JP2005281150A - Method for increasing treating effect in radiotherapy and chemotherapy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for increasing treating effect in radiotherapy and chemotherapy in treating lesions induced by radiotherapy and/or chemotherapy and to obtain a pharmaceutical composition. <P>SOLUTION: The composition increases treating effect in radiotherapy and chemotherapy in malignant or benign diseases during proliferation, having low frequency of sequelae of treatment, giving tumor control in high possibility, by administering a therapeutically effective amount of a histone-deacetylation enzyme inhibitor. The method for increasing treating effect in radiotherapy and chemotherapy is provided. The compound simultaneously stimulates epithelial cell reproduction, inhibits fibroblast proliferation, reduces collagen deposition, controls a fibrotic growth factor, brings inflammation-inducing cytokinins to be sedate, has ability of regulating the expression of a cell cycle gene, a tumor control factor and a cancer gene, is useful for increasing treating effect in radiotherapy and chemotherapy, reduces skin swelling and inflammation, promotes epithelial healing in mucosa and dermis, reduces mouth dryness, prevents/reduces degree of seriousness of vola-palm syndrome, prevents tissue fibrotic disease, ulceration, necrosis and tumorigenesis and increases tumor growth inhibition and tumor treatment efficiency. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to the treatment of damage induced by radiation and / or chemotherapy. More particularly, the invention relates to methods and pharmaceutical compositions for increasing therapeutic effects in radiation and chemotherapy.

  Radiation therapy with or without combined or continuous chemotherapy includes head and neck cancer, breast cancer, skin cancer, anogenital cancer, and keloids, dermatomas, hemangiomas, arteriovenous malformations, It is an effective method for certain non-malignant diseases such as unexplained histiocytic proliferation. However, therapeutic benefits include radiation and chemotherapy-induced mucosal epithelial damage and skin irradiation syndrome (CRS), including acute reactions of tissue swelling, mucositis, dermatitis, exfoliation, and ulceration, and Limited by long-term effects of tissue / dermatofibrosis, necrosis, and the development of life-threatening sequelae of sarcoma, squamous cell and basal cell carcinoma (Non-Patent Document 1). In fact, the skin is affected in all forms of external radiation therapy of internal organs, mucositis is a side effect of chemotherapy and radiation therapy, as an oral cavity, esophagus, gastrointestinal tract, bladder, vagina, rectum, lung, nasal cavity, It can occur in the ears and eyes. Although the use of steroids or non-steroidal anti-inflammatory agents is the most common treatment for tissue damage induced by radiation and chemotherapy, the results are still unsatisfactory. How to selectively reduce dermatologic conditions without sacrificing the tumor killing effects of radiation therapy and chemotherapy is a long-standing goal in radiation and medical oncology.

Peter, R. U., “The cutaneous, radiation syndrome.”, Advances in the treatment of radiation injuries, 237-240, Oxford: Elsevier, 1996.

  Drugs previously tested for the management of radiation and chemotherapy-induced damage include antioxidants (vitamin E and superoxide dismutase), anti-inflammatory drugs (corticosteroids, cortisine, D-penicillamine, and TNF- α antagonist antibodies), and anti-fibrogenic agents (interferons, TGF-β antagonists, and angiotensin converting enzyme inhibitors). Of these, none can simultaneously improve acute dermatitis and mucositis, prevent the development of fibrosis, and reduce late stage tumor formation. Furthermore, toxicity, side effects, tumor protection potential, and lack of anti-tumor effects are troublesome.

  The present invention provides a method and method for increasing the therapeutic effect in radiation therapy and / or chemotherapy by causing a low frequency of therapeutic complications or sequelae in a subject in need and generating tumor control with a high probability. A pharmaceutical composition is provided. The pharmaceutical composition comprises a therapeutically effective amount of a histone hyperacetylating agent and a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt thereof. The method includes administering a pharmaceutical composition to a subject in need.

  The purpose of the methods and pharmaceutical compositions of the present invention is to simultaneously (1) increase suppression of cell growth during tumor or proliferation in a host in need of radiation therapy and / or chemotherapy, and (2) radiation and / or Or preventing the occurrence of chemotherapy-induced mucositis, dermatitis, ulceration, fibrosis, dry mouth, plantar-palmar syndrome, and complications or sequelae of tumorigenesis .

  Surprisingly, according to the present invention, pharmaceutical compositions comprising histone deacetylase inhibitors reduce radiation-induced normal tissue fibrosis and are induced with radiation in mucositis and dermatitis Promotes wound healing, promotes chemotherapy-induced tissue necrosis, prevents radiation-related tumor formation, exhibits direct anti-tumor effects, enhances tumor radiation sensitization, and radiation therapy and others It was revealed that the anticancer drug of the present invention synergistically inhibits tumor cell growth.

  The compounds of the present invention can be administered intravenously, enterally, parenterally, intramuscularly, intranasally, subcutaneously, topically, or orally. Dosage is based on the effective concentration observed in in vitro and in vivo studies. The diverse effective utility of the compounds of the present invention makes it useful for cytokines, interleukins, anti-cancer agents, or anti-neoplastic agents, anti-angiogenic agents, chemotherapeutic agents, antibodies, conjugated antibodies, immunostimulants, antibiotics, Further evidence is shown by the fact that it can be administered in combination or combination with retinoic acid, tyrosine kinase inhibitors, hormone antagonists, or growth stimulants.

  In the pharmaceutical composition according to the present invention, (1) treatment in chemotherapy and / or radiation therapy for proliferative malignant or non-malignant diseases with a low frequency of sequelae of treatment and high possibility of tumor control. A pharmaceutical composition with an enhanced effect, characterized in that it comprises a histone hyperacetylating agent and a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt thereof.

  Also, in the pharmaceutical composition according to the present invention, (2) the increased therapeutic effect simultaneously enhances tumor radiation sensitization or sensitizes the tumor to chemotherapy and increases tumor growth inhibition Promotes wound healing in mucositis and dermatitis, prevents / reduces severity of plantar-palm syndrome, reduces tissue fibrosis, protects normal tissue from cell death, prevents dry mouth And the pharmaceutical composition according to (1) above, which suppresses tumor formation.

  In the pharmaceutical composition according to the present invention, (3) the hyperacetylating agent is the pharmaceutical composition according to the above (1), which is a histone deacetylase inhibitor.

  In the pharmaceutical composition according to the present invention, (4) the radiation therapy is the pharmaceutical composition according to the above (1), wherein the radiation therapy is teleradiation therapy, brachytherapy, or ionizing radiation. .

  In the pharmaceutical composition according to the present invention, (5) proliferative malignant diseases are melanoma, Kaposi sarcoma, osteosarcoma, neuroblastoma, rhabdomyosarcoma, Ewing sarcoma, soft tissue sarcoma, skin cancer, Lymphoma, leukemia, breast cancer, germ cell tumor, undifferentiated neuroectodermal tumor, brain glioma, brain meningioma, head and neck cancer, thyroid cancer, thymic cancer, cervical cancer, anal cancer, colorectal cancer, The pharmaceutical according to (1) above, which is selected from the group consisting of prostate cancer, lung cancer, hepatocellular carcinoma, bile duct cancer, gastric cancer, pancreatic cancer, esophageal cancer, virus-related tumor, and disease derived from having undergone bone marrow transplantation It is a composition.

  In the pharmaceutical composition according to the present invention, (6) non-malignant diseases are pterygium, Graves' ophthalmopathy, orbital pseudotumor, macular degeneration, keloid, wart, keratophyte cell tumor, hemangioma, Venous malformations, bursitis, tendonitis, tendonoma, Peony disease, vascular stenosis, ameloblastoma, aneurysmal bone cyst, ectopic bone formation, gynecomastia, ovarian castration, parotitis , Eczema, atopic dermatitis, psoriasis, brachial periarthritis, epicondylitis, knee arthropathy, sulphodenitis, gourd, autoimmune inflammatory arthritis, unexplained histiocytosis, and organ transplantation The pharmaceutical composition according to (1) above, which is selected from the group consisting of diseases derived from the above.

  In the pharmaceutical composition according to the present invention, (7) the histone hyperacetylating agent is trichostatin A or trichostatin C. .

  Further, in the pharmaceutical composition according to the present invention, (8) the histone hyperacetylating agent is selected from the group consisting of oxamflatin, trapoxin A, FR901228, apicidin, HC-toxin, WF27082, and clamidosine, 1) The pharmaceutical composition described above.

  In the pharmaceutical composition according to the present invention, (9) the histone hyperacetylating agent is selected from the group consisting of salicylic hydroxamic acid, suberoylanilide hydroxamic acid, and azelaic acid bishydroxamic acid. 1) The pharmaceutical composition described above.

