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WO2004006887A2 - Compositions et methodes pour le traitement de troubles cutanes - Google Patents

Compositions et methodes pour le traitement de troubles cutanes Download PDF

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Publication number
WO2004006887A2
WO2004006887A2 PCT/IL2003/000576 IL0300576W WO2004006887A2 WO 2004006887 A2 WO2004006887 A2 WO 2004006887A2 IL 0300576 W IL0300576 W IL 0300576W WO 2004006887 A2 WO2004006887 A2 WO 2004006887A2
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WIPO (PCT)
Prior art keywords
vitamin
metabolite
nicotinamide
cadpr
group
Prior art date
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PCT/IL2003/000576
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English (en)
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WO2004006887A3 (fr
Inventor
Olga Bloch
Avikam Harel
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Dermipsor Ltd
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Dermipsor Ltd
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Filing date
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Priority claimed from US10/192,842 external-priority patent/US20030032617A1/en
Application filed by Dermipsor Ltd filed Critical Dermipsor Ltd
Priority to EP03764101A priority Critical patent/EP1539100A4/fr
Priority to CA002492248A priority patent/CA2492248A1/fr
Priority to AU2003242965A priority patent/AU2003242965B2/en
Publication of WO2004006887A2 publication Critical patent/WO2004006887A2/fr
Publication of WO2004006887A3 publication Critical patent/WO2004006887A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A61K8/673—Vitamin B group
    • A61K8/675—Vitamin B3 or vitamin B3 active, e.g. nicotinamide, nicotinic acid, nicotinyl aldehyde
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the present invention relates to compositions, kits and methods for treating skin disorders and, more particularly, to compositions, kits and methods for treating hyperproliferative epidermal pathologies and other conditions of the epidermis.
  • compositions for treating skin disorders are known. For example, there are many reports of successful treatment of psoriasis and other related skin disorders in humans, following oral or topical treatment with vitamin D3 and its analogues [6].
  • cADPR cyclic ADP ribose
  • Nicotinamide (NA) is a water-soluble derivative of vitamin B, whose physiological active forms are nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH).
  • the physiological active forms of NA serve as coenzyme in a variety of important metabolic reactions.
  • NA nicotinamide
  • NA is also known as a weak free-radical scavenger, inhibitor of poly- ADP -ribose synthetase and inducible nitric oxide synthase in pancreatic islets [4].
  • NA is also known as a weak free-radical scavenger, inhibitor of poly- ADP -ribose synthetase and inducible nitric oxide synthase in pancreatic islets [4].
  • NA is also known as a weak free-radical scavenger, inhibitor of poly- ADP -ribose synthetase and inducible nitric oxide synthase in pancreatic islets [4].
  • NA is also known as a weak free-radical scavenger, inhibitor of poly- ADP -ribose synthetase and inducible nitric oxide synthase in pancreatic islets [4].
  • NA is also known as a weak free-radical s
  • U.S. Patent No. 4,505,896 discloses compositions and methods for the treatment of acne vulgaris.
  • the compositions disclosed in this patent include nicotinic acid or nicotinamide and, optionally, another chemical agents such as sulfur, salicylic acid and Vitamin A acid, which are known to be effective in treating acne.
  • the compositions and methods disclosed in U.S. Patent No. 4,505,896 are specifically directed toward the treatment of acne vulgaris, which is an inflammatory disease and not a hyperproliferative benign (e.g., psoriasis) or malignant skin disorders.
  • compositions for treating skin conditions which include derivatives of nicotinic acid or nicotinamide and, in particular, methyl nicotinate, as the active ingredient. These compositions are topically applies and are directed toward the treatment of acne and other skin conditions such as fine lines and age spots, burns, etc.
  • U.S. Patent No. 4,505,896 U.S. Patent No. 6,248,763 fails to teach compositions and methods for the treatment of hyperproliferative skin disorders.
  • the prior art teaches various roles and uses of NA and/or combinations thereof with various agents
  • the prior art clearly fails to teach the effects of NA on epidermal cells and, in particular, on the proliferation and differentiation of these cells and hence fails to teach uses of NA or its derivatives in the treatment of hype ⁇ roliferative skin disorders and as an anti-oxidant in epidermal cells.
  • the prior art f rther fails to teach uses of Vitamin D3, atRA and cADPR and their agonistic derivatives in the treatment of hype ⁇ roliferative skin disorders.
  • nicotinamide and/or other agents that are known to be associated with cell differentiation and/or proliferation can exert anti-proliferative effects in various epidermal cell associated pathologies.
  • Vitamins A and D3 exert synergistic effect on epidermal cell proliferation
  • nicotinamide is highly effective as an anti-oxidant against auto-oxidative agents.
  • a method of treating a benign or malignant hype ⁇ roliferative epidermal pathology in a subject in need thereof comprises administering to the subject a therapeutically effective amount of an agent selected from the group consisting of nicotinamide, a nicotinamide agonist, a nicotinamide derivative, a nicotinamide metabolite and prodrugs thereof.
  • Another method treating a benign or malignant hype ⁇ roliferative epidermal pathology in a subject in need thereof.
  • the method comprises administering to the subject a therapeutically effective amount of an agent selected from the group consisting of cyclic adenosine diphosphate-ribose (cADPR), a cADPR derivative, a cADPR metabolite, a cADPR agonist and prodrugs thereof.
  • an agent selected from the group consisting of cyclic adenosine diphosphate-ribose (cADPR), a cADPR derivative, a cADPR metabolite, a cADPR agonist and prodrugs thereof.
  • nicotinamide in combination with cADPR results in a synergistic effect with respect to inhibiting proliferation of epidermal cells, and hence, according to yet another aspect of the present invention there is provided a method of treating a benign or malignant hype ⁇ roliferative epidermal pathology in a subject in need thereof.
  • This method comprises administering to the subject a therapeutically effective amount of an agent selected from the group consisting of nicotinamide, a nicotinamide agonist, a nicotinamide derivative, a nicotinamide metabolite and prodrugs thereof, in combination with a therapeutically effective amount of an agent selected from the group consisting of cyclic adenosine diphosphate-ribose (cADPR), a cADPR derivative, a cADPR metabolite, a cADPR agonist and prodrugs thereof.
  • an agent selected from the group consisting of cyclic adenosine diphosphate-ribose (cADPR), a cADPR derivative, a cADPR metabolite, a cADPR agonist and prodrugs thereof.
  • Each of the above methods can further comprise administering to the subject, in combination with the agent(s) described above, a therapeutically effective amount of an agent selected from the group consisting of Vitamin D3, a Vitamin D3 metabolite, a Vitamin D3 agonist, a Vitamin D3 derivative and prodrugs thereof.
