peptide is modified, e.g., it may be acylated. In particular embodiments, the peptide PKC alpha inhibitors are N- acylated by an acyl group. According to some embodiments, the acyl group is derived from a C4-C24 fatty acid. According to particular embodiments the acyl group is derived from a C12-C20 fatty acid, such as C14 acyl (myristoyl) or C16 acyl (palmitoyl).

According to additional embodiments the carboxy terminus is modified, e.g., it may be amidated, reduced to alcohol or esterified.

In various embodiments, the peptide PKC alpha inhibitors typically contain between 6-25 amino acids, or between 6 and 12 amino acids, but may be longer or shorter in length. In various embodiments a peptide PKC alpha inhibitor may range in length from 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, 6 to 20, 6 to 15, or 6 to 10 amino acids. In one embodiment the peptide includes 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids. Each possibility represents a separate embodiment of the present invention.

In general, peptide PKCa inhibitors include the common motif sequence Phe-Ala- Arg-Lys-Gly-Ala, alternatively, in another embodiment, PKCa inhibitors include the common motif sequence Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser.

A "peptide-multimer" according to the present invention refers to a construct comprising at least two, covalently linked, peptides. The at least two peptides may be identical or different. A peptide-multimer may further include a permeability moiety and/or N or C terminal modifications.

While the peptide PKC alpha inhibitors a may be defined by exact sequence or motif sequences, one skilled in the art would understand that peptides that have similar sequences may have similar functions. Therefore, peptides having substantially the same sequence or having a sequence that is substantially identical or similar to a PKC inhibitor disclosed above, are intended to be encompassed. As used herein, the term "substantially the same sequence" includes a peptide including a sequence that has at least 60+% (meaning sixty percent or more), preferably 70+%, more preferably 80+%, and most preferably 90+%, 95+%, or 98+% sequence identity with the sequences defined in the present invention.

A further indication that two peptides are substantially identical is that one peptide is immunologically cross reactive with that of the second. Thus, a peptide is typically substantially identical to a second peptide, for example, where the two peptides differ only by conservative substitutions.

The terms "identical" or percent "identity" in the context of two peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.

The phrase "substantially identical," in the context of peptides, refers to two or more sequences or subsequences that have at least 60+%, preferably 80+%, most preferably 90-95+% amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.

As is generally known in the art, optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman ((1981) Adv Appl Math 2:482), by the homology alignment algorithm of Needleman & Wunsch ((1970) J Mol Biol 48 :443), by the search for similarity method of Pearson & Lipman ((1988) Proc Natl Acad Sci USA 85:2444), by computerized implementations of these algorithms by visual inspection, or other effective methods. The pharmaceutical composition according to the present invention may be administered as a stand-alone treatment or in addition to a treatment with any other therapeutic agent. According to a specific embodiment, PKC alpha inhibitors according to the present invention are administered to a subject in need thereof as part of a treatment regimen in conjunction with at least one anti-cancer agent. The pharmaceutical composition according to the present invention may be administered together with the other agent or separately.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include melanoma, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, or endometrial cancer.

The pharmaceutical composition according to the present invention may be administered together with an anti-neoplastic composition. According to a specific embodiment the anti-neoplastic composition comprises at least one chemotherapeutic agent. The chemotherapy agent, which could be administered together with the peptide inhibitor according to the present invention, or separately, may comprise any such agent known in the art exhibiting anticancer activity, including but not limited to: mitoxantrone, topoisomerase inhibitors, spindle poison vincas: vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine, gemcitabin; podophyllotoxins: etoposide, irinotecan, topotecan, dacarbazin; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxifen, leuprolide, flutamide, and megestrol acetate.

According to a specific embodiment, the chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L- asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressant, adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone analog. According to another embodiment, the chemotherapeutic agent is selected from the group consisting of 5-fiuorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel and doxetaxel.

In various embodiments, peptide PKC alpha inhibitors may be administered by any suitable means, including topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, intravenous, and/or intralesional administration in order to treat the subject. However, in exemplary embodiments, the peptides are formulated for topical application, such as in the form of a liquid, cream, gel, ointment, foam spray or the like.

Therapeutic formulations of the PKC alpha inhibitor used in accordance with the present disclosure are prepared, for example, by mixing a PKC inhibitor having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients and/or stabilizers (see, for example: Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, acetate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (for example, Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In exemplary embodiments, the PKC alpha inhibitors are formulated in a cream. The inhibitors of PKC iso forms are ideal for topical treatment of skin radio-to xicity.