  In the pharmaceutical composition according to the present invention, (10) the histone hyperacetylating agent is azelaic acid-1-hydroxamate-9-anilide, M-carboxycinnamic acid bishydroxamide, 6- (3 -A chlorophenylureido) carpoic hydroxamic acid, MW2796, and a pharmaceutical composition according to (1) above, selected from the group consisting of MW2996.

  In the pharmaceutical composition according to the present invention, (11) the histone excess acetylating agent is sodium butyrate, isovalerate, valerate, 4-phenylbutyrate, sodium phenylbutyrate, propionate, butrimide, isobutyramide, phenylacetate, The pharmaceutical composition according to (1) above, which is selected from the group consisting of 3-bromopropionate, valproic acid, and tributyrin.

  In the pharmaceutical composition according to the present invention, (12) the histone hyperacetylating agent is MS-27-275 or a 3′-amino derivative thereof as described in (1) above. It is characterized by.

  In addition, the pharmaceutical composition according to the present invention is characterized in that (13) the histone hyperacetylating agent is depudecin or scriptoid, the pharmaceutical composition according to the above (1).

  The pharmaceutical composition according to the present invention is (14) the pharmaceutical composition according to the above (1), which is administered parenterally.

  Further, in the pharmaceutical composition according to the present invention, (15) cream, ointment, gel, paste, powder, lotion, patch, suppository, liposome composition, suspension, mouthwash, enema, injection solution, Alternatively, the pharmaceutical composition according to (1) above, which is an infusion.

  In the pharmaceutical composition according to the present invention, (16) the pharmaceutical composition is present in an amount of an excess acetylating agent from 0.001% to 100% by weight of the composition. It is characterized by being a pharmaceutical composition.

  In the pharmaceutical composition according to the present invention, (17) a second substance selected from the group consisting of cytokines, interleukins, anticancer agents, or antineoplastic agents, antiangiogenic agents, chemotherapeutic agents, antibodies The pharmaceutical composition according to (1) above, further comprising a conjugated antibody, an immunostimulant, an antibiotic, a retinoic acid, a tyrosine kinase inhibitor, a hormone antagonist, and a growth stimulant.

  In the pharmaceutical composition according to the present invention, (18) the conjugated antibody is a pharmaceutical composition according to the above (17) selected from the group consisting of trastuzumab, c225, rituximab, and cetuximab. Features.

  In the pharmaceutical composition according to the present invention, (19) the chemotherapeutic agent is an alkylating agent, a purine analog, a pyrimidine analog, a vinca alkaloid, a vinca-like alkaloid, an etoposide, an etoposide-like drug, a corticosteroid, The pharmaceutical composition according to (17) above, which is selected from the group consisting of nitrosourea, antimetabolite, platinum-based cytotoxic agent, antiandrogen, and antiestrogen.

  In the pharmaceutical composition according to the present invention, (20) the anti-angiogenic agent is selected from the group consisting of thalidomide, SU5416, SU6668, thrombospondin-1, endostatin, and angiostatin, 17) A pharmaceutical composition as described above.

  In the pharmaceutical composition according to the present invention, (21) the antibiotic is selected from the group consisting of ganciclovir, acyclovir, and famciclovir. Features.

  In accordance with the present invention, administration of a therapeutically effective amount of a histone deacetylase inhibitor results in radiation for proliferative malignant or non-malignant diseases with low frequency of treatment sequelae and high likelihood of tumor control Compositions and methods are provided that increase the therapeutic effect in therapy and chemotherapy.

  The invention will be more fully understood and further advantages will become apparent when reference is made to the following description of the invention and the accompanying drawings.

  Release of cytokines (such as tumor necrosis factor (TNF-α)) and growth factors (such as transforming growth factor (TGF-β)) in affected tissues after radiation and / or chemotherapeutic agents induce damage Promotes recruitment and proliferation of fibroblasts, but inhibits epithelial cell growth and perpetuates and increases inflammatory responses (Hill et al., Int. J. Radiat. Oncol. Biol. Phys., 49: 353-365 Seong et al., Int. J. Radiat. Oncol. Biol. Phys., 46: 639-643, 2000; Wang et al., Am. J. Pathol., 153: 1531-1540, 1998; Fedorocko et al., Int. J. Radiat. Biol., 78: 305-313, 2002; Martin et al., Int. J. Radiat. Oncol. Biol. Phys., 47: 277-290, 2000; Randall et al., Int. J. Radiat. Biol., 70: 351-360, 1996; Border et al., N. Engl. J. Med., 331: 1286-1292, 1994; Singer Et al., N. Engl. J. Med., 341: 738-746, 1999). In particular, the amplified damage is responsive to radiation by persistent secretion of TNF-α and TGF-β from epithelial, endothelial, and connective tissue cells, and it is likely in genetic programming of cell differentiation and proliferation Caused by modification, leading to histological modifications characterized by CRS (Zhou et al., Int. J. Radiat. Biol., 77: 763-772, 2001; Skwarchuk et al., Int. J. Radiat. Oncol Biol. Phys., 42: 169-178, 1998; Sivan et al., Int. J. Radiat. Oncol. Biol. Phys., 53: 385-393, 2002; Delanian et al., Radiother. Oncol. 47, 255-261, 1998). Chronic activation of the TGF-β pathway also stimulates late tumorigenesis (Wieser et al., Curr. Opin. Oncol., 13: 70-77, 2001; Massague et al., Cell, 103: 295-309) ,the year of 2000). Thus, CRS and radiation-induced carcinogenesis can be considered a genetic disorder of the wound healing process after radiation.

  Compounds of the class of gene modulators, histone deacetylases (HDACs), activate and repress subsets of genes by remodeling chromatin structure through altered states in histone acetylation (Marks et al., J. Natl. Cancer Inst., 92: 1210-1216, 2000; Kramer et al., Trends Endocrinol. Metab., 12: 294-300, 2001). Histone hyperacetylation is the up-regulation of cell cycle inhibitors (p21Cip1, p27Kip1 and p16INK4), down-regulation of oncogenes (Myc and Bcl-2), inflammatory cytokines (interleukin (IL) -1, IL-8, TNF-α and TGF-β) are suppressed or produce no change (GAPDH and γ-actin) (Lagger et al., EMBO J., 21: 2672-2681, 2002; Richon et al., Clin. Cancer Res. 8: 662-667, 2002; Richon et al., Proc. Natl. Acad. Sci. USA., 97: 10014-10019, 2000; Van Lint et al., Gene Expr., 5: 245 -243, 1996; Huang et al., Cytokine, 9: 27-36, 1997; Mishra et al., Proc. Natl. Acad. Sci. USA., 98: 2628-2633, 2001; J. Neurosurg., 83: 672-681, 1995; Segain et al., Gut, 47: 397-403, 2000; Leoni et al., Proc. Natl. Acad. Sci. USA., 99: 2995-3000, 2002). In addition to inducing histone hyperacetylation, HDAC inhibitors also induce hyperacetylation of non-histone proteins such as liposomal S3, p53 or Rel-A subunits of NF-κB, and protein kinase C ( PKC) activity, inhibit protein isoprenylation, reduce DNA methylation and bind to nuclear receptors (Webb et al., J. Biol. Chem., 274: 14280-14287, 1999; Chen; Et al., Science, 293: 1653-1657, 2001). HDAC inhibitors have been shown to induce cell cycle arrest, cell differentiation, and apoptotic cell death in tumor cells and to reduce inflammation and fibrosis in inflammatory diseases (Warrell et al., Natl. Cancer Inst., 90: 1621-1625, 1998; Vigushin et al., Clin. Cancer Res., 7: 971-976, 2001; Saunders et al., Cancer Res., 59: 399-404, 1999; Gottlicher et al., EMBO J., 20: 6969-6978, 2001; Rombouts et al., Acta Gastroenterol. Belg., 64: 239-246, 2001). Although the effects of HDAC inhibitors induce large amounts of histone acetylation, they result in apoptotic cell death, terminal differentiation, and growth arrest in tumor cells, but no toxicity in normal cells (Garber et al., J. Natl. Cancer Inst., 94: 793-795, 2002). Furthermore, modulation of chromatin structure by HDAC inhibitors can further sensitize tumors in which the cells are inherently resistant to radiation and can also sensitize tumor cells to chemotherapy (Ferrandina et al., Oncol. Res., 12: 429-440, 2001; Miller et al., Int. J. Radiat. Biol., 72: 211-218, 1997; Biade et al., Int. J. Radiat. Biol., 77: 1033 -Page 1042, 2001).