  • each of the above methods can further comprise administering to the subject, in combination with the agent(s) described above, a therapeutically effective amount of an agent selected from the group consisting of Vitamin A, a Vitamin A metabolite, a Vitamin A agonist, a Vitamin A derivative and prodrugs thereof.
  • additional methods of treating a benign or malignant hype ⁇ roliferative epidermal pathology in a subject in need thereof comprise administering to the subject a therapeutically effective amount of an agent selected from the group consisting of nicotinamide, a nicotinamide agonist, a nicotinamide derivative, a nicotinamide metabolite and prodrugs thereof, or a therapeutically effective amount of an agent selected from the group consisting of cyclic adenosine diphosphate-ribose (cADPR), a cADPR derivative, a cADPR metabolite, a cADPR agonist and prodrugs thereof, in combination with a therapeutically effective amount of an agent selected from the group consisting of Vitamin D3, a Vitamin D3 metabolite, a Vitamin D3 agonist, a Vitamin D3 derivative and prodrugs thereof, or in combination with a therapeutically effective amount of an agent selected from the group consisting of Vitamin D3, a Vitamin D3 metabolite,
  • compositions identified for use in the treatment of a benign or malignant hype ⁇ roliferative epidermal pathology and/or for use in the treatment of a condition whereby increasing anti-oxidative properties of epidermal cells is advantageous.
  • a condition can be, for example, aging or cancer.
  • compositions of the present invention comprise, as an active ingredient or as a combination of active ingredients, a therapeutically effective amount of an agent selected from the group consisting of nicotinamide, a nicotinamide agonist, a nicotinamide derivative, a nicotinamide metabolite and prodrugs thereof, and/or a therapeutically effective amount of an agent selected from the group consisting of cyclic adenosine diphosphate-ribose (cADPR), a cADPR derivative, a cADPR metabolite, a cADPR agonist and prodrugs thereof, and a pharmaceutically, cosmetically or cosmeceutically acceptable carrier.
  • an agent selected from the group consisting of nicotinamide, a nicotinamide agonist, a nicotinamide derivative, a nicotinamide metabolite and prodrugs thereof and/or a therapeutically effective amount of an agent selected from the group consisting of cyclic
  • compositions can further comprise, in combination with the agent(s) described above, a therapeutically effective amount of an agent selected from the group consisting of Vitamin D3, a Vitamin D3 metabolite, a Vitamin D3 agonist, a Vitamin D3 derivative and prodrugs thereof.
  • each of the above pharmaceutical compositions can further comprise, in combination with the agent(s) described above, a therapeutically effective amount of an agent selected from the group consisting of Vitamin A, a Vitamin A metabolite, a Vitamin A agonist, a Vitamin A derivative and prodrugs thereof.
  • the therapeutically effective amount of nicotinamide preferably ranges between 1 mM and 50 mM, most preferably, between 1 mM and 10 mM.
  • the therapeutically effective amount of cADPR preferably ranges between 10 ⁇ M and 100 ⁇ M.
  • compositions of the present invention can optionally be packaged in a container and identified in print in or on the container, for use in the treatment of a benign and/or a malignant hype ⁇ roliferative epidermal pathology.
  • the pharmaceutical, cosmetic or cosmeceutical compositions of the present mvention can be packaged in a container and identified in print in or on the container for use in the treatment of a condition whereby increasing anti-oxidative properties of epidermal cells is advantageous.
  • pharmaceutical, cosmetic or cosmeceutical kits which comprise a therapeutically effective amount of an agent selected from the group consisting of nicotinamide, a nicotinamide agonist, a nicotinamide derivative, a nicotinamide metabolite and prodrugs thereof, and/or a therapeutically effective amount of an agent selected from the group consisting of cyclic adenosine diphosphate-ribose (cADPR), a cADPR derivative, a cADPR metabolite, a cADPR agonist and prodrugs thereof.
  • cADPR cyclic adenosine diphosphate-ribose
  • Each of the above pharmaceutical, cosmetic or cosmeceutical kits can further comprise a therapeutically effective amount of an agent selected from the group consisting of Vitamin D3, a Vitamin D3 metabolite, a Vitamin D3 agonist, a Vitamin D3 derivative and prodrugs thereof.
  • each of the above pharmaceutical, cosmetic or cosmeceutical kits can further comprise a therapeutically effective amount of an agent selected from the group consisting of Vitamin A, a Vitamin A metabolite, a Vitamin A agonist, a Vitamin A derivative and prodrugs thereof.
  • the agents are packaged individually within the kit.
  • methods of increasing anti-oxidative properties of epidermal cells comprise contacting the cells with an effective amount of an agent selected from the group consisting of nicotinamide, a nicotinamide agonist, a nicotinamide derivative, a nicotinamide metabolite and prodrugs thereof and/or with an effective amount of an agent selected from the group consisting of cyclic adenosine diphosphate-ribose (cADPR), a cADPR derivative, a cADPR metabolite, a cADPR agonist and prodrugs thereof.
  • an agent selected from the group consisting of nicotinamide, a nicotinamide agonist, a nicotinamide derivative, a nicotinamide metabolite and prodrugs thereof comprise contacting the cells with an effective amount of an agent selected from the group consisting of cyclic adenosine diphosphate-ribose (cADPR), a cADPR derivative,
  • the agent is nicotinamide and the effective amount ranges between 1 mM and 50 mM.
  • Other methods of increasing anti-oxidative properties of epidermal cells comprise contacting the cells with one of the agents described above, in combination with an effective amount of an agent selected from the group consisting of Vitamin D3, a Vitamin D3 metabolite, a
  • Vitamin D3 agonist a Vitamin D3 derivative and prodrugs thereof or an effective amount of an agent selected from the group consisting of Vitamin A, a Vitamin A metabolite, a Vitamin A agonist, a Vitamin A derivative and prodrugs thereof.
  • methods of inhibiting proliferation of benign or malignant hype ⁇ roliferative epidermal cells comprise contacting the cells with a therapeutically effective amount of an agent selected from the group consisting of nicotinamide, a nicotinamide agonist, a nicotinamide derivative, a nicotinamide metabolite and prodrugs thereof and/or with a therapeutically effective amount of an agent selected from the group consisting of cyclic adenosine diphosphate-ribose (cADPR), a cADPR derivative, a cADPR metabolite, a cADPR agonist and prodrugs thereof.
  • an agent selected from the group consisting of nicotinamide, a nicotinamide agonist, a nicotinamide derivative, a nicotinamide metabolite and prodrugs thereof comprise contacting the cells with a therapeutically effective amount of an agent selected from the group consisting of nicotinamide, a nicotin
  • Other methods of inhibiting proliferation of benign or malignant hype ⁇ roliferative epidermal cells comprise contacting the cells with one of the agents described above, in combination with a therapeutically effective amount of a second agent selected from the group consisting of Vitamin D3, a Vitamin D3 metabolite, a Vitamin D3 agonist, a Vitamin D3 derivative and prodrugs thereof or a second agent selected from the group consisting of Vitamin A, a Vitamin A metabolite, a Vitamin A agonist, a Vitamin A derivative and prodrugs thereof
  • the hype ⁇ roliferative benign epidermal pathology is selected from the group consisting of psoriasis, ichythyiosis, common warts, keratoacanthoma, seborrhoic keratosis and seborrhea.