Exemplary formulations for topical administration are disclosed in US20120190611. However, one skilled in the art would understand that alterations of the formulation may be made while retaining the essential characteristics of the cream, such as viscosity, stabilization, non-toxicity and the like. Also, one skilled in the art would recognize that the formulation may be used as a vehicle for any of the peptide PKC inhibitors of the present disclosure.

In another embodiment, an article of manufacture, such as a kit containing materials useful for carrying out the treatment method of the disclosure is provided. In various embodiments, the kit includes a PKC alpha inhibitor as disclosed herein, and instructions for administering the inhibitor to the subject.

The term "instructions" or "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products, and the like.

As disclosed herein, the peptide inhibitor of PKC alpha may be formulated for a specific route of administration. As such, the kit may include a formulation including a peptide inhibitor of PKC that is contained in a suitable container, such as, for example, tubes, bottles, vials, syringes, and the like. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that is effective for treating skin or GI toxicities, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one component in the formulation is an inhibitor of PKC alpha. The label or package insert indicates that the composition is used for treating skin or GI toxicity in a subject suffering therefrom with specific guidance regarding dosing amounts and intervals for providing the formulation including an inhibitor of PKC alpha. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It will be understood, that the specific dose level and frequency of dosage for any particular subject in need of treatment may be varied and will depend upon a variety of factors including the activity of the PKC inhibitor employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, the severity of the particular condition, and the host undergoing therapy. The exact formulation and dosage can be chosen by the individual physician in view of the patient's condition (Fingl et al. "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1 (1975)).

Thus, depending on the severity and responsiveness of the condition to be treated, dosing can be a single or repetitive administration, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disorder is achieved.

In particular embodiments, the PKC alpha inhibitory peptide is provided in the composition at a concentration of between 0.001 and 100 μg/ml (about 0.02 μg-2 mg/day). For example, the concentration may be between 0.001 and 100, 0.01 and 50, 0.01 and 10, 0.01 and 1, and 0.01 and 0.5 μg/ml (about 0.02 μg and 2 mg, 0.2 μg and 1 mg ,0.2 μg and 200 μg, 0.2 μg and 20 μg, and 0.2 and 1 μg/day).

In one exemplary dosing protocol, the method comprises administering a peptide PKC alpha inhibitor to the subject topically, for example as a cream or gel via a dressing. According to some embodiments a cream or gel formulation is one of the formulations disclosed in US2012190611. The peptide is topically applied at a concentration of from about 1 μg/ml to about 1000 μg/ml (about 20μg to 20mg per day), 1 μg/ml to about 500 μg/ml (about 20 μg to lOmg per day), 1 μg/ml to about 100 μg/ml (about 20 μg to about 2mg per day), 1 μg/ml to about 10 μg/ml (about 20 μg to about 200 μg per day), or 10 μg/ml to about 100 μg/ml (about 200 μg to about 2mg per day). The peptide is administered until the condition is treated or as necessary according to the anti-cancer therapy regimen.

In another dosing protocol, the method comprises administering a peptide PKC alpha inhibitor to the subject parentally, for example subcutaneously or intravenously. The peptide is applied in a concentration of from about 1 μg/ml to about 1000 μg/ml (about 20μg to 20mg per day), 1 μg/ml to about 500 μg/ml (about 20 μg to lOmg per day), 1 μg/ml to about 100 μg/ml (about 20 μg to about 2mg per day), 1 μg/ml to about 10 μg/ml (about 20 μg to about 200 μg per day), or 10 μg/ml to about 100 μg/ml (about 200 μg to about 2mg per day). The peptide is administered according to some embodiments in an aqueous solution or buffer. According to some embodiments, the aqueous solution is phosphate buffer. According to other embodiments, aqueous solution is Dulbecco's Phosphate Buffered Saline (DPBS) comprising 0.2 g/L potassium chloride, 0.2 g/L potassium dihydrogen phosphate and 8 g/L sodium chloride, which does not contain calcium and magnesium cations. This is also designated herein as the aqueous solution is DPBS ^_/~ containing no calcium or magnesium cations.

DPBS -/- as used herein is Dulbecco's Phosphate Buffered Saline of the formula:

The "-/-"designation after DPBS means that it contains no calcium or magnesium.

DPBS as used herein has the following formula:

A "+/- "designation after DPBS means that it contains calcium but not magnesium.

A "-/+ "designation after DPBS means that it contains no calcium but does contain magnesium.