  Therefore, by remodeling chromatin in normal and tumor cells differentially, expression of tumor suppressors, oncogenes, pro-inflammatory cytokines (TNF-α), and fibrogenic growth factor (TGF-β) can be simultaneously performed Based on their ability to work together, selectively, and epigenetically, pharmaceutical compositions containing HDAC inhibitors only reduce mucosal / skin damage induced by radiation and / or chemotherapy Rather, it may provide an effective treatment that enhances the anti-tumor effect that maximizes the therapeutic efficiency of radiation therapy and / or chemotherapy.

  The active compound used to carry out the invention is generally a histone hyperacetylating agent such as a histone deacetylase inhibitor. Many such compounds are known. See, for example, P. Dulski, “Histone Deacetylase as Target for Antiprotozoal Agents”, WO 97/11366 (March 27, 1997). Examples of such compounds include, but are not limited to:

  A. Trichostatin A and its analogs: eg, Trichostatin A (TSA); and Trichostatin C (Koghe et al., 1998, Biochem. Pharmacol. 56: 1359-1364) (Trichostatin B is isolated But has not been shown to be an HDAC inhibitor).

  B. Peptides: For example, oxamflatin [(2E-5- [3-[(phenylsulfonyl) aminolphenyl 1] -pent-2-en-4-inohydroxamic acid (Kim et al., Oncogene, 18: 2461) -2470 (1999)); Trapoxin A (TPX) -cyclic tetrapeptide (cyclo- (L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-) Oxo-9,10-epoxy-decanoyl)) (Kijima et al., J. Biol. Chem. 268, 22429-22435 (1993)); FR901228, depsipeptide (Nakajima et al., Ex. Cell Res. 241, 126) -133 (1998); FR225497, cyclic tetrapeptide (H. Mori et al., International Publication No. 00/08048 (February 17, 2000); apicidin, cyclic tetrapeptide [cyclo (NO -Methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)]] (Darkin-Rattra y et al., Proc. Natl. Acad. Sci. USA. 93, 13143-13147 (1996); apicidin 1a, apicidin Ib, apicidin Ic, apicidin IIa, and apicidin IIb (P. Dulski et al., International Publication No. 97/11366); HC-toxin, cyclic tetrapeptide (Bosch et al., Plant Cell 7, 1941-1950 (1995)); WF27082, cyclic tetrapeptide (WO 98/48825); And chlamydocin (Bosch et al., Supra).

  C. Hydroxamic acid-based hybrid polar compound (HPC): For example, salicylihydroxamic acid (SBHA) (Andrews et al., International J. Parasitology 30, 761-768 (2000) Suberoylanilide hydroxamic acid (SAHA) (Richon et al., Proc. Natl. Acad. Sci. USA 95, 3003-3007 (1998)); azelaic bishydroxamic acid: ABHA) (Andrews et al., Supra); azelaic-1-hydroxamate-9-anilide (AAHA) (Qiu et al., Mol. Biol. Cell 11, 2069-2083). (2000)); M-carboxycinnamic acid bishydroxamide (CBHA) (Ricon et al., Supra); 6- (3-chlorophenylureido) 6- (3-chlorophenylureido) ) carpoic hydroxa mic acid: 3-Cl-UCHA) (Richon et al., supra); MW2796 (Andrews et al., supra); and MW2996 (Andrews et al., supra). Analogs that are not effective as HDAC inhibitors include hexamethylene bisacetamide (HBMA) (Richon et al., 1998, PNAS, 95: 3003-3007); and diethyl bis (pentamethylene-N, N-dimethylcarboxamide) malonate ( (EMBA) (Richon et al., 1998, PNAS, 95: 3003-3007).

  D. Single chain fatty acid (SCFA) compound: For example, sodium butyrate (Cousens et al., J. Biol. Chem. 254, 1716- 1723 (1979)); isovalerate (McBain et al., Biochem. Pharm. 53) Volume: 1357-1368 (1997)); Valproic acid; Valerate (McBain et al., Supra); 4-Phenylbutyrate (4-PBA) (Lea and Tulsyan, Anticancer Research, 15, 879-873 (1995) Phenylbutyrate (PB) (Wang et al., Cancer Research, 59, 2766-2799 (1999)); propionate (McBain et al., Supra); butrymide (Lea and Tulsyan, supra); Isobutyramide (Lea and Tulsyan, supra); phenylacetate (Lea and Tulsyan, supra); 3-bromopropionate (Lea and Tulsyan, supra); and tributyrin (Guan et al., Cancer Research, 60, 749-755). (the year of 2000).

  E. Benzamide derivatives: For example, MS-27-275 [N- (2-aminophenyl) -4- [N- (pyridin-3-yl-methoxycarbonyl) aminomethyl] benzamide] (Saito et al., Proc. Natl. Acad. Sci. USA. 96, 4592-4597 (1999)); and 3'-amino derivatives of MS-27-275 (Saito et al., Supra).

  F. Other inhibitors: For example, depudecin [analogues thereof (mono-MTM-depudecin and depudecin-bisether) do not inhibit HDAC] (Kwon et al., 1998, PNAS 95: 3356-3361) And scriptaid (Su et al., 2000, Cancer Research, 60: 3137-3142).

  The description in this application is to increase the therapeutic effect in radiation therapy and chemotherapy for proliferative malignancies or non-malignant diseases because the sequelae of treatment occur with a low frequency but with a high probability of tumor control. Are directed to valproic acid, trichostatin A, and phenylbutyrate, but are not limited to these examples and are not intended to limit the scope of the invention. Also disclosed are pharmaceutical compositions and uses of compounds of valproic acid, trichostatin A and phenylbutyrate.

  In the course of the experiment, valproic acid, trichostatin A, and phenylbutyrate as histone deacetylase inhibitors reduce skin swelling, promote exfoliation healing in mucositis and dermatitis, reduce fibrosis, and It was found not only to prevent tumor formation in irradiated tissue / skin, but also to show potent effects in inhibiting tumor cell growth and enhancing tumor radiation sensitization.

  Histone deacetylase inhibitors can be provided in the form of pharmaceutically acceptable salts. Such pharmaceutically acceptable salts may be used as long as they do not adversely affect the desired pharmacological effect of the compound. Selection and production can be performed by one skilled in the art. Examples of pharmaceutically acceptable salts include alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, salts with organic bases such as ammonium salts, or And salts having an organic base such as triethylamine salt or ethanolamine salt.

  The histone deacetylase inhibitor of the present invention can be administered orally or parenterally. For oral administration, they can be administered in the form of soft and hard capsules, tablets, granules, powders, solutions, suspensions, mouthwashes, and the like. In the case of parenteral administration, they can be administered in the form of creams, ointments, gels, lotions, patches, suppositories, liposomal compositions, injection solutions, infusions, enemas, and so on. Membrane absorption can be maintained in the form of a solid, viscous liquid, or suspension. The selection of methods for the preparation of these formulations and excipients, disintegrants or suspensions can be easily made by those skilled in the art. In addition to valproic acid, trichostatin A, or phenylbutyrate, the histone deacetylase inhibitor of the present invention may further comprise a composition having anti-inflammatory or anti-tumor activity, or other hyperacetylation Agents and pharmaceutically acceptable carriers or pharmaceutically acceptable salts thereof may be included.

  As will be appreciated by those skilled in the art, effective dosages include routes of administration, excipient usage, and other therapeutic treatments such as the use of cytokines, interleukins, anticancer agents, or anti-neoplastic agents, anti-angiogenic agents. , Chemotherapeutic agents, antibodies, conjugated antibodies, immunostimulants, antibiotics, retinoic acid, tyrosine kinase inhibitors, hormone antagonists, or the possibility of simultaneous use with growth stimulants. The effective amount and treatment regimen for any particular subject (eg, human, dog, or cat) is the activity, age, weight, general health, sex, diet, time of administration of the particular compound used. Usually between 0.001% and 100% by weight of the composition, regardless of the means of administration, depending on a variety of other factors, including the rate of excretion, severity and course of the disease, and patient predisposition to the disease Up to. The active compounds of the invention can optionally be administered together with other compounds useful in increasing the therapeutic effect in radiation therapy and / or chemotherapy for proliferative malignancies or non-malignant diseases. Other compounds can optionally be administered simultaneously. As used herein, the term “simultaneously” means sufficiently close to the time that the combined effect occurs (ie, “simultaneously” occurs simultaneously or within a short period of time before and after each other). It may be two or more events). As used herein, administration of two or more compounds “simultaneously” or “in combination” is the time when the presence of one modifies the biological effect of the other with two compounds Is administered close enough. The two compounds can be administered simultaneously or sequentially. Simultaneous administration can be accomplished by mixing the compounds prior to administration, or by administering the compounds at the same time in time but using different anatomical sites or different routes of administration. It can be carried out.