  • the hype ⁇ roliferative malignant epidermal pathology is selected from the group consisting of squamous-cell carcinoma (SCC), basal cell carcinoma (BCC) and a non-melanoma skin cancer (NMSC).
  • SCC squamous-cell carcinoma
  • BCC basal cell carcinoma
  • NMSC non-melanoma skin cancer
  • Vitamin D3 metabolite is l ,25-dihydroxy- vitamin D3.
  • a therapeutically effective amount of l ⁇ ,25-dihydroxy- vitamin D3 is l ,25-dihydroxy- vitamin D3.
  • D3 ranges between 1 nM and 200 nM.
  • Vitamin A metabolite is an all-trans-retinoic acid.
  • a therapeutically effective amount of all-trans-retinoic acid ranges between 0.1 nM and 10 nM.
  • the Vitamin A agonist is a retinoic acid receptor agonist.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing methods, pharmaceutical, cosmetic or cosmeceutical compositions and pharmaceutical, cosmetic or cosmeceutical kits for treating various benign and malignant proliferative pathologies and for increasing anti-oxidative properties of epidermal cells, using highly efficient agents or combinations of agents that exert synergistic effects.
  • FIGs. l(a-b) show the anti-proliferative effect of NA on HaCat and A431 cell proliferation ( Figure la) and on cultured human epidermal keratinocytes ( Figure lb);
  • FIG. 2 shows the anti-proliferative effect of a D3 metabolite (l ⁇ 25(OH) 2 D3) on HaCat and A431 cell proliferation;
  • FIG. 3 shows the anti-proliferative effect of cADPR on HaCat and A431 cell proliferation
  • FIG. 4 shows the anti-proliferative effect of a Vitamin A metabolite (atRA) on HaCat and A431 cell proliferation
  • FIGs. 5(a-b) show the anti-proliferative effect of a combination of NA and a D3 metabolite (l ⁇ 25(OH) 2 D3) on HaCat cell line proliferation ( Figure 5a) and the synergistic effect of this combination as compared with the anti-proliferative effects of each of these compounds separately (NA and l ⁇ 25(OH) 2 D3), on this cell line, shown as the effect of the combined treatment minus the effect of each of the compounds ( Figure 5b); FIGs.
  • FIG. 6(a-b) show the anti-proliferative effect of a combination of NAand a D3 metabolite (l ⁇ 25(OH) 2 D3) on A431 cell line proliferation ( Figure 6a) and the synergistic effect of this combination as compared with the anti-proliferative effects of each of these compounds separately (NA and l ⁇ 25(OH) 2 D3), on this cell line, shown as the effect of the combined treatment minus the effect of each of the compounds ( Figure 6b);
  • FIGs. 7(a-b) show the anti-proliferative effect of a combination of NA and cADPR on HaCat cell line proliferation ( Figure 7a) and the synergistic effect of this combination as compared with the anti-proliferative effects of each of these compounds separately (NA and cADPR), on this cell line, shown as the effect of the combined treatment minus the effect of each of the compounds ( Figure 7b);
  • FIGs. 8(a-b) show the anti-proliferative effect of a combination of NA and cADPR on A431 cell line proliferation (Figure 7a) and the synergistic effect of this combination as compared with the anti-proliferative effects of each of these compounds separately (NA and cADPR), on this cell line, shown as the effect of the combined treatment minus the effect of each of the compounds ( Figure 7b);
  • FIGs. 9(a-b) show the anti-proliferative effect of a combination of NA and a Vitamin A metabolite (atRA) on HaCat cell line proliferation ( Figure 9a) and the synergistic effect of this combination as compared with the anti-proliferative effects of each of these compounds separately (NA and atRA), on this cell line, shown as the effect of the combined treatment minus the effect of each of the compounds ( Figure 9b);
  • FIGs. l ⁇ (a-b) show the anti-proliferative effect of a combination of NA and a Vitamin A metabolite (atRA) on A431 cell line proliferation ( Figure 10a) and the synergistic effect of this combination as compared with the anti- proliferative effects of each of these compounds separately (NA and atRA), on this cell line, shown as the effect of the combined treatment minus the effect of each of the compounds ( Figure 10b);
  • FIG. 11 shows the effect of NA on involucrin and keratin klO expression in HaCat cells;
  • FIG. 12 shows the effect of NA on basal and envelope comified cell expression in HaCat cell line
  • FIG.13 shows the effect of NA on apoptosis level in HaCat and A431 cell lines
  • FIG. 14 shows the resistance of HaCat cells treated for long-term period with NA to oxidative stress induced by hydrogen peroxide (H 2 0 2 ).
  • the present invention is of pharmaceutical, cosmetic or cosmeceutical compositions, pharmaceutical cosmetic or cosmeceutical kits and methods, which can be used in the treatment of skin disorders.
  • the present invention can be used in the treatment of benign and malignant proliferative epidermal pathologies, and in the treatment of conditions that require increasing anti-oxidative properties of epidermal cells, such as, for example, aging.
  • nicotinamide and/or other agents that are known to be associated with cell differentiation and/or proliferation can exert anti-proliferative effects in various epidermal cell associated pathologies. It was further hypothesized that using such agents in combination with other agents that are known to affect certain skin disorders, such as Vitamins D3 and A and their analogs, could result in enhanced anti-proliferative activity of the agents. While reducing the present invention to practice, as is demonstrated in the
  • HaCat cell line a spontaneously immortalized human keratinocyte
  • HaCat cells serves as a model for highly proliferative epidermis, such as, but not limited to, psoriatic epidermis [9], and as a model for effects of external modulators of epidermal differentiation [10]
  • an epidermal carcinoma cell line which is referred to herein as "A431 cell line” or “A431 cells”, which bear the mutated alleles of p53, and serves as a model for testing anti-cancerogenic drugs [11] and hence as a model for malignant hype ⁇ roliferative pathologies.
  • the nicotinamide agent is either nicotinamide itself, or any nicotinamide analog that is known to act similarly thereto, such as, but not limited to, a nicotinamide agonist, a nicotinamide derivative or a nicotinamide metabolite.
  • the nicotinamide agent can further be a prodrug of each of the nicotinamide agents described.
  • the cADPR agent is either cADPR itself or any cADPR analog that is known to act similarly thereto, such as, but not limited to, a cADPR agonist, a cADPR derivative or a cADPR metabolite.