A "+/+ "designation after DPBS means that it contains both calcium but not magnesium. According to other embodiments, the aqueous solution is acetate buffer, which according to some embodiments comprises 8.2 g of sodium acetate anhydrous and 300 μΐ of glacial acetic acid in 1 liter of sterile water.

The peptide is administered at least once daily, weekly, biweekly, or monthly until the condition is treated or as necessary according to the anti-cancer therapy regimen. Each possibility represents a separate embodiment of the present invention.

It has been discovered by the present inventors that PKCa inhibition has a remarkable effect on different aspects of skin inflammation associated with radiation and EGFR inhibition therapy. The downregulation by PKCa inhibition of the activation of resident skin immune cells, such as keratinocytes and macrophages, which in turn lead to reduction in the expression of activation markers and the secretion of cytokines and chemokines, attenuates the recruitment and activation of additional immune cells through blood vessels, and prevents the development of severe inflammation, which is usually associated with radiation exposed skin and skin toxicity associated with EGFR inhibition therapy.

Altogether, PKCa inhibition has a beneficial effect on radiation toxicity, including overall survival, GI toxicities and skin toxicities. As such, PKCa inhibitors, according to the present invention, are used as protective agents against radiation exposure and additional skin toxicities associated with anti-cancer therapy.

The following examples are provided to further illustrate the embodiments of the present disclosure, but are not intended to limit the scope. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLES

Peptides

Peptides were synthesized using solid phase synthesis techniques known in the art and purified using conventional known methods.

A non limitative list of peptides which were designed, synthesized and tested is presented in Table 1 : Table 1.

Example 1 - PKCa inhibitor treatment protected irradiated mice from GI injury and increase survival.

Eleven week old BALB/C mice (each weighting about 25gr) were subjected to local accumulative 30Gy dose of ionizing radiation (X-rays, Kimtron Polaris 320kVp 10mA) of two consecutive days. Following irradiation, mice were treated once daily with 1 or 10μg/ml PKCa inhibitor of SEQ ID NO: 7 (in 1 ml of acetate buffer) or with vehicle (1 ml acetate buffer) alone as control, for 14 days. Mortality rate was observed on a daily basis for a whole period of the treatment. The results (percentage of survived mice at the end of the experiment), shown in Figure 1, demonstrate an increase in survival rate in PKCa inhibitor MPDY-1 treated groups. In the group treated with the PKCa inhibitor, 75% (lμg/ml dose) and 100% (10μg/ml dose) survival rate was observed in comparison to only 37% survival in the control group. These results were further corroborated by the PKCa inhibitor MPDY1- protective effects observed on gastrointestinal system. In control group, irradiated mice suffered from low food intake as well as diarrhea few days post irradiation. These observations of GI abnormalities were in correlation with overall survival of mice. These results indicate a protective role for PKCa inhibition from radiation-induced gastrointestinal toxicities. Example 2 - Prevention of radiation-induced hyperplasia

Eleven week old BALB/C mice (each weighting about 25gr) were subjected to local 20Gy dose of ionizing radiation (X-rays, Kimtron Polaris 320kVp 10mA), to the right hind legs. Following irradiation, mice were treated once daily with 1 or lC^g/ml PKCa inhibitor MPDY-1 in 1 ml acetate buffer (1 and 10 μg/day, directly to the mouse leg) or with vehicle alone (acetate buffer) as control, for 21 days. Skin biopsies of the irradiated areas were removed and submitted to paraffin embedding, followed by skin section preparation and subsequent staining with hematoxylin and eosin (H&E), with Masson Trichrom for collagen or for K15 for epidermal stem cells.

It was demonstrated that topical treatment of PKCa inhibitor MPDY-1 significantly prevented radiation-induced hyperplasia of the epidermis. In the control group, epidermal hyperplasia and hair follicle abnormalities were observed in 67% of mice while in the PKCa inhibitor MPDY-1 treated group only 17% of mice exhibited hyperplasia as shown in Figures 2 and 3.

In addition, MPDY-1 prevents radiation-induced collagen degradation in a dose dependent manner. When administered in 10 μg/ml dose (about 10μg/day, 400μg/Kg in 1 ml acetate buffer), MPDY-1 preserves proper collagen structure in at least 50% of irradiated samples as opposed to none in control (Figure 4).

MPDY-1 also protects epidermal K15 positive stem cells from radiation-induced death in a dose dependent manner. Treatment with 10 μg/ml MPDY-1 (SEQ ID NO: 7, in 1 ml acetate buffer) preserves normal K15 expression in at least 60% of irradiated samples, as opposed to none in control (Figure 5).