  As used herein, proliferating malignancies are melanoma, Kaposi's sarcoma, osteosarcoma, neuroblastoma, rhabdomyosarcoma, Ewing sarcoma, soft tissue sarcoma, skin cancer, lymphoma, leukemia, breast cancer, Germ cell tumor, undifferentiated neuroectodermal tumor, brain glioma, brain meningioma, head and neck cancer, thyroid cancer, thymic cancer, cervical cancer, anal cancer, colorectal cancer, prostate cancer, lung cancer, liver Includes cell cancer, cholangiocarcinoma, gastric cancer, pancreatic cancer, esophageal cancer, virus-related tumors, and diseases resulting from bone marrow transplantation.

  As used herein, non-malignant diseases include pterygium, Graves' ophthalmopathy, orbital pseudotumor, macular degeneration, keloid, wart, keratophyte cell tumor, hemangioma, arteriovenous malformation, bursa Inflammation, tendonitis, tendonoma, Peony disease, vascular stenosis, ameloblastoma, aneurysmal bone cyst, ectopic bone formation, gynecomastia, ovarian castration, parotitis, eczema, atopic dermatitis Diseases resulting from psoriasis, brachial perioarthritis, epicondyleitis, knee arthritis, dysplasia, hail pox, autoimmune inflammatory arthritis, unexplained histiocytosis, and organ transplantation Include.

  In order to more fully understand the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not intended to limit the invention in any way. All references cited herein are specifically incorporated by reference in their entirety.

Various topical compositions-oily ointments, creams and gels
A. Preparation of oily ointment of phenylbutyrate
470 g white petrolatum (Riedel-de Haen), 25 g paraffin wax 50/52 (regional supplier), and 5 g 4-phenylbutyrate (Merck) are mixed in a beaker and heated to 70 ° C to paste Formed. The paste was stirred at 400 rpm for 1 hour and then cooled at room temperature.

B. Preparation of oily ointment of phenylbutyrate
65 g white petrolatum (Riedel-de Haen), 15 g cetyl alcohol (Riedel-de Haen), 260 g soft paraffin (Merck), 155 g liquid paraffin (Merck), and 5 g 4-phenylbutyrate (Merck) Mix in a beaker and heat to 70 ° C. to form a paste. The paste was stirred at 400 rpm for 1 hour and then cooled at room temperature.

C. Preparation of cream of phenylbutyrate Component I:
70 g Tefose 63 (R), 20 g Superpolystate (R), 10 g Coster 5000 (R), 15 g Myriyol 318 (R), 15 g Coster 5088 (R), and 15 g GMS SE ( ®) (all commercially available from local suppliers) were mixed in a beaker and heated at 70 ° C.

Ingredient II:
5.739 g sodium 4-phenylbutyrate (Triple Crown America, Inc.), 0.125 g methylparaben (Merck), 0.075 g propylparaben (Merck), and 149.061 g deionized water were mixed in a beaker to obtain 70 Heated at ° C.

  Component II was slowly added to Component I followed by stirring at 400 rpm for 5 minutes to form a mixture. 2% StabilezeQM® (prepared by dissolving 2 g StabilezeQM® in 98 g deionized water, heating and stirring at 70 ° C. to form a paste, followed by cooling at room temperature ) Was added to the mixture and stirred for 5 minutes. The pH of the mixture was adjusted to 5.34 using 0.85% phosphoric acid (Merck) and stirred at 600 rpm for 20 minutes. The mixture was cooled at room temperature.

D. Preparation of phenyl butyrate gel Component I:
10 g StabilezeQM® and 232.035 g deionized water were mixed in a beaker and heated at 70 ° C.

Ingredient II:
5.739 g sodium 4-phenylbutyrate (Triple Crown America, Inc.), 0.125 g methylparaben (Merck), 0.075 g propylparaben (Merck), 232.035 g deionized water, and 20 g 10% NaOH in a beaker And heated at 70 ° C.

  Component II was slowly added to Component I followed by stirring at 400 rpm for 20 minutes to form a mixture. The mixture was cooled at room temperature.

E. Preparation of a gel of phenylbutyrate Component I:
10 g StabilezeQM® and 380.561 g deionized water were mixed in a beaker and heated at 70 ° C.

Ingredient II:
5.739 g sodium 4-phenylbutyrate (Triple Crown America, Inc.), 0.125 g methylparaben (Merck), 0.075 g propylparaben (Merck), 83.5 g 1,2-propanediol, and 20 g 10% NaOH Were mixed in a beaker and heated at 70 ° C.

  Component II was slowly added to Component I followed by stirring at 400 rpm for 20 minutes to form a mixture. The mixture was cooled at room temperature.

F. Preparation of sustained-release preparations of phenylbutyrate
Two formulations were prepared with the compositions listed in Table 1.

^＊PF-127（登録商標）は、組成物の基剤であり、カルボキシメチルセルロースナトリウムは濃化剤である。 (Table 1) Composition of two sustained release formulations

^* PF-127 (registered trademark) is the base of the composition and sodium carboxymethylcellulose is a thickener.

Preparation of G. Phenylbutyrate Liposome Formulation In this liposome formulation, egg phosphatidylcholine (EPC) and cholesterol were used as primary lipid components in equimolar or different molar concentrations. Various liposomes arranged with 4-phenylbutyrate were obtained by changing the lipid: phenylbutyrate ratio. Liposomes were prepared by thin film hydration, and were physically evaluated after aligning the size by membrane protrusion.

Preparation of H. trichostatin A ointment
472.5g white petrolatum (Riedel-de Haen), 27g paraffin wax 50/52 (regional supplier), and 0.5g trichostatin A (Sigma) mixed in a beaker and heated to 70 ° C to paste Formed. The paste was stirred at 400 rpm for 1 hour and then cooled at room temperature.

I. Preparation of trichostatin A oily ointment
67.5 g white petrolatum (Riedel-de Haen), 16 g cetyl alcohol (Riedel-de Haen), 260.5 g soft paraffin (Merck), 155.5 g liquid paraffin (Merck), and 0.5 g trichostatin A (Sigma ) Were mixed in a beaker and heated at 70 ° C. to form a paste. The paste was stirred at 400 rpm for 1 hour and then cooled at room temperature.

Preparation of cream of J. valproic acid Component I:
70 g Tefose 63 (R), 20 g Superpolystate (R), 10 g Coster 5000 (R), 15 g Myriyol 318 (R), 15 g Coster 5088 (R), and 15 g GMS SE ( ®) (all commercially available from local suppliers) were mixed in a beaker and heated at 70 ° C.

Ingredient II:
5.739 g of parproic acid (Sigma), 0.125 g of methylparaben (Merck), 0.075 g of propylparaben (Merck), and 149.061 g of deionized water were mixed in a beaker and heated at 70 ° C.

  Component II was slowly added to Component I followed by stirring at 400 rpm for 5 minutes to form a mixture. 2% StabilezeQM® (prepared by dissolving 2 g StabilezeQM® in 98 g deionized water, heating and stirring at 70 ° C. to form a paste, followed by cooling at room temperature ) Was added to the mixture and stirred for 5 minutes. The pH of the mixture was adjusted to 5.34 using 0.85% phosphoric acid (Merck) and stirred at 600 rpm for 20 minutes. The mixture was cooled at room temperature.

Several phenylbutyrate (PB) formulations were characterized, as shown in Table 2, where topical phenylbutyrate (PB) treatment induces histone hyperacetylation in vivo .

^＊cはクリーム、gはゲル、oは軟膏を示す；
¹傾きは皮膚を介した各薬剤の一時間当たりの浸透量を示す（μg/cm²/時間）；
²Y切片（Intercept）（μg/cm²）は、負の数ならば、皮膚を介した各薬剤の潜在的な浸透量を示し、正の数ならば、試料採取時間が長すぎて正確な値を得ることができないことを示す；
³滞留時間は、薬剤が皮膚に浸透するためにかかる平均時間数（時間）を示す；
⁴定数（×10^-5cm²/秒）は、薬剤拡散定数を示す；
⁵Cs（×10⁵mg/cm³）は、皮膚表面における薬剤濃度を示す；
⁶μg/cm²は、皮膚1cm²当たりを介した各薬剤の平均浸透量を示す；
⁷mg/cm³は、皮膚における平均濃度を示す。 Table 2: Formulation characteristics and pharmacokinetic parameters

^* C represents cream, g represents gel, o represents ointment;
¹ slope shows the amount of permeation per hour through each skin (μg / cm ² / hour);
² Y-intercept (μg / cm ² ), if negative, indicates potential penetration of each drug through the skin; if positive, sampling time is too long and accurate Indicates that the value cannot be obtained;
³ Residence time indicates the average number of hours it takes for the drug to penetrate the skin;
⁴ constants (× 10 ⁻⁵ cm ² / sec) indicate drug diffusion constants;
⁵ Cs (× 10 ⁵ mg / cm ³ ) indicates the drug concentration on the skin surface;
^{6 μg} / cm ² shows the average amount of penetration of the drug through the skin 1 cm ² per;
⁷ mg / cm ³ indicates the average concentration in the skin.