  • the cADPR agent can further be a prodrug of each of the cADPR agents described.
  • hypo ⁇ roliferative epidermal pathology includes any disease, condition or syndrome that is characterized by a higher than normal level of proliferation of epidermal cells, and, as a rule, also by abnormal differentiation.
  • the hype ⁇ roliferative epidermal pathology may be malignant or benign, as is discussed hereinabove and is demonstrated in detail in the Examples section that follows.
  • malignant hype ⁇ roliferative epidermal pathologies that are treatable by the methods of the present invention include, without limitation, squamous-cell carcinoma (SCC), basal-cell carcinoma (BCC) and other non-melanoma skin cancers (NMSCs).
  • SCC squamous-cell carcinoma
  • BCC basal-cell carcinoma
  • NMSCs non-melanoma skin cancers
  • benign hype ⁇ roliferative epidermal pathologies that are treatable by the methods of the present invention include, without limitation, psoriasis, common warts, keratoacanthoma, seborrhoic keratosis, seborrhea and ichthyosis.
  • the term "treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a pathology, substantially ameliorating clinical symptoms of a pathology or substantially preventing the appearance of clinical symptoms of a pathology. These effects may be manifested, for example, by a decrease in the rate of proliferation, an improved differentiation or a combination thereof and/or by complete elimination of the abnormal proliferation and differentiation of the epidermal cells in the treated subject.
  • administering describes a method for bringing a nicotinamide agent, a cADPR agent, or any other agent or combination of agents described herein, and epidermal cells affected by the pathology together in such a manner that the agent can affect the proliferation and/or differentiation of these cells.
  • the administration according to the present invention is accomplished either by topical application or by subcutaneous administration of the agent or the combination of agents.
  • terapéuticaally effective amount describes an amount administered to an individual, which is sufficient to abrogate, substantially inhibit, slow or reverse the progression of an epidermal pathology, to substantially ameliorate clinical symptoms of an epidermal pathology or substantially prevent the appearance of clinical symptoms of an epidermal pathology.
  • the phrase "therapeutically effective amount” defined above describes an amount of an agent or a combination of agents administered to an individual, which improves, in a measurable manner, the differentiation of the epidermal cells, a feature which is determined, for example, by the indirect immunofiuorescence analysis of keratin 10 and involucrin expression and/or by determination of the level of envelope cornified formation [13].
  • this phrase describes an administered amount of an agent or a combination of agents which can decrease, to a measurable amount, the proliferation of the cells, a feature which is determined, for example, by measurement of the activity of mitochondrial dehydrogenase enzymes of living cells (MTT assay) [14] and by counting of basal cells level [15].
  • the method of treating a benign or malignant hype ⁇ roliferative epidermal pathology in a subject in need thereof is effected by administering to the subject a therapeutically effective amount of a nicotinamide agent, as described hereinabove, in combination with a therapeutically effective amount of a cADPR agent, as described hereinabove.
  • the method of treating a benign or malignant hype ⁇ roliferative epidermal pathology in a subject in need thereof is effected by administering to the subject a therapeutically effective amount of a nicotinamide agent, as described hereinabove, in combination with a therapeutically effective amount of a Vitamin D3 agent.
  • the Vitamin D3 agent is any analog of Vitamin D3, such as, but not limited to, a Vitamin D3 metabolite, a Vitamin D3 agonist and a Vitamin D3 derivative.
  • the Vitamin D3 agent can further be a prodrug of each of the above agents.
  • this method of the present invention is effected by administering to the subject a therapeutically effective amount of nicotinamide, in combination with l ⁇ ,25-dihydroxy- Vitamin D3, which is also referred to herein as "l ⁇ ,25(OH) 2 D3", and is known as a metabolite of Vitamin D3.
  • Vitamin D3 and its analogs are known as useful agents in the treatment of psoriasis and other related skin diseases [6].
  • Vitamin D3 metabolites as well as agonists, derivatives and prodrugs of Vitamin D3 are expected to act similarly when used in the context of the present invention.
  • vitamin D3 which can be used in the context of present invention include, without limitation, 25-hydroxycholecalciferol (25 OH D3) and 24R, 25-dihydroxycholecalciferol (24R, 25(OH) 2 D3).
  • Vitamin D3 agents are also expected to exert synergistic effect on the proliferation and/or differentiation of epidermal cells, when used in combination with cADPR and its related compounds described hereinabove.
  • the method of this aspect of the present invention is effected by administering to the subject a therapeutically effective amount of a nicotinamide agent, as described hereinabove, in combination with a therapeutically effective amount of a Vitamin A agent.
  • the Vitamin A agent is any analog of Vitamin A, such as, but not limited to, a Vitamin A metabolite, a Vitamin A agonist and a Vitamin A derivative.
  • the Vitamin A agent can further be a prodrug of each of the above agents.
  • this method of the present invention is effected by administering to the subject a therapeutically effective amount of nicotinamide, in combination with all-trans-retinoic acid, which is also referred to herein as "atRA".
  • atRA all-trans-retinoic acid
  • All-trans-retinoic acid is a well-known metabolite of Vitamin A. As this compound is a FDA approved drug, it is widely used in a variety of therapeutic applications, including skin disorders. However, the presently known methods that utilize atRA or other Vitamin A agents, are limited by the skin irritations that are often caused by of these compounds.
  • Vitamin A metabolites as well as agonists, derivatives and prodrugs of Vitamin A are expected to act similarly when used in the context of the present invention.
  • Vitamin A agents are expected to exert synergistic effect on the proliferation and/or differentiation of epidermal cells, when used in combination with cADPR and its related compounds described hereinabove.
  • Representative examples of other Vitamin A agents that are useful in the context of the present invention include, without limitation, the well-known variety of retinoic acid receptor (RAR) agonists.
  • RAR retinoic acid receptor
  • the Vitamin A agonist comprises a retinoic acid receptor agonist.
  • agents for the prapose of convenience, and unless otherwise defined, the term "agent” or “agents” is used hereinafter to describe a NA agent or a cADPR agent, as is defined hereinabove.
  • the phrase “combination of agents” is used hereinafter to describe all the optional combinations of agents that can be used in the context of the present invention, such as, but not limited to, a combination of a NA agent and a cADPR agent, a combination of a NA agent and a Vitamin D3 agent or a Vitamin A agent, a combination of a cADPR agent and a Vitamin D3 agent or a Vitamin A agent, a combination of a NA agent, a cADPR agent and a Vitamin D3 agent or a Vitamin A agent, and more.
  • nicotinamide and cADPR were both found highly active in the treatment of hype ⁇ roliferative epidermal pathologies, as is demonstrated herein, it is expected that like nicotinamide, cADPR or any analog thereof, as defined hereinabove, would also exert an anti-oxidative effect on epidermal cells by increasing the anti-oxidative properties of the cells. Moreover, it is expected that all the combinations of agents described and defined hereinabove would exert synergistic anti-oxidative effects.