Example 3 - Prevention of radiation-induced skin-fibrosis

Eleven week old BALB/C mice (each weighting about 25gr) were subjected to accumulative localized radiation of 40Gy for 4 days to buttock area, using the equipment described in Example 1. Irradiated mice were treated topically daily for 6 days, with PKCa inhibitory peptide of SEQ ID NO: 7 ^g/ml (about ^g/day, 40μg/Kg), or 10μg/ml (about 10μg/day, 400μg/Kg) or DPBS ^_/~ (i,.e., without calcium or magnesium). Skin biopsies of the affected buttock areas were removed and paraffin embedded for histological analysis. Masson Trichrome staining of the slides was performed and assessed according to a protocol well known in the art. The results indicate that, while sections of radiated skin exhibited dermis fibrosis, the PKC alpha inhibitor treated group presented collagen deposition and overall dermal structure and appendages similar to non-irradiated controls.

Example 4 - Prevention of hair follicle damage in PKCa depleted mice

Eleven week old BALB/C WT (wild type) mice (each weighting about 25gr) and PKCa depleted mice (KO PKCa, each weighting about 25gr) were subjected to local accumulative 30-40 Gy dose of ionizing radiation (X-rays, Kimtron Polaris 320kVp 10mA) of two consecutive days. 14 days following irradiation, mice were sacrificed and skin samples of the hind leg area were removed for analysis.

PKCa depletion prevented damage to collagen fibers and the formation of fibrotic tissue in the dermis. The overall structure and cellularity of the dermis as well as structure of sebaceous glands and hair follicles were similar to non-irradiated controls (Figure 6).

Example 5 - Direct effect of PKCa inhibitors on cutaneous components

Mice derived immunogenic cells (keratinocytes, splenocytes, and bone-marrow derived dendritic cells and macrophages) were immunologically stimulated using various factors (tumor necrosis factor alpha (TNFa), interleukin 17 (IL-17) and lipopolysaccharide (LPS)) in the absence or presence of PKCa inhibitors (SEQ ID NOs: 1, 9, 1, 5 and 8) in DPBS ^{~ ~}, followed by measurements of secreted cytokines, chemokines and systemic factors by utilizing Luminex fluorometric system. Keratinocytes were derived from newborn C57BL/6J mice skin. The cells were incubated for 5 days in 24 wells plates. Cells were then treated with DPBS^_/", LPS/TNFo/IL-17 or LPS/TNFo/IL-17 +PKCa inhibitors. Medium containing secreted cytokines was collected after 48 hr and analyzed using a Luminex system.

As shown in Figures 7-16 and in table 2, inhibition of PKCa significantly reduced secretion of cytokines associated with radiation- induced response, including IL-la, IL-Ιβ, IL-6 and TNFa. in all skin cells associated with immune response, such as primary keratinocytes, splenocytes, bone-marrow derived dendritic cells and macrophages. Table 2. The influence of PKC alpha inhibitors on TNF alpha and IL-6 secretion in response to LPS stimulation:

Example 6 - PKCa inhibitors as additive to anti-cancer treatment

For live cell viability test, HaCaT cells were cultured in 96 wells plates for 4 days and treated with the epidermal growth factor receptor (EGFR) inhibitor Erbitux (1 μg/ml, 10 μg/ml or 20 μg/ml) with or without MPDY-1 (1 μg/ml). 48 hours post treatment, cells viability was measured by means of Fluorescein diacetate assay (FDA - Sigma). MDPY-1 addition to Erbitux did not affect the cell-killing activity of the antibody.

Furthermore, it was shown that the reduction of cytokine secretion induced by MPDY-1 was not interrupted by Erbitux as shown in Figures 17 for G-CSF and 18 for GM- CSF. For this experiment, HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY-1 (1 μg/ml) and Cetuximab (lmg/ml, as Erbitux®, commercially available from ImClone LLC, a wholly owned subsidiary of Eli Lilly and Company, New York, NY USA and Bristol-Myers Squibb Company, Princeton, NJ USA) ( for two hours, followed by activation with TNFa (10 ng/ml). 48 hours post activation, medium was collected from plates and the cytokine levels were measured using Multiplex ELISA Arrays assay.