  Among various formulations, PB cream (Tri-c-02-3) with good stability, low skin irritation rate, long shelf life and high skin penetration rate was selected for testing (Figure 1A). In order to determine what is the appropriate local PB dose for treating irradiated skin, the amount of histone hyperacetylation in the nucleus is used as a marker to indicate the extent of drug penetration and the topical drug It was shown whether the concentration had a sufficient biological effect. Western blot analysis for acetylated histones in irradiated skin 6 hours after irradiation (40 Gy single fraction) showed that the acetylated form of histone H3 was moderately increased in the control and vehicle-treated groups, but not irradiated. Immediately after, it was shown that it was significantly increased by topical treatment with 200 mg of 1% PB cream per irradiated skin surface (FIG. 1B). Coomassie blue stained gel sections show that the protein loading is comparable. Immunofluorescence staining further showed that histone hyperacetylation in irradiated skin was also apparent in the deeper subcutaneous layer 6 hours after drug treatment (ie, the concentration of drug in skin tests). Consistent with the peak) (FIGS. 1A and 1C-1F). Acetylated H3 in the nucleus was used as a marker to indicate the extent of drug penetration.

Histone Deacetylase Inhibitors purchased adult female Sprague-Dawley (SD) rats from the Taiwan National Science Commission Animal Center, which are effective in reducing normal tissue damage induced by rapid irradiation , The body weight was 250 g to 300 g at the time of irradiation. Each rat was housed in a cage alone and fed food and water. Before irradiation, anesthesia was performed by intraperitoneal injection of 50 mg / kg of pentobarbital. The skin of the entire buttocks area was completely shaved, and the outline of the irradiated area having a diameter of 2 cm was drawn with a marking pen immediately before irradiation. An electron beam of 6MeV energy generated by a linear accelerator was used. On day 0, a dose of up to 40 Gy at 4 Gy / min was delivered to the preparation area. Each group treated with histone deacetylase inhibitor was further divided into three subgroups (5 each): first subgroup treated with vehicle after skin irradiation; after skin irradiation A second subgroup treated with a histone deacetylase inhibitor; and a third subgroup treated with skin irradiation alone. After irradiation with either petrolatum (negative control), madecassol ointment (positive control), or excipients, ie 1% phenylbutyrate cream, 1% valproate cream or 0.1% trichostatin A ointment It was applied topically to the irradiated skin twice daily from day 1 to day 90. The average dose for each treatment in the respective groups are petrolatum skin 1 cm ² per 16 mg, Madekazoru skin 1 cm ² per 16 mg, phenylbutyrate skin 1 cm ² per 50mg, valproate skin 1 cm ² per 50mg, trichostatin A was 5 mg per cm ² of skin, and an equal amount of vehicle base for the control group. The overall skin response was evaluated for all rats. Acute skin reaction was assessed and scored every other day up to 90 days after irradiation using a modified skin scoring system (Abe Y. and Urano M., Fraction size-dependent acute skin reaction of mice after multiple twice -a-day doses., International Journal of Radiation Oncology, Biology, Physics., 18 (2): 359-64, 1990). Where 0 = normal, 0.5 = slight hair removal, 1.0 = hair removal in about 50% of the irradiated area, 1.5 = hair removal in areas above 50%, 2.0 = complete hair removal, 2.5 = clear in areas above 50% Complete hair removal with edema or dry detachment, 3.0 = wet detachment in small area, 3.5 = wet detachment in most area.

  The skin score increases with increasing severity of skin reactions. The average skin reaction score in each group is shown in FIG. On day 11, the score of skin reaction in the group treated with madecazole (positive control) or histone deacetylase inhibitor was below that in the negative control group or vehicle control group. Up to day 21, in most areas in the negative control group or excipient group, hair loss progressed to wet stripping, whereas it was improved in the Madecazole group or histone deacetylase inhibitor group, and Epithelial healing began quickly.

H & E histology of irradiated skin correlates with accelerated healing of wounds by HDAC inhibitors, while valproic acid, trichostatin A, and phenylbutyrate are structurally unrelated histone deacetylase inhibitors All of these showed similar effects in the suppression of radiation-induced skin damage, including acute dermatitis and exfoliation, and late fibrosis, ulceration and necrosis. As shown in FIG. 3, the group treated with a histone deacetylase inhibitor for 180 days was more effective compared to the vehicle group at 180 days and the control group (normal skin and acute reaction on day 7). It has a thick epidermis with many cell layers but a thin dermis (measured from the epidermis to the subcutaneous fat layer) with little collagen deposition.

Changes in radiation-induced histology after treatment with an HDAC inhibitor indicate that the occurrence of radiation-induced damage that correlates with suppression in inflammation and fibrogenic cytokine expression is a radiation-induced transient change, and Inhibition of radiation-induced damage by HDAC inhibitors due to sustained upregulation of pro-inflammatory cytokines such as TNF-α and fibrogenic growth factors such as TGF-β1 and TGF-β2 Has been shown to correlate with suppression of TNF-α and TGF-β expression.

  The timing of peak expression levels of TGF-β1, TGF-β2, and TNF-α expression induced by irradiation correlated with the onset of radiation-induced damage development (FIG. 4). Multiple cytokine RNase protection assays containing a set of templates that generate 32P-labeled antisense RNA probes hybridized with target mRNA for TGF-β1, TGF-β2, TGF-β3, TNF-α, and internal control GAPDH TGF-β1, TGF-β2, TGF-β3 and TNF-α mRNA levels were evaluated using a kit (Riboquant; Pharmingen, Sign Diego, Calif.). After hybridization of the labeled probe to the target RNA, the unprotected RNA was digested with ribonuclease (RNase), the protected RNA fragment was dissolved in a 6% polyacrylamide gel and recorded by phosphorimaging (Molecular Dynamics Corp. Sunnyvale, California). The amount of each mRNA species was quantified using a densitometer and normalized to the internal control GAPDH.

  The average skin score for skin reaction from 5 rats in each group was calculated. The average level of cytokine / growth factor mRNA obtained from the three skin samples in each group was normalized to the internal control GAPDH and expressed as a ratio to the average level in the time-matched control group. Statistical significance at the p <0.05 level for differences in mean skin score and mean mRNA levels, respectively, between treated and control rats using the Mann-Whitney test (Stata Statistical Software, College Station, Texas) Sex was judged.

  In the phenylbutyrate (PB) treated group, TGF-β1, TGF-β2, and TNF-α perturbations were maximal 6 hours after irradiation, but levels were substantially suppressed after 14 days. Even when topical PB treatment was discontinued on day 90, suppression was still sustained at 12 months. In the petrolatum and vehicle control groups, TGF-β1, TGF-β2, and TNF-α mRNA levels in irradiated skin increased and fluctuated above non-irradiated control levels for one year. At 6 hours after irradiation, the first peak reached 2 to 3 times greater than the non-irradiated control level, reached a second peak 10.5 to 16 times larger around 14 to 28 days after irradiation, and 9 months after irradiation And reached a third peak 13 to 14 times larger. Subsequently, the level decreased to 2 to 3 times the normal level by 12 months after irradiation. TGF-β1, TGF-β2, and TNF-α mRNA levels at the first peak at 6 hours in the PB group were higher than those in the petrolatum and vehicle control groups, but on day 14 Decreased to levels lower than those in the petrolatum group and vehicle group and recovered to non-irradiated control levels from day 28-35.

  TGF-β1 and TGF-β2 show similar cellular effects while TGF-β3 shows the opposite effect in inhibiting epithelial cell growth and promoting skin fibroblast proliferation. Changes in TGF-β3 mRNA levels showed a slight, transient 2-fold increase on day 14 in all irradiated groups, followed by a progressive decrease to non-irradiated control levels or below. I understood. No significant difference in TGF-β3 mRNA levels was observed between irradiated groups treated with petrolatum, vehicle or PB.