  • epidermal cells preferably human epidermal cells.
  • a method according to this aspect of the present invention is effected by contacting the cells with a NA agent and/or a cADPR agent.
  • a method according to this aspect of the present invention is effected by contacting the cells with a NA agent in combination with a Vitamin D3 agent or a Vitamin A agent.
  • a method according to this aspect of the present invention is effected by contacting the cells with a cADPR agent in combination with a Vitamin D3 agent or a Vitamin A agent.
  • the phrase "effective amount” and “therapeutically effective amount” describes an amount of the agent that is sufficient to substantially increase the anti-oxidative properties of affected cells and hence, for example, abrogate, substantially inhibit, slow or reverse the progression of oxidative processes in epidermal cells, substantially ameliorate aging and oxidative-induced symptoms or injuries of epidermal cells or substantially prevent the appearance of clinical symptoms associated with aging of epidermal cells.
  • the method according to this aspect of the present invention is effected by contacting the cells with nicotinamide.
  • the effective amount of nicotinamide preferably ranges between about 1 mM and about 50 mM, more preferably between about 1 mM and about 20 mM and most preferably between about 5 mM and about 15 mM.
  • Epidermal cells that are treatable by these methods of the present invention include, for example, epidermal cells with symptoms of skin aging (dryness, roughness, burning and atrophy of the skin, itching, cold intolerance, wrinkles, hepe ⁇ ilosity, alopecia), and epidermal cells that are involved in natural or oxidative stress-inducing aging processes.
  • skin cells characterized by increasing sensitivity to oxidative injury, such as cells with predisposition to initiation of tumors can also be treatable by these methods. Increasing the anti-oxidative properties of such cells provides for anti-cancer protection of these cells.
  • compositions which are identified for use in the treatment of benign or malignant hype ⁇ roliferative pathologies and/or for use in the treatment of conditions whereby increasing anti-oxidative properties of epidermal cell is advantageous.
  • compositions can be either pharmaceutical compositions for therapeutic uses, and/or cosmetic or cosmeceutical compositions.
  • compositions there are provided pharmaceutical, cosmetic or cosmeceutical compositions.
  • compositions are identified for use in the treatment of benign or malignant hype ⁇ roliferative pathologies, as is detailed hereinabove, and/or in the treatment of conditions whereby increasing anti-oxidative properties of epidermal cell is advantageous.
  • Conditions whereby increasing anti-oxidative properties of epidermal cell is advantageous or, in other words, conditions that require a treatment in which anti-oxidative properties of epidermal cell are increased include, for example, aging of epidermal cells and cancer.
  • aging of epidermal cells and “aging of the skin” describes all the symptoms associated with physiological aging of the epidermis such as, but not limited to, wrinkles, loss of elasticity, decreased metabolism, dryness, roughness, burning and atrophy of skin, itching, hepe ⁇ ilosity and alopecia.
  • the treatment of a condition such as aging of the epidermal cells includes, according to the present invention, treatment of the symptoms described hereinabove with respect to aging of epidermal cells. This treatment further includes prevention of these symptoms, and in particular aging signs, before they occur.
  • anti-cancer protection describes a condition in which epidermal cells are characterized by increased sensitivity to oxidative injury, such as cells with predisposition to initiation of tumors, and therefore require anti-cancer protection.
  • the treatment of such a condition includes increasing the anti-oxidative properties of epidermal cells that are in need for anti-cancer protection, as described hereinabove. Since the formation of cancer tumors in epidermal cells typically occurs as a result of oxidative processes, increasing the anti-oxidative properties of epidermal cells, can serve for protecting these cells from processes that lead to cancer.
  • the pharmaceutical, cosmetic or cosmeceutical compositions of the present invention comprise, as an active ingredient, a therapeutically effective amount, as this phrase is defined hereinabove, of a nicotinamide agent, as described hereinabove and/or a therapeutically effective amount of a cADPR agent, as described hereinabove, and a pharmaceutically, cosmetically or cosmeceutically acceptable carrier.
  • a pharmaceutical, cosmetic or cosmeceutical composition according to this embodiment of the present invention, comprises as an active ingredient, either nicotinamide, agonist, derivative or metabolite thereof or prodrugs thereof, cADPR, agonist, derivative or metabolite thereof or prodrugs thereof or a combination thereof.
  • a pharmaceutical, cosmetic or cosmeceutical composition according to this embodiment of the present invention, comprises a combination of active ingredients and hence comprises both a NA agent and a cADPR agent, which combination exerts synergistic effect on proliferation and differentiation of various epidermal cells, as is described hereinabove.
  • the therapeutically effective amount of nicotinamide ranges between about ImM and about 50 mM. Preferably, it ranges between about 0.5 mM and about 20 mM. More preferably, it ranges between about 1 mM and about 10 mM. Most preferably, it ranges between about 2.5 mM and about 5 mM.
  • the therapeutically effective amount of cADPR preferably ranges between about 1 ⁇ M and 100 ⁇ M, more preferably between about lO ⁇ M and about 50 ⁇ M and most preferably between about 25 ⁇ M and about 50 ⁇ M.
  • a preferred pharmaceutical, cosmetic or cosmeceutical composition according to the present invention therefore comprises nicotinamide at a final concentration that ranges between about 2.5 mM and about 5 mM and cADPR at a final concentration that ranges between about 25 ⁇ M and about 50 ⁇ M.
  • nicotinamide at a final concentration that ranges between about 2.5 mM and about 5 mM
  • cADPR at a final concentration that ranges between about 25 ⁇ M and about 50 ⁇ M.
  • the pharmaceutical, cosmetic or cosmeceutical compositions include, as a combination of active ingredients, a therapeutically effective amount of either a NA agent, as described hereinabove, or a cADPR agent, as described hereinabove, in combination with a therapeutically effective amount, as this phrase is defined hereinabove, of a Vitamin A agent or a Vitamin D3 agents, as these agents are described in detail hereinabove.
  • a pharmaceutical, cosmetic or cosmeceutical composition according to this embodiment of the present invention comprises a therapeutically effective amount of a NA agent, a therapeutically effective amount of a Vitamin D3 agent and a pharmaceutically, cosmetically or cosmeceutically acceptable carrier.
  • a preferred pharmaceutical, cosmetic or cosmeceutical composition in this particular comprises therapeutically effective amounts of nicotinamide and a Vitamin D3 metabolite.
  • the vitamin D3 metabolite is l ⁇ ,25 dihydroxy- vitamin D 3 .
  • a preferred concentration of the NA in this composition is within the ranges defined hereinabove.