In an additional assay, the influence of Erbitux on cytokine secretion induced by LPS and modulated by MPDY- 1 was tested. HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY-1 (1 μg/ml) and Cetuximab (Erbitux) ((Erb - 1 μg/ml) for two hours , followed by activation with LPS (10 μg/ml). 48 hours post activation, medium was collected from the plates and cytokine profile was measured using Multiplex ELISA Arrays assay. As demonstrated in figures 19 and 20 (for G-CSF and TNFa secretion respectively), reduction of cytokine secretion induced by MPDY-1 was not interrupted by Erbitux. In two additional assays, the results of which are demonstrated in Figures 21-24, it was shown that the reduction of cytokines secretion induced by LPS or TNFa and inhibited by MPDY-1 was not interrupted by common chemo therapeutic drugs. In first assay it was shown that reduction of IL-6 (Fig. 21) and IL-lb Fig 22) secretion by MPDY-1 was not interrupted by Epirubicin (Epi), a Epirubicin is a chemotherapeutic drug used in different types of epithelial cancer. It was tested as a stand-alone agent or in combination with MPDY- 1. HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY- 1 (1 μg/ml) and Epirubicin (Epi 100 μΜ) for two hours, followed by activation with LPS (10 μg/ml). 48 hours post activation, medium was collected from plates and cytokines were measured using Multiplex ELISA Arrays assay.

In a second assay, it was further shown that reduction of IL-6 secretion induces by MPDY-1 was not interrupted by the chemotherapeutic drug Cyclophosphamide. HaCaT cells were cultured on 24 wells plates for 2 days. Cells were treated with MPDY-1 (1 μg/ml) and cyclophosphamide (CPA-100 μΜ) for two hours, followed by activation with TNFa (10 ng/ml, Fig. 23) or LPS (10 μg/ml, Fig 24). 48 hours post activation, medium was collected from plates and cytokines were measured using Multiplex ELISA Arrays assay.

Example 7 - An exemplary protocol of a phase II proof of concept trial in human subjects

An open-Label, randomized study to evaluate the safety and efficacy of MPDY-1 (SEQ ID NO: 7), in reducing radiation dermatitis is conducted in breast cancer patients undergoing adjuvant radiation therapy.

The objectives of the trial are: i) to determine if SEQ ID NO: 7 (also denoted MPDY- 1 or HO/02/02), when topically administered at 20μg per day in 20 ml of DPBS ^{~ ~}, can delay the onset or reduce the development of grade 1-4 radiation dermatitis in breast cancer patients undergoing adjuvant radiation therapy when compared to standard of care (SoC) (Aloe Vera), and ii) to determine the safety profile of SEQ ID NO: 7 20μg dose.

Primary Endpoints: Degree of radiation dermatitis experienced during radiation therapy and the follow-up period; and time to onset and duration of radiation dermatitis grade 1-4.

Secondary Endpoints: adverse events (AEs) and serious adverse events (SAEs) assessment and incidence rate; Time to resolution of signs and symptoms of radiation dermatitis; and patient's evaluation of itch, pain, tingling and burning within the radiation fields assessed with a 0-10 cm visual analogue scale (VAS).

Primary Outcome Measure: number of patients using SEQ ID NO: 7 20μg that developed grade 1-4 radiation dermatitis during adjuvant radiation therapy for breast cancer compared to SoC (Aloe Vera) commonly used as standard of care treatment.

Secondary Outcome Measure: frequency and severity of adverse events reported by the patients and assessed clinically by NCI CTC v2.0

The trial is conducted in a single site using 2: 1, Active : SoC (Aloe Vera), as per Randomization Coding list.

The maximal study duration time is 13 weeks in total, including screening, treatment, boost and optional follow up, as follows:

• Screening - Up to 30 Days

• Prophylctic treatment -3- days

• Radiation Therapy (25 Fractions) - 35 days

• Boost Radiation (8 Fractions) - 10 days

• Post boost treatment - 7 days

• Follow-up (Optional) - 7 days post last HO/02/02 20μg vs. SoC treatment.

The two treatment arms are:

Arm 1: Study drug - SEQ ID NO: 7 20μgin DPBS7^" topically administered over gauze to the wound site ) applied daily topically for 15 minutes on treatment fields;

Arm 2: SoC -Aloe Vera Gel (New ESI^® Paraben-free Aloe Vera Gel, pure to 97%). In addition, as a control, sterile and deionized water over gauze is applied daily, topically for 15 minutes on treatment fields to create the same cooling effect as in treatment arm 1).

A total of up to 90 patients are enrolled into the study. Patients are females, age over 18, diagnosed with breast cancer requiring breast and regional radiation therapy for adjuvant treatment. All patients begin study prophylactic treatment (as per Arml / Arm2 allocation) within 3 days prior to the first radiation therapy fraction.