Immunofluorescence of fibrogenic growth factor TGF-β in irradiated skin was suppressed by a histone deacetylase inhibitor. The same pathological section as in Example 4, anti-TGF-β1 antibiotic, anti-TGF-β2 antibiotic was used. The material was subjected to immunofluorescence. As shown in FIG. 5, TGF-β protein, a potent fibrogenic factor, is upregulated by irradiation, and on days 7 and 180 in the acute reaction group and vehicle treatment group, respectively, and keratinocytes in the epidermis and Although both dermal myofibroblasts are highly expressed in fibrotic skin, TGF-β expression was effectively suppressed in the PB-treated group on day 180.

Immunohistochemistry of the proinflammatory cytokine TNF-α in irradiated skin is suppressed by histone deacetylase inhibitors
On day 270, 3 out of 5 rats in the petrolatum treated group and 4 out of 5 rats in the vehicle treated group compared to 0 out of 5 rats in the PB treated group Chronic ulceration, necrosis, blistering, and inflammatory cell infiltration were demonstrated. The reduction in delayed radiation-induced skin damage by local PB was consistent with suppression of TNF-α expression (Figure 6).

Topical phenylbutyrate (PB) treatment prevents delayed radiation-induced cutaneous tumorigenesis, and many studies have shown that chronic overexpression of TGF-β stimulates neoplastic growth. The decrease in TGF-β expression caused by PB treatment can reduce the incidence of late radiation-induced tumor formation. Newly-developed skin and skin tumors were found to increase over time in the irradiated control group that did not receive PB treatment, but no tumor was seen in the PB-treated irradiated group (Fig. 7). At 90 weeks, 15% (6/39) of the cumulative tumor formation rate was observed in the irradiation group without PB treatment compared to 0% (0/42) in the irradiation group treated with PB . The histology of radiation-induced tumors included fibroma, spindle cell sarcoma, basal cell carcinoma, and squamous cell carcinoma.

Topical phenylbutyrate (PB) is derived from syngeneic cancer cells 1MEA7R.1 and CT-26 (purchased from American Type Culture Collection, Manassas, VA), which have a direct antitumor effect on skin tumor growth , in female BALB / c Mice were injected subcutaneously in the flank region. The maximum tumor size was assumed to be 0.5 cm. Topical HDAC inhibitors and excipients were applied at a dose of 200 mg / mouse twice a day for 4 weeks to cover the entire tumor surface and surrounding skin. Tumor size was calculated weekly with calipers according to the formula ab2 / 2, where a and b are the larger diameter (cm) and the smaller diameter (cm), respectively.

  Topical PB formulations have been shown to have anti-tumor effects. BNL 1MEA7R.1 and CT-26 cancer cells that showed growth inhibition by PB in vitro by up-regulation of p21Cip1 (FIG. 8A) were inoculated on the back of syngeneic mice. Skin tumors were grown to a maximum dimension of 0.5 cm (FIG. 8D) and 200 mg / mouse PB was applied topically to the tumor surface twice a day. By 4 weeks, the tumor sizes of 1MEA7R.1 and CT-26 cancer in the placebo group were approximately 6 and 1.6 times larger than those in the PB treated group, respectively (FIGS. 8B-8C). Skin tumors in the PB-treated group grew slowly without skin ulceration (Figure 8F), whereas tumors in the control or placebo group grew rapidly and showed necrotic appearance and skin ulceration (Figure 8E). Five weeks after treatment, discontinuation of topical PB resulted in loss of tumor growth inhibition, and within 2 weeks, these tumors reached the same size as tumors in the control or placebo groups.

Similar effects of structurally unrelated anti-tumor HDAC inhibitors in the treatment of radiation-induced normal tissue damage and in the inhibition of late radiation-induced tumorigenesis
In addition to PB, other structurally unrelated anti-tumor HDAC inhibitors such as trichostatin A (an antifungal agent) and parproic acid (an anti-seizure agent) also cause radiation-induced damage and tumor formation. Also reduced radiation-induced expression of TGF-β1, TGF-β2, and TNF-α (FIG. 9 and Table 3).

  Total RNA was isolated from frozen skin samples using Trigent (Molecular Research Center Inc., Cincinnati, Ohio). Total RNA (30 μg) was electrophoresed on a denaturing formaldehyde-agarose gel, blotted onto Hybond N (Amersham, Amersham, UK) and fixed by ultraviolet (UV) irradiation. Membranes were incubated with 32P labeled probe in Rapid-hyb buffer (Amersham) as described below. In order to prepare probes for rat TGF-β1 and TGF-β2, their full-length coding sequences were transferred to specific forward primers (TGF-β1, 5′-CGGGTGGCAGGCGAGAGC-3 ′ (SEQ ID NO: 1); and TGF -β2, 5'-CATGCACTACTGTGTGCT-3 '(SEQ ID NO: 2)) and reverse primer (TGF-β1, 5'-GGAATTGTTGCTATATTTCTGC-3' (SEQ ID NO: 3); and TGF-β2, 5'-CCGAGGACTTTAGCTGCA-3 '(SEQ ID NO: 4)) was amplified by reverse transcription polymerase chain reaction. A pair of TNF-α and GAPDH templates from the RNase protection assay kit (Riboquant; Pharmingen) was used to generate 32P-labeled antisense RNA probes that hybridized with mRNA for TNF-α and GAPDH.

Table 3 Summary of histological findings in irradiated skin treated with various HDAC inhibitors

  Histological findings on skin preparations obtained from 5 irradiated rats in each group are shown as follows: A, subepithelial swelling; B, dry exfoliation in areas above 50%; C, 50% Wet exfoliation in areas above; D, atopy of epidermis in most areas; E, atopy of skin appendages in most areas; F, increased thickness of epidermis with more cell layers; G, collagen deposition Increased, vascular density, and increased dermis thickness; H, chronic ulceration, necrosis, blistering, and more increased inflammatory cell infiltration; I, TGF-β1, TGF-β2, and TNF-α Down-regulation; J, prevention of delayed radiation-induced tumor formation; K, evidence of a direct anti-tumor effect on skin tumor growth. “+” To “++++++” means that a preparation obtained from 1 to 5 rats showed a reaction or an effect as indicated.

Valproic acid uses radiation enhancement ratio (RER), which enhances tumor radiation sensitization, to calculate the ratio of radiation dose that shows the same effect.
RER = (radiation dose without drug treatment at 10% survival rate) / (radiation dose with drug treatment at 10% survival rate).
Valproic acid (0.1 mM or 0.5 mM) was added to CNE2 nasopharyngeal carcinoma cells 60 minutes before irradiation with doses of 2 Gy to 16 Gy and then washed away. One week later, colonies containing more than 50 cells were counted. Plate efficiency was the same as in the control group and the PB-treated group. The radiation enhancement ratio (RER) of 0.1 mM or 0.5 mM valproic acid was increased to 1.2 and 1.7, respectively, at 10% survival (FIG. 10).

Co-administration of trichostatin A (TSA), ganciclovir (GCV), and radiation (RT), an HDAC inhibitor as a gene regulator that selectively kills Epstein-Barr virus (EBV) -infected cancer cells , EBV thymidine The kinase's activity to kill EBV-related tumors by ganciclovir (GCV) may be upregulated, which inhibits cellular DNA polymerase and causes EBV thymidine kinase to cause cell death in the S phase of the cell cycle Can be phosphorylated. Because HDAC inhibitors also act as anti-proliferative and radiation sensitizers, TSA, GCV, and radiation (RT) combination therapy is a nasopharyngeal cancer, Hodgkin's disease lymphoma, X-related lymphocytes in immunosuppressed children It may have therapeutic benefits for EBV-related tumors such as proliferative diseases, AIDS-related non-Hodgkin lymphoma, and smooth muscle tumors.

For the EBV thymidine kinase activity assay, an equal volume of cytosolic fraction was added to the reaction buffer (50 mM Tris-HCl (pH 7.4), 1 mg / ml BSA, 3 mM creatine phosphate, 11.2 U / 30 min at 37 ° C. for 30 min. Incubated with ml creatine phosphokinase, 0.1 μM [3H-8] -GCV (specific activity 13.5 Ci / mmol), 2.5 mM ATP, 2.5 mM MgCl ₂ , 10 mM NaF, 10 mM DTT). The reaction mixture was added onto Whatman R DE-81 ion exchange chromatography paper, which was then washed 3 times with 1.5 mM NH ₄ COOH to remove the unphosphorylated form of [3H-8] -GCV. . After air drying, these papers were incubated overnight with a liquid scintillation cocktail. The phosphorylated form of [3H-8] -GCV bound to Whatman R DE-81 ion exchange chromatography paper was measured with an LS6500 scintillation counter (Beckman).