  • a preferred ratio between the NA and the Vitamin D3 metabolite ranges between about 5000:1 and about 500,000:1 and preferred final concentrations of the Vitamin D3 metabolite typically ranges between 1 nM and 10000 nM, and more preferably between about 0.01 ⁇ M (10 nM) and about 1 ⁇ M (1000 nM).
  • a pharmaceutical, cosmetic or cosmeceutical composition according to this embodiment of the present invention comprises a therapeutically effective amount of a NA agent, a therapeutically effective amount of a Vitamin A agent, as these phrases are defined hereinabove, and a pharmaceutically, cosmetically or cosmeceutically acceptable carrier.
  • a preferred pharmaceutical, cosmetic or cosmeceutical composition of this particular comprises therapeutically effective amounts of nicotinamide and a Vitamin A metabolite.
  • the vitamin A metabolite is all-trans-retinoic acid.
  • a preferred concentration of the NA in this composition is within the ranges defined hereinabove.
  • a preferred ratio between the NA and the Vitamin A metabolite ranges between about 5 x 10 5 :1 and about 25 x 10 7 :1.
  • Preferred final concentrations of the Vitamin A metabolite typically ranges between about 0.1 nM and about 100 nM, and more preferably between about 1 nM and about 10 nM.
  • compositions of the present invention include a pharmaceutically, cosmetically or cosmeceutically acceptable carrier.
  • cosmetically acceptable carrier and cosmeceutically acceptable carrier refer to a carrier or a diluent that does not cause significant irritation to the skin and does not abrogate the biological activity and properties of the applied active agent.
  • cosmetically or cosmeceutically acceptable carriers that are useful in the context of the present invention include, without limitation, emulsions, creams, aqueous solutions, oils, ointments, pastes, gels, lotions, milks, foams, suspensions and powders.
  • the cosmetically or cosmeceutically acceptable carrier of the present invention may include, for example, a thickener, an emollient, an emulsifier, a humectant, a surfactant, a suspending agent, a film forming agent, a foam building agent, a preservative, an antifoaming agent, a fragrance, a lower monoalcoholic polyol, a high boiling point solvent, a propellant, a colorant, a pigment or mixtures thereof.
  • the final cosmetic or cosmeceutical composition of the present invention may be, for example, in the form of an oil, a gel, a solid stick, a lotion, a cream, a milk, an aerosol, a spray, an ointment or a fatty ointment and a powder.
  • the cosmetic and cosmeceutical compositions of the present invention are preferably topically applied on the treated epidermal cells.
  • the phrase "pharmaceutically acceptable carrier”, which is also refened to herein interchangeably as “physiologically acceptable carrier” describes a canier, an excipient or a diluent that does not cause significant irritation to a subject, and particularly to the skin of a subject.
  • preferred carriers in the pharmaceutical compositions of the present invention are also dermatological acceptable carriers.
  • the carrier does not abrogate the biological activity and properties of the administered compound or combination of compounds.
  • the carrier is typically added to facilitate the administration of the active ingredient(s).
  • excipient describes an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the pharmaceutical compositions of the present invention are administered either topically or subcutaneously.
  • compositions for topical administration are preferably in the form of cream, gel, solution, salve, lotion, ointment or fatty ointment.
  • Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the compounds (agents) of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the agents of the present invention can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this pu ⁇ ose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the agents for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuos infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water
  • the compound(s) of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions of the present invention may also be formulated for local administration, such as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the composition may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives such as sparingly soluble salts.
  • suitable polymeric or hydrophobic materials for example, as an emulsion in an acceptable oil
  • ion exchange resins for example, as an emulsion in an acceptable oil
  • sparingly soluble derivatives such as sparingly soluble salts.
  • Compositions for topical administration may include, but are not limited to, lotions, suspensions, ointments gels, creams, drops, liquids, sprays emulsions and powders, as is described hereinabove.
  • compositions herein described may also comprise suitable solid of gel phase carriers or excipients.
  • suitable solid of gel phase carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.
  • agents in the claimed compositions of the present invention may be provided as physiologically acceptable salts wherein the agent may form the negatively or the positively charged species.
  • salts in which the agent forms the positively charged moiety include, without limitation, quaternary ammonium (defined elsewhere herein), salts such as the hydrochlori.de, sulfate, carbonate, lactate, tartrate, maleate, succinate, etc, wherein the nitrogen of the quaternary ammonium group is a nitrogen of a compound of the present invention which reacts with an appropriate acid.
  • Salts in which the agent forms the negatively charged species include, without limitation, the sodium, potassium, calcium and magnesium salts formed by the reaction of a carboxylic acid group in the molecule with the appropriate base (e.g., sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca(OH) 2 ), etc.).
  • Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended pu ⁇ ose, as is discussed and defined hereinabove. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the therapeutically effective amount or dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 5 o as determined in cell culture (e.g., the concentration of the test compound, which achieves a half-maximal inhibition of the epidermal cells proliferation). Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the agents described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC 50 and the LD 50 (lethal dose causing death in 50 % of the tested animals) for a subject compound.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the antiproliferative effects, termed the minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90 % inhibition of a kinase may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.
  • Dosage intervals can also be determined using the MEC value. Preparations should be administered using a regimen, which maintains plasma levels above the MEC for 10-90 % of the time, preferable between 30-90 % and most preferably 50-90 %.
  • the effective local concentration of the drug may not be related to plasma concentration. In such cases, other procedures known in the art can be employed to determine the effective local concentration.
  • dosing can also be a single administration of a slow release composition described hereinabove, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • the amount of an agent or a combination of agents to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • compositions comprising the agents of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of a benign and/or a malignant hype ⁇ roliferative epidermal pathology.
  • compositions comprising the agents of the invention formulated in a compatible pharmaceutical, cosmetic or cosmeceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of a condition whereby increasing anti-oxidative properties of epidermal cells is advantageous.
  • kits comprising any of the agents or combinations of agents described hereinabove. Whenever a combination of agents is present in the kits of the present invention, the agents are individually packaged in the kit.
  • the pharmaceutical, cosmetic and cosmeceutical kits are identified, in print, for use in the treatment of a benign and/or a malignant hype ⁇ roliferative epidermal pathologies and/or in the treatment of a condition whereby increasing anti-oxidative properties of epidermal cells is advantageous.
  • the high efficacy of the agents or the combinations of agents of the present invention in treating hype ⁇ roliferative epidermal pathologies is mainly attributed to the high capability of these agents or combination of agents to inhibit the proliferation of epidermal cells.
  • the cells may be malignant epidermal cells such as, but not limited to, cells from squamous cell carcinoma (SCC) basal cell carcinoma (BCC) or other non-melanoma skin cancers (NMSCs) or alternatively may be hype ⁇ roliferative benign cells, such as human keratinocytes from psoriatic skin, and keratinocytes from keratoacanthoma, common warts or seborrhoic keratoses lesions.