The radiotherapy is given to the breast and lymphatic drainage areas as for the discretion of the treating radiation oncologist. Radiotherapy is delivered daily with 2.0 Gy/fraction in 25 fractions over 5 weeks using linear accelerator by Varian, either Clinac 2100CD., Clinac 600CD , True Beam or NOVALIS. Patients are examined at baseline and consecutively in weekly intervals during radiotherapy, days 5 and 10 within boost radiotherapy, 7 days post last radiotherapy and single optional visit 7 days post last study drug, SEQ ID NO: 7 20μg vs. SoC (Aloe Vera) treatment.

Study treatment discontinued by principle investigator (PI) when radiation dermatitis symptoms are as follows:

1. Wet desquamation, any grade 3, any signs of unexpected side effects as for the discretion of the treating radiation oncologist.

2. Consent is withdrawn.

Patients who discontinue the study treatment for reasons other than consent withdrawal, are asked to continue and have daily skin assessment until complete skin recovery or as per PI decision (rescue therapy).

Screening phase (Days -30 to -3):

The screening phase commences after obtaining informed consent and is continued no longer than 28 days before SEQ ID NO: 7 20μg vs. SoC (Aloe Vera) treatment.

Demographic, Medical History, Concomitant Medications and inclusion / exclusion information is collected. Physical examination, vital signs, blood tests (Hematology and Biochemistry and Serum Pregnancy (if applicable)) are performed.

Former Hematology and Biochemistry results are valid up to 21 days prior to Day -3 visit / first prophylactic SEQ ID NO: 7 20μg vs. SoC (Aloe Vera) treatment and therefore should not be taken during this visit.

Treatment Phase (Pre First Radiation Therapy, Days -3 to 0):

All screening evaluations are completed no longer then first prophylactic SEQ ID NO: 7 20μg vs. SoC (Aloe Vera) treatment.

After meeting all inclusion criteria and ruling out all exclusion criteria, patients are randomized and allocated into one of two study arms.

Study drug, SEQ ID NO: 7 20μg in DPBS7^" or SoC (Aloe Vera), is provided by the company as required per protocol from screening, throughout the duration of the study and to be applied either at home or at the clinic once daily.

All eligible patients are asked to be treated with prophylaxis by SEQ ID NO: 7 20μg or SoC (Aloe Vera) in addition to sterile water wet gauze as per specific randomization list for 3 days prior to first radiation treatment. Sterile and deionized water over gauze is applied daily in addition to SoC (Aloe Vera treatment), topically for 15 minutes on treatment fields to create the same cooling effect as in treatment arm 1.

The area to be irradiated is documented before the first study drug vs. SoC (Aloe Vera) exposure. Skin assessment is visually performed (for any skin changes, including erythema, rash, dry desquamation, wet desquamation and area in the breast or supraclavicular region with the side effect) by Pi/study nurse according to the National Cancer Institute skin radiation toxicity criteria (grade 1-4) version 2.0, April 30, 1999. Baseline photo documentation should be made prior to HO/02/02 20μg or SoC (Aloe Vera) exposure.

Treatment phase (Post First Radiation Therapy Days 1 to 35 and boost Days 36 to 45):

The clinical volume for radiotherapy is the breast and regional lymph nodes. Radiotherapy is delivered daily with 2.0 Gy/fraction in 25 fractions over 5 weeks and up to 8 boost radiotherapy fractions using linear accelerators by Varian.

Newly and ongoing skin assessment are visually performed by Pi/study nurse according to criteria mentioned above. Skin assessment and photos are taken during the study visits as follows:

1. Day 1 (+1 day)

2. Day 12 (+3 days)

3. Day 24 (+1 day) or first boost day

4. Boost - Day 5

5. Boost - Last fraction of radiation therapy

6. Post boost - 7 (+3) days post radiation.

7. Follow up visit (optional) - 7 (±2) days post last study drug vs. SoC treatment.

Ongoing adverse events are recorded and monitored until complete resolution or until PI/Co-PI deems it to be no longer medically indicated.

Blood tests (Hematology and Biochemistry) are performed on radiotherapy last fraction and as per PI instructions during the study.

Patients are asked to fulfill a VAS questionnaire in regarding to pain, burning, itching and tingling within radiation field assessed with a 0-10 cm visual analogue scale (VAS) on days: 12 (+3) and 25 (+1, or first boost treatment day), Boost day 4 (+1). Follow UP:

A single follow up visit is optionally performed for safety and efficacy assessment 7 (±2) days post last study drug SEQ ID NO: 7 2C^g vs. SoC (Aloe Vera) treatment.