  As shown in FIG. 11A, EBV thymidine kinase activity was upregulated by 48 hours of 1 nM TSA treatment. The combination of TSA, GCV, and radiation maximized selective killing of EBV positive cells, but never occurred in EBV negative cells (FIG. 11B). Doubly cells (EBV +) and HL-60 (EBV-) are treated with ganciclovir (GCV) (10 μg / ml) and / or trichostatin A (TSA) (1 nM) for 48 hours, followed by 1 Gy radiation (RT) Irradiated or not. Viable cells were counted after 1 week.

Animal model chemotherapy for the treatment of radiation-induced mucositis using topical phenylbutyrate Mucositis induced by chemotherapy and a hamster model of radiation-induced mucositis were made. The reproducibility of the model was confirmed by the coincidence of ulcerative mucositis appearance between day 12 and day 15 after irradiation. Using this model, the efficiency of topical phenylbutyrate gels and solutions was tested for their ability to modify the course of radiation-induced mucositis. In order to cause severe mucositis around the 15th day, an acute irradiation dose of 40 Gy was applied on the 0th day. The use of acute irradiation to induce mucositis is preferred over the use of either radiation or chemotherapy taken for these original studies. Because acute models have little systemic toxicity, animals rarely die. This fact made it possible to use smaller groups in the original study. Elucidation of the effectiveness of many compounds using an acute model has been successful. Thus, the rapid irradiation model is suitable as an initial protocol for screening a diverse family of compounds.

  Mucositis was induced using an acute irradiation protocol. On day 0, a single line dose (40 Gy / dose) of radiation was applied to all animals. Radiation was generated with a 250 kilovolt potential (15 mM) source at a focal length of 50 cm and cured with a 0.35 mm Cu filtration system. Irradiation targeted the left buccal pouch mucosa at a rate of 121.5 cGy / min. Prior to irradiation, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (80 mg / kg). The left buccal sac was turned over, fixed, and isolated using an induction shield.

  The study used 20 hamsters, arbitrarily divided into 4 groups of 5 animals per group. Each group was assigned a different treatment as follows: group 1 was 20 μl PBS / hamster; group 2 was 200 μg sodium phenylbutyrate / 20 μl PBS / hamster; group 3 was 20 mg placebo (gel base) / hamster; Group 4 is 20 mg phenyl butyrate gel / hamster. From day 15 onwards, each substance was applied topically once daily for 10 consecutive days to the radiation-induced mucositis area of the test hamster. Image analyzer (Life Science Resources VISTA, version 30) showing mucositis wound area on clean plastic sheet on days 15, 17, 19, 21, 23, and 25 Quantified by use of The half-stop time (CT50) of the mucositis wound area was measured by linear regression using GraphPad Prism (Graph Pad Software, USA), and an unpaired Student t test was performed at each time point. And applied for comparison between placebo group. At p <0.05, the difference was considered statistically significant.

  As shown in Table 4, a significant increase in mucositis wound closure (p <0.01) was observed in group 2 (200 μg sodium phenylbutyrate / 20 μl PBS / hamster) on days 21, 23, and 25, and Observed in group 4 (20 μg phenylbutyrate gel / hamster) on days 17, 19, 21, 23 and 25. CT50 is also significantly significant for Group 2 up to 6.5 ± 0.2 days (compared to 7.7 ± 0.2 days in Group 1) and up to 8.9 ± 0.2 days in Group 4 (compared to 10.5 ± 1.3 days in Group 3) Decreased (p <0.01).

差は、^＊p＜0.05、^＊＊p＜0.01で有意と考えられる。 TABLE 4 Mucositis wound healing treated with phenylbutyrate (PB)

Differences are considered significant at ^* p <0.05 and ^** p <0.01.

Other Embodiments All of the features disclosed herein can be used in any combination. Each characteristic disclosed in this specification may be replaced by an alternative characteristic serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

  From the above description, those skilled in the art can easily ascertain the essential features of the present invention and make various changes and modifications to the present invention without departing from its concept and scope. And can be adapted to the conditions. For example, compounds that are structurally and functionally similar to the histone deacetylase inhibitors described above can also be used in the practice of the present invention. Accordingly, other aspects are also within the scope of the appended claims.

FIG. 2 shows a pharmacological study of the delivery of various PB formulations through the skin. FIG. 6 shows Western blot analysis for acetylated H3 in irradiated skin (40 Gy single ratio) treated with or without phenylbutyrate cream (PB) for 6 hours after irradiation. Lane 1, normal skin without irradiation; Lane 2, irradiated skin without treatment; Lane 3, irradiated skin treated with vehicle; Lane 4, irradiated skin treated with 1% PB cream. FIG. 6 shows immunofluorescence staining of acetylated H3 in irradiated skin treated with or without phenylbutyrate cream (PB) for 6 hours after irradiation. C shows normal skin without irradiation; D shows irradiated skin without treatment; E shows irradiated skin treated with excipients; F shows irradiated skin treated with 1% PB cream. Show. It is a figure which shows the acute skin reaction score which shows the time curve of the average skin score after 40 Gy irradiation. It is a figure which shows the H & E histology photograph which shows that a local histone deacetylase inhibitor has an effect in suppressing the skin damage induced by irradiation. A, D, G, and J are H & E histology at 40 × field; B, E, H, and K are H & E histology at 100 × field; C, F, I, and L Is an H & E histology with 200x field of view. A to C are from normal skin. D to F are those of an acute reaction on the 7th day after irradiation, and indicate subepithelial tissue swelling (white arrows). GI is from the 180 day excipient group, showing thin epithelial tissue, thick dermis with subepithelial tissue swelling, increased blood vessel and skin appendage density, and more collagen deposition. The black arrow indicates that the subcutaneous fat layer in the vehicle group was replaced by fibrous tissue and appendages. JL is from the group treated with histone deacetylase inhibitors on day 180, thicker epidermis with 10-30 cell layers (black arrows), less subepithelial tissue swelling, It indicates that the dermis with less collagen deposition is thin, and that there are fewer skin appendages. FIG. 6 shows changes in the expression level of TGF-β1 after irradiation treated with or without a local HDAC inhibitor (phenylbutyrate (PB)). Transient variation in TGF-β1 mRNA levels in skin after irradiation is normalized to the internal control GAPDE and expressed as a ratio to the level in the non-irradiated control sample. Each point represents the average mRNA level of 5 samples in the same group of petrolatum, excipient, or phenylbutyrate (PB). The arrows indicate that drug treatment was discontinued after 90 days ( ^* p <0.05, ^** p <0.001 compared with PB treatment and vehicle group). FIG. 6 shows changes in the expression level of TGF-β2 after irradiation treated with or without a local HDAC inhibitor (phenylbutyrate (PB)). Transient variation in TGF-β2 mRNA levels in skin after irradiation is normalized to the internal control GAPDE and expressed as a ratio to the level in the non-irradiated control sample. Each point represents the average mRNA level of 5 samples in the same group of petrolatum, excipient, or phenylbutyrate (PB). The arrows indicate that drug treatment was discontinued after 90 days ( ^* p <0.05, ^** p <0.001 compared with PB treatment and vehicle group). FIG. 6 shows changes in the expression level of TGF-β3 after irradiation treated with or without a local HDAC inhibitor (phenylbutyrate (PB)). Transient variation in TGF-β3 mRNA levels in skin after irradiation is normalized to the internal control GAPDE and expressed as a ratio to the level in the non-irradiated control sample. Each point represents the average mRNA level of 5 samples in the same group of petrolatum, excipient, or phenylbutyrate (PB). The arrows indicate that drug treatment was discontinued after 90 days ( ^* p <0.05, ^** p <0.001 compared with PB treatment and vehicle group). It is a figure which shows the change of the expression level of TNF- (alpha) after the irradiation treated with the local HDAC inhibitor (phenyl butyrate (PB)) or without. Transient variation in TGF-α mRNA levels in skin after irradiation is normalized to the internal control GAPDE and expressed as a ratio to the level in the non-irradiated control sample. Each point represents the average mRNA level of 5 samples in the same group of petrolatum, excipient, or phenylbutyrate (PB). The arrows indicate that drug treatment was discontinued after 90 days ( ^* p <0.05, ^** p <0.001 compared with PB treatment and vehicle group). It is a photograph of immunofluorescence using anti-TGF-β1 and 2 antibodies, and shows that the expression of TGF-β, a fibrogenic growth factor, was suppressed by the histone deacetylase inhibitor in Example 4. FIG. A is from normal skin stained with TGF-β. B is a photograph of acute dermatitis on day 7 after irradiation without any drug treatment, indicating that TGF-β was upregulated. C is from the vehicle group 180 days after irradiation, and TGF-β expression increases and persists with time, both in the epidermal keratinocytes and in the dermal myoblasts. It is expressed at high levels in fibrogenic skin. D is from the PB-treated group 180 days after irradiation, and local PB effectively suppresses TGF-β expression and indicates that collagen fiber accumulation in the dermis is small. This correlates well with the large number of cell layers in the epithelium because TGF-β promotes glandular fibroblast proliferation but inhibits epithelial cell growth. It is the photograph of the immunohistochemistry using the anti- TNF- (alpha) antibody which shows that the expression of TNF- (alpha) which is a proinflammatory cytokine was suppressed with the histone deacetylase inhibitor. A is from normal skin for TNF-α staining. B is in the PB-treated group 270 days after irradiation, and the histone deacetylase inhibitor efficiently suppresses TNF-α expression. This correlates well with less inflammatory cell infiltration and no chronic ulceration. C is irradiated skin treated with petrolatum, indicating that TNF-α was upregulated in the subcutaneous tissue showing ulceration on day 270 (arrows indicate necrotic wounds). D shows irradiated skin treated with vehicle, indicating that TNF-α was upregulated in subcutaneous tissue showing severe inflammatory cell infiltration on day 270. It is a figure which shows suppression of the skin tumor formation after the irradiation by an HDAC inhibitor. Newly developed skin or skin tumors increased with time after irradiation after 50 weeks in the control group that did not receive PB treatment, but none of the PB treated group developed tumors. FIG. 5 shows that local phenylbutyrate (PB) directly inhibits tumor growth, CT- during treatment with p21Cipl protein, a cell cycle inhibitor at BNL 1MEA7R.1, and 4 mM PB. Figure 2 shows a time course analysis of the upregulation level of 26 cancer cells. FIG. 4 shows that local phenylbutyrate (PB) directly inhibits tumor growth and shows a growth inhibition curve. FIG. 5 shows that local phenylbutyrate (PB) directly inhibits tumor growth, showing tumor growth inhibition in vivo. D shows the initial tumor size of 1MEA7R.1 approximately 0.5 cm below the skin in the dimensions before treatment. E is a tumor treated with placebo or vehicle at 4 weeks. F is a PB-treated tumor at 4 weeks. Northern blot assay showing that upregulation of TGF-β1,2 and TNF-α expression in skin after irradiation is suppressed by structurally unrelated HDAC inhibitors (trichostatin A and valproic acid) . Shows enhancement of tumor radiation sensitization by valproic acid. Points represent the average of two different experiments performed in triplicate; SD is less than 10%. Epstein-Barr virus (EBV) -positive Daudi lymphoma cells are selectively destroyed, but EBV-negative HL-60 leukemia cells are not destroyed by low doses of a combination of HDAC inhibitors, antibiotics and radiation Indicates. A shows that 48 h of 1 nM TSA upregulated EBV thymidine kinase activity in EBV-positive Daudi cells. B shows that the combination of TSA, GCV, and radiation results in maximal death of EBV positive cells. Bars represent the average of three different experiments performed in triplicate.