  • SCC squamous cell carcinoma
  • BCC basal cell carcinoma
  • NMSCs non-melanoma skin cancers
  • the cells may also be from other benign skin disorders such as ichthyosis.
  • a method according to this aspect of the present invention is effected by contacting the cells with a NA agent and/or a cADPR agent.
  • a method according to this aspect of the present invention is effected by contacting the cells with a NA agent in combination with a Vitamin D3 agent or a Vitamin A agent.
  • a method according to this aspect of the present invention is effected by contacting the cells with a cADPR agent in combination with a Vitamin D3 agent or a Vitamin A agent.
  • the immortalized human keratinocyte HaCat cells were routinely cultured in 75 cm 2 flasks using Eagle's minimal essential medium (MEM- EAGLE) supplemented with 5 % fetal calf serum (FCS) and 1 % antibiotics
  • Epidermal carcinoma A431 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % FCS and antibiotics as above.
  • DMEM Dulbecco's modified Eagle's medium
  • Nicotinamide (NA); cyclic adenosine diphosphate-ribose (cADPR); calcitriol (l ⁇ , 25-dihyroxy- vitamin D3); all trans retinoic acid (atRA; Vitamin A acid; Tretinoin); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); propidium iodide; dimethylsulphoxide (DMSO); bovine serum albumin (BSA); sucrose; trisodium citrate; igepal CA-630 (NP-40); Tris-(hydroxymethyl)- aminomethane; trypsin; trypsin inhibitor; ribonuclease A; spermin- tetrahydrochloride; sodium dodecylsulfate (SDS); ⁇ -mercaptoethanol and hydrogen peroxide (H 2 0 2 ), were all obtained from Sigma (USA).
  • DMSO dimethylsulphoxide
  • MEM-EAGLE Eagle's minimal essential medium
  • DMEM fetal calf serum
  • FCS fetal calf serum
  • PBS Dulbecco's phosphate buffered saline
  • trypsin 0.05 %-EDTA solution were obtained from Biological Industries (Israel).
  • Keratinocyte Growth Medium ® -2 Bullet Kit ® CC-3107 was received from BioWhittaker, Inc. A Cambrex Company, Clonetics, USA).
  • Anti-human cytokeratin 10 (NCL-CK10) and involucrin (NCL-LNV) mouse monoclonal antibodies were obtained from Novocastra Laboratories Ltd. (UK) and CyTM 2-conjugated goat anti-mouse IgG was obtained from Jackson Immunoresearch Laboratories, Inc. (USA).
  • HaCat and A431 cells were propagated in 25 cm or 75 cm tissue culture flasks (Coming, USA) and 24-well and 96-well tissue culture plates (Corning,
  • NA or a combination of NA and another agent NA and cADPR; NA and Vitamin D3 metabolite; NA with Vitamin A metabolite
  • Late differentiation processes in HaCat cells treated with nicotinamide were measured by determining the Comified cell envelope formation, according to the procedure described in Sun T-T, Green, H: Differentiation of the epidermal keratinocytes in cell culture: formation of comified envelope, Cell, 9:511-521,
  • DNA labeling and flow cytometry analysis HaCat and A431 cells were seeded in 25 cm tissue culture flasks and incubated for 72 hours with 0, 5, 10 and 20 mM of NA. Cells treated with 5 % ethanol served as positive control of apoptosis.
  • the nuclei for flow cytometry analysis of DNA were prepared by a detergent trypsin method with propidium iodide, according to the procedure described in Lars L Rindelov: A detergent trypsin method for the preparation of nuclei for FACS DNA analysis, Cytometry 3(5)323-327, 1983.
  • the cells (10 6 per tube) were washed with PBS.
  • the cell pellet was re-suspended in 40 ⁇ l citrate buffer (pH 7.6) supplemented with 250 mM sucrose, 40 mM trisodium citrate and 5 % DMSO.
  • the re-suspended cells were then incubated in 450 ⁇ l solution of trypsin (0.15 mg/ml, pH 7.6) for 10 minutes, and thereafter with trypsin inhibitor and ribonuclease A for another 10 minutes.
  • trypsin (0.15 mg/ml, pH 7.6)
  • trypsin inhibitor and ribonuclease A for another 10 minutes.
  • a hundred (100) ⁇ g/ml of fluorochrome solution containing propidium iodide were then added to nuclei.
  • the tubes were placed in the dark and the flow cytometry analysis was carried out in fluorescence-activated cell sorter (FACScan; Becton Dickinson, CA). The level of apoptosis was determined using the Cell Quest Program of Becton Dickonson. Each experiment was repeated three times.
  • Results are presented as mean ⁇ standard deviation of the mean (mean ⁇ SD). Statistical significance (P ⁇ 0.05) was derived by Student's t-test.
  • A431 cells were incubated with varying amounts of NA for a period of 72 hours.
  • the cells proliferation was estimated by the MTT method, described hereinabove, and was expressed as the percent from control (untreated cells).
  • cADPR As shown in Figure 3, cADPR, at concentrations of 25 and 50 ⁇ M, exerted effective inhibition of cell proliferation.
  • HaCat cells and A431 cells were incubated with varying amounts of Vitamin D3 metabolite (l ⁇ 25(OH) 2 D3) or Vitamin A metabolite (atRA) for 72 hours.
  • Vitamin D3 metabolite l ⁇ 25(OH) 2 D3
  • Vitamin A metabolite atRA
  • HaCat cells and A431 cells were incubated with the Vitamin D3 metabolite l ⁇ 25(OH) 2 D3 alone and with a combination of l ⁇ 25(OH) 2 D3 and NA, for a period of 72 hours.
  • Figures 5a and 6a present the results obtained with the above combination in HaCat and A431 cell lines, respectively.
  • Figures 5b and 6b present a deduction of the anti-proliferative effects of l ⁇ 25(OH) 2 D3 and NA, when applied separately on the cell lines as described hereinabove, from the anti-proliferative effect of the combination of NA and l ⁇
  • the anti-proliferative effect of the combination of NA and l ⁇ 25(OH) 2 D3 is substantially higher than the summation of the anti-proliferative effects of each of these compounds separately.
  • a combination of 100 nM l ⁇ 25(OH) 2 D3 and 5 mM NA was used in HaCat cells, enhancement of 12 % was observed in the inhibition of cell proliferation.
  • a combination of 10 nM of l ⁇ 25(OH) 2 D3 and 5 mM NA was used in A431 cells, enhancement of 20 % was observed in the inhibition of cell proliferation.
  • FIG. 7a and 8a present the results obtained in HaCat cells and in A431 cells, respectively.
  • Figures 7b and 8b present the respective deduction of the antiproliferative effects of cADPR and NA, when applied separately on each of the cell lines as described hereinabove, from the anti-proliferative effect of the combination, presented in Figures 5a and 6a.