Ongoing skin assessment are visually performed by Pi/study nurse as indicated above and are monitored until complete resolution or until PI/Co-PI deems it to be no longer medically indicated.

Planned Sample Size: Up to 90 patients. This is a proof of concept study which is designed to provide preliminary evidence of clinical efficacy of SEQ ID NO: 7 20 μg vs. SoC (Aloe Vera) in prevention and treatment radiation-induced dermatitis in patients undergoing breast cancer radiation therapy.

Inclusion Criteria:

1. Female patients 18 years old and above.

2. Histology confirmed unilateral breast cancer following lumpectomy

3. Planned to receive 50 Gy, whole breast XRT and regional lymph nodes radiation.

4. ECOG performance status 0-2

5. Completed Chemotherapy 3 weeks prior to XRT (if applicable)

6. Patient should be available for the entire study period, and be able and willing to adhere to protocol requirements;

7. Patient must sign an informed consent form prior to undergoing any study-related procedures.

Exclusion Criteria

1. Known uncontrolled diabetes

2. Prior radiation to breast

3. Known connective tissue disorder

4. Known skin disease over the treated breast

5. Prior burn over treated area

6. Evidence of infection or inflammation of breast to be treated.

7. Receiving biological therapy or hormone therapy (other than Herceptin) during radiation treatment/study duration and 4 weeks prior to study entry.

8. Pre-existing skin breakdown within the planned radiotherapy field at the time of study entry. 9. Pregnancy, planned pregnancy, lactation or inadequate contraception as judged by the Investigator.

10. Participation in another investigational drug or vaccine trial concurrently or within 30 days.

11. Use of any other topical or systemic treatments aimed at radiation dermatitis.

12. Use of a prescription or over-the-counter medication that contains hydrocortisone or any other cortisone or corticosteroid containing preparation.

Safety Parameters:

1. Skin Assessment

2. Clinical laboratory parameters

3. AEs / SAEs incidence rate and severity (according to CTCAE version 4.0)

While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow.

All references cited herein are hereby incorporated by reference in their entireties for all purposes.

Claims

1. A method of preventing, ameliorating or treating damage to an organ or tissue of a subject, caused by radiation, comprising administering to the subject a pharmaceutical composition comprising at least one PKC alpha inhibitory peptide of 6-24 amino acids comprising a sequence selected from the group consisting of:

Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser (SEQ ID NO: 1); and

Phe-Ala-Arg-Lys-Gly-Ala (SEQ ID NO: 2), or an analog, salt or derivative thereof; thereby preventing, ameliorating or treating the damage to the organ or tissue.

2. The method of claim 1 wherein the subject suffers from a disease or disorder which is treated by irradiation.

3. The method of claim 2 wherein the disease is a cancer.

4. The method of claim 1 wherein the subject was exposed to accidental ionizing radiation.

5. The method of claim 1 wherein the damage is to at least one tissue or organ selected from the group consisting of: skin, gastrointestinal track, bone marrow, reproductive system, muscle and brain.

6. The method of claim 5 wherein the damage includes skin toxicity.

7. The method of claim 6 wherein the skin toxicity comprises radiation dermatitis

8. The method of claim 9 wherein the damage includes toxicity to the gastrointestinal tract.

9. The method of claim 1 wherein the damage includes injury to hair follicles.

10. The method according to any one of claimsl-9, wherein the PKCa inhibitory peptide consists of 8-15 amino acids comprising the sequence Thr-Leu-Asn-Pro-Gln-Trp-Glu- Ser (SEQ ID NO: 1), or an analog, salt or derivative thereof.

11. The method according to any one of claimsl-9, wherein the PKCa inhibitory peptide consists of 6-12 amino acids comprising a sequence selected from the group consisting of:

Phe-Ala-Arg-Lys-Gly-Ala (SEQ ID NO: 2); Phe- Ala- Arg-Lys-Gly- Ala-Leu (SEQ ID NO: 3); Phe- Ala- Arg-Lys-Gly- Ala-Leu- Arg (SEQ ID NO: 4); Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (SEQ ID NO: 5);

Phe- Ala- Arg-Lys-Gly- Ala- Arg-Gln (SEQ ID NO: 6); or an analog, salt or derivative thereof.

12. The method according to any one of claimsl-9, wherein the PKC alpha inhibitory peptide is selected from the group consisting of:

H-Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser-OH (SEQ ID NO: 1);

H-Phe-Ala-Arg-Lys-Gly-Ala-Leu-OH (SEQ ID NO: 3); and

13. The method according to any one of claims 1-12 wherein the PKC alpha inhibitory peptide is conjugated with at least one permeability moiety.