Claims (21)

  A pharmaceutical composition having an increased therapeutic effect in chemotherapy and / or radiation therapy for proliferative malignant or non-malignant diseases with a low frequency of treatment sequelae and high likelihood of tumor control A pharmaceutical composition comprising a hyperacetylating agent and a pharmaceutically acceptable carrier or a pharmaceutically acceptable salt thereof.   Increased therapeutic effects simultaneously enhance tumor radiation sensitization or sensitize tumors to chemotherapy, increase tumor growth inhibition, promote wound healing in mucositis and dermatitis, and plantar -The pharmaceutical according to claim 1, wherein the severity of palm syndrome is prevented / reduced, tissue fibrosis is reduced, normal tissue is protected from cell death, dry mouth is prevented, and tumor formation is inhibited. Composition.   2. The pharmaceutical composition according to claim 1, wherein the hyperacetylating agent is a histone deacetylase inhibitor.   2. The pharmaceutical composition according to claim 1, wherein the radiation therapy is teleradiation therapy, brachytherapy, or ionizing radiation.   Proliferative malignancies include melanoma, Kaposi sarcoma, osteosarcoma, neuroblastoma, rhabdomyosarcoma, Ewing sarcoma, soft tissue sarcoma, skin cancer, lymphoma, leukemia, breast cancer, germ cell tumor, undifferentiated neuroectodermal Tumor, brain glioma, brain meningioma, head and neck cancer, thyroid cancer, thymic cancer, cervical cancer, anal cancer, colorectal cancer, prostate cancer, lung cancer, hepatocellular carcinoma, bile duct cancer, stomach cancer, pancreatic cancer, 2. The pharmaceutical composition according to claim 1, wherein the composition is selected from the group consisting of esophageal cancer, virus-related tumor, and disease resulting from having undergone bone marrow transplantation.   Non-malignant diseases include pterygium, Graves' eye disease, orbital pseudotumor, macular degeneration, keloid, warts, keratophyte, hemangioma, arteriovenous malformation, bursitis, tendonitis, tendonoma, Peony disease, vascular stenosis, enamel epithelioma, aneurysmal bone cyst, ectopic bone formation, gynecomastia, ovarian castration, parotitis, eczema, atopic dermatitis, psoriasis, brachial shoulder joint Claims selected from the group consisting of inflammation, epicondylaritis, knee arthritis, dysplasia, hail pox, autoimmune inflammatory arthritis, unexplained histiocytosis, and diseases resulting from organ transplantation The pharmaceutical composition according to 1.   2. The pharmaceutical composition according to claim 1, wherein the histone hyperacetylating agent is trichostatin A or trichostatin C.   2. The pharmaceutical composition of claim 1, wherein the histone hyperacetylating agent is selected from the group consisting of oxamflatin, trapoxin A, FR901228, apicidin, HC-toxin, WF27082, and chlamycin.   2. The pharmaceutical composition of claim 1, wherein the histone hyperacetylating agent is selected from the group consisting of salicylic hydroxamic acid, suberoylanilide hydroxamic acid, and azelaic acid bishydroxamic acid.   Histone hyperacetylating agents are from azelaic acid-1-hydroxamate-9-anilide, M-carboxycinnamic acid bishydroxamide, 6- (3-chlorophenylureido) carpoic hydroxamic acid, MW2796, and MW2996 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is selected from the group consisting of:   The histone hyperacetylating agent is selected from the group consisting of sodium butyrate, isovalerate, valerate, 4-phenylbutyrate, sodium phenylbutyrate, propionate, butrimide, isobutyramide, phenylacetate, 3-bromopropionate, valproic acid, and tributyrin 2. The pharmaceutical composition according to claim 1, which is selected.   2. The pharmaceutical composition according to claim 1, wherein the histone hyperacetylating agent is MS-27-275 or a 3′-amino derivative thereof.   2. The pharmaceutical composition according to claim 1, wherein the histone hyperacetylating agent is depudecin or scriptaid.   2. The pharmaceutical composition according to claim 1, which is administered parenterally.   2. The pharmaceutical composition of claim 1, which is a cream, ointment, gel, paste, powder, lotion, patch, suppository, liposome composition, suspension, mouthwash, enema, injection solution, or infusion.   2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is present in an amount of a hyperacetylating agent from 0.001% to 100% by weight of the composition.   A second substance selected from the group consisting of cytokines, interleukins, anticancer agents, or anti-neoplastic agents, antiangiogenic agents, chemotherapeutic agents, antibodies, conjugated antibodies, immunostimulants, antibiotics, retinoic acid, tyrosine kinase 2. The pharmaceutical composition of claim 1, further comprising an inhibitor, a hormone antagonist, and a growth stimulator.   18. The pharmaceutical composition of claim 17, wherein the conjugated antibody is selected from the group consisting of trastuzumab, c225, rituximab, and cetuximab.   Chemotherapeutic agents are alkylating agents, purine analogs, pyrimidine analogs, vinca alkaloids, vinca-like alkaloids, etoposide, etoposide-like drugs, corticosteroids, nitrosoureas, antimetabolites, platinum-based cytotoxic agents, antiandrogens 18. The pharmaceutical composition of claim 17, selected from the group consisting of: and estrogen.   18. The pharmaceutical composition of claim 17, wherein the anti-angiogenic agent is selected from the group consisting of thalidomide, SU5416, SU6668, thrombospondin-1, endostatin, and angiostatin.   18. The pharmaceutical composition of claim 17, wherein the antibiotic is selected from the group consisting of ganciclovir, acyclovir, and famciclovir.

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