  • a combination of 50 ⁇ M cADPR and 5 mM NA exerted a synergistic effect of more than 20 % in inhibiting proliferation as compared with the effect of each of these agents alone, at the same concentrations, in HaCat cells.
  • a combination of 25 ⁇ M cADPR and 2.5 mM NA exerted a synergism of above 16 % in inhibiting proliferation of A431 cells, as compared with the inhibition of each of these components alone.
  • HaCat cells and A431 cells were incubated with 5 mM NA and 2.5 mM NA, respectively, and with varying concentrations of atRA (0.1 nM - 1 ⁇ M).
  • Figures 9a and 10a present the antiproliferative effect of this combination in HaCat cells and in A431 cells, respectively, while Figures 9b and 10b demonstrate the respective synergistic effect of this combination in theses cell lines.
  • Figures 9b and 10b demonstrate the respective synergistic effect of this combination in theses cell lines.
  • a synergistic anti-proliferative effect of more than 25 % was observed in A431 cells with a combination that included a concentration of 0.1 ⁇ M atRA (Fig. 10b) and a synergism of more than 20 % was observed in HaCat cells with a combination that included a concentration of 10 ⁇ M of atRA ( Figure 9b).
  • Effect ofNA on cell differentiation and apoptosis
  • NA on differentiation was determined by indirect immunofluorescence of keratin K10 and involucrin and by comified envelope formation, as described above, and the results are presented in Figure 11 and Figure 12, respectively.
  • the NA treatment simulated both expressions of keratin 10 (K10) and involucrin, which are markers of early and late differentiation processes of the epidermal cells, respectively.
  • the NA treatment also affected the ratio between the amount of cells and envelope comified cells. A higher proportion of enveloped comified cells, which are more differentiated cells, in the tested cells was observed.
  • the effect of NA on the level of apoptosis was also determined in HaCat and A431 cells. As is shown in Figure 13, the determined apoptosis levels show that NA becomes cytotoxic to the cells at a concentration of 30 mM in A431 cells and at a concentration of 50 mM in HaCat cells. These results are significant since they demonstrate that the effect of NA on cell proliferation, as is expressed, for example, in Figure 1, is effectively exerted by NA concentrations that are lower than the cytotoxic level of NA, namely, at concentrations lower than the concentrations that are toxic to cells.

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Abstract

La présente invention concerne des méthodes et des compositions destinées à être utilisées dans le traitement d'une pathologie proliférative bénine et/ou maligne. Lesdites méthodes consistent à administrer du nicotinamide ou ses analogues et/ou de la cADPR ou ses analogues, éventuellement en combinaison avec un analogue de la vitamine D3 ou avec un analogue de la vitamine A.
PCT/IL2003/000576 2002-07-11 2003-07-10 Compositions et methodes pour le traitement de troubles cutanes Ceased WO2004006887A2 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006120682A2 (fr) 2005-05-10 2006-11-16 Dermipsor Ltd. Compositions et methodes pour traiter des maladies epidermiques hyperproliferatives
JP2008540513A (ja) * 2005-05-10 2008-11-20 ダーミプソル リミテッド スキンケアのための組成物及び方法
EP1765341A4 (fr) * 2004-06-04 2010-04-14 Chemgenex Pharmaceuticals Inc Procedes de traitement de maladies proliferatives cellulaires au moyen de naphtalimide et d'inhibiteurs de parp-1
US20120022013A1 (en) * 2002-08-09 2012-01-26 President And Fellows Of Harvard College Methods and compositions for extending the life span and increasing the stress resistance of cells and organisms
US9597347B2 (en) 2003-12-29 2017-03-21 President And Fellows Of Harvard College Compositions for treating obesity and insulin resistance disorders
US9877981B2 (en) 2012-10-09 2018-01-30 President And Fellows Of Harvard College NAD biosynthesis and precursors for the treatment and prevention of cancer and proliferation
WO2020029441A1 (fr) * 2018-08-07 2020-02-13 浙江大学 Application d'une composition de nicotinamide dans la préparation d'un médicament pour le traitement d'une réaction cutanée main-pied induite par le sorafénib

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US20120022013A1 (en) * 2002-08-09 2012-01-26 President And Fellows Of Harvard College Methods and compositions for extending the life span and increasing the stress resistance of cells and organisms
US9597347B2 (en) 2003-12-29 2017-03-21 President And Fellows Of Harvard College Compositions for treating obesity and insulin resistance disorders
EP1765341A4 (fr) * 2004-06-04 2010-04-14 Chemgenex Pharmaceuticals Inc Procedes de traitement de maladies proliferatives cellulaires au moyen de naphtalimide et d'inhibiteurs de parp-1
US8034788B2 (en) 2005-05-10 2011-10-11 Dermipsor Ltd. Composition and methods for skin care
EP1879449A4 (fr) * 2005-05-10 2009-12-16 Dermipsor Ltd Compositions et procedes de soin de la peau
EP1879595A4 (fr) * 2005-05-10 2009-07-15 Dermipsor Ltd Compositions et methodes pour traiter des maladies epidermiques hyperproliferatives
WO2006120682A2 (fr) 2005-05-10 2006-11-16 Dermipsor Ltd. Compositions et methodes pour traiter des maladies epidermiques hyperproliferatives
JP2008540514A (ja) * 2005-05-10 2008-11-20 ダーミプソル リミテッド 過剰増殖表皮疾患の治療用組成物及び方法
AU2006245283B2 (en) * 2005-05-10 2012-11-01 Dermipsor Ltd. Compositions and methods for treating hyperproliferative epidermal diseases
US9173835B2 (en) 2005-05-10 2015-11-03 Dermipsor Ltd. Compositions and methods for treating hyperproliferative epidermal diseases
JP2008540513A (ja) * 2005-05-10 2008-11-20 ダーミプソル リミテッド スキンケアのための組成物及び方法
US9603861B2 (en) 2005-05-10 2017-03-28 Dermipsor Ltd. Compositions and methods for treating hyperproliferative epidermal diseases
US9877981B2 (en) 2012-10-09 2018-01-30 President And Fellows Of Harvard College NAD biosynthesis and precursors for the treatment and prevention of cancer and proliferation
WO2020029441A1 (fr) * 2018-08-07 2020-02-13 浙江大学 Application d'une composition de nicotinamide dans la préparation d'un médicament pour le traitement d'une réaction cutanée main-pied induite par le sorafénib
US11278542B2 (en) 2018-08-07 2022-03-22 Zhejiang University Use of nicotinamide composition in preparation of drug for treating hand-foot skin reaction induced by sorafenib

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AU2003242965A1 (en) 2004-02-02
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WO2004006887A3 (fr) 2004-03-18
EP1539100A4 (fr) 2006-01-18

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