14. The method according to claim 13, wherein the permeability moiety is connected, by a covalent bond, to the N-terminus of the peptide.

15. The method according to claim 14 wherein the permeability moiety is a hydrophobic moiety selected from a fatty acid, a steroid and a bulky aromatic or aliphatic compound.

16. The method according to claim 15 wherein the fatty acid comprises an aliphatic tail of 3-12 carbons.

17. The method according to claim 15 wherein the fatty acid is selected from the group consisting of: myristic acid, palmitic acid and cholesterol.

18. The method according to claim 17 wherein the peptide conjugate is selected from the group consisting of:

Myristoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH (SEQ ID NO: 7);

Palmitoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH (SEQ ID NO: 8); and

19. The method according to any one of claims 1-18 wherein the PKC alpha inhibitory peptide comprises at least one modification selected from the group consisting of: amino- terminus modification and carboxy- terminus modification.

20. The method of claim 19, wherein the at least one modification is selected from the group consisting of: N-terminus acylation, C-terminus amidation and modification of the C-terminal acid to an alcohol.

21. The method according to any one of claims 1-20 wherein the PKC alpha inhibitory peptide is an analog of a peptide selected from the group consisting of: SEQ ID NOs: 1-9, comprising at least one peptide-terminal modification selected from the group consisting of: N-terminus acylation, C-terminus amidation and modification of the C- terminal acid to an alcohol.

22. The method according to claim 1 wherein the PKC alpha inhibitor is a peptide multimer of 12-60 amino acids, comprising at least two, identical or different, sequences.

23. The method of claim 22 wherein the peptide multimer comprises a permeability moiety.

24. The method according to any one of claims 1 -23, wherein the PKC alpha inhibitory peptide is administered prior to exposure to radiation.

25. The method according to any one of claims 1 -23, wherein the PKC alpha inhibitory peptide is administered after exposure to radiation.

26. The method according to any one of claims 1-25, wherein the pharmaceutical composition is administered by a route selected from the group consisting of: topical, transdermal, parenteral, nasal and oral.

27. The method according to any one of claims 1-25, wherein the pharmaceutical composition is administered topically.

28. The method according to any one of claims 1-25, wherein the pharmaceutical composition is administered parenterally.

29. A kit comprising a pharmaceutical composition according to claim 1 and instructions for administering the PKC alpha inhibitory peptide.

30. A method of improving the survival of a subject exposed to radiation or treated with a chemotherapy or biological therapy against cancer, comprising administering a pharmaceutical composition comprising at least one PKCa inhibitory peptide, thereby improving the survival of the subject.

31. A method of preventing, ameliorating or treating damage caused to an organ or tissue of a subject by anti-cancer therapy, comprising administering a pharmaceutical composition comprising at least one PKC alpha inhibitory peptide of 6-24 amino acids comprising a sequence selected from the group consisting of:

Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser (SEQ ID NO: 1); and

32. The method of claim 31 wherein the anti-cancer therapy comprises a treatment selected from the group consisting of: radiotherapy, chemotherapy and biological therapy.

33. The method of claim 33 wherein the biological therapy comprises administration of an epidermal growth factor receptor (EGFR) inhibitor.

34. The method of claim 32, wherein the pharmaceutical composition is administered in conjunction with chemotherapy.

35. The method of claim 31, wherein the PKC alpha inhibitory peptide is selected from the group consisting of:

H-Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser-OH (SEQ ID NO: 1);

H-Phe-Ala-Arg-Lys-Gly-Ala-Leu-OH (SEQ ID NO: 3); and

H-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH (SEQ ID NO: 5);

Myristoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH (SEQ ID NO: 7);

Palmitoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH (SEQ ID NO: 8); and

36. The method according to any one of claims 31-35, wherein the pharmaceutical composition is administered by a route selected from the group consisting of: topical, transdermal, parenteral, nasal and oral.

37. The method according to any one of claims 31-35, wherein the pharmaceutical composition is administered topically.

38. The method according to any one of claims 31-35, wherein the pharmaceutical composition is administered parenterally.

39. A pharmaceutical composition comprising at least one PKCa inhibitory peptide of 6- 24 amino acids comprising a sequence selected from the group consisting of:

Thr-Leu-Asn-Pro-Gln-Trp-Glu-Ser (SEQ ID NO: 1); and

Phe-Ala-Arg-Lys-Gly-Ala (SEQ ID NO: 2), or an analog, salt or derivative thereof; for preventing, ameliorating or treating damage caused to an organ or tissue of a subject, by radiation.