MX2013004423A - Use of cysteamine and derivatives thereof to suppress tumor metastases. - Google Patents

Abstract

The present disclosure is directed to methods for inhibiting or suppressing metastasis of a tumor in a mammalian subject using a cysteamine product, e.g., cysteamine or cystamine or derivatives thereof. Also described herein is a method for treating pancreatic cancer in a mammalian subject by administering a cysteamine product described herein.

Description

USE OF CISTEAMINE AND DERIVATIVES OF THE SAME PAR EXTRACT TUMOR METASTASES Field of the invention The present invention relates to the use of cysteamine or cystamine or derivatives thereof to treat, inhibit or suppress metastasis of a tumor. The present invention also relates to the use of cysteamine or cystamine or derivatives thereof to treat pancreatic cancer.

Background Cysteamine (HS-CH2-CH2-NH2) is a small sulfhydryl compound that is able to cross cell membranes easily due to its small size. Cysteamine plays a role in the formation of the glutathione protein precursor (GSH), and is currently approved by the FDA for use in the treatment of cystinosis, an intra-lysosomal cystine storage disorder. In cystinosis, cysteamine acts by converting cystine to cysteine and mixed disulfide of cysteine-cysteamine, both of which are then able to leave the Msosome through the cysteine and lysine transporters respectively (Gahl et al., N Engl J Med 2002; 347 (2): 111-21). Within the cytosol, the mixed disulfide can be reduced by its reaction with glutathione and the released cysteine can be used for further synthesis of GSH. Treatment with cysteamine has been shown to result in decreased intracellular cystine levels in circulating leukocytes (Dohil et al., J. Pedlatr 148 (6): 764-9, 2006).

Cysteamine is also discussed in (Prescott et al., Lancet 1972: 2 (7778): 652; Prescott et al., Br Med J 1 978; 1 (61 16): 856-7; Mitchell et al., Clin Pharmacol Ther 1974; 16 (4): 676-84; Toxicol Appl Pharmacol 7979 48 (2): 221-8; Qiu et al., World J Gastroenterol 13: 4328-32, 2007. Unfortunately, sustained concentrations of cysteamine necessary for therapeutic effect are difficult to maintain due to the rapid metabolism and elimination of cysteamine from the body, with almost all the cysteamine administered converted to taurine in a matter of hours.These difficulties are transferred to patients in the form of high dosage levels and frequencies , with all the consequential unpleasant side effects associated with cysteamine (eg, gastrointestinal distension and body odor) See insert package for CYSTAGON® (cysteamine bitartrate) International Publication No. WO 2007/079670 describes cysteamine enteric products coated and a method to reduce the dosage frequency of cysteamine.

Cysteamine is treated in the international patent applications nos. WO 2009/070781 and WO 2007/089670, and U.S. Patent Publications Nos. 201 10070272, 200900481 54 and 20050245433.

In the field of cancer, studies of anti-cancer effects of cysteamine have been reported with respect to the development and proliferation of cancer. Cysteamine prevented the development of metaplasia and carcinogenesis of mammary tumor and gastric cancer induced chemically and by radiation (6-8). Cysteamine by itself or conjugated with nanoparticles or other compounds suppress the proliferation of cancer cells derived from neural neoplastic tumors (9, hepatocellular carcinoma SMIVIC-7721 (10), breast cancer (11), and melanoma cell lines (12) in vitro .

BRIEF DESCRIPTION OF THE INVENTION In one aspect, a method for inhibiting or suppressing metastasis of a mammalian subject comprising administering cysteamine, cystamine or pharmaceutically acceptable salts thereof to the subject in an effective amount to inhibit tumor metastasis is disclosed herein.

In another aspect, a method for treating pancreatic cancer in a mammalian subject comprising administering cysteamine, cystamine or pharmaceutically acceptable salts thereof to the subject in an effective amount to treat cancer is disclosed herein.

In various embodiments, the cysteamine, cystamine or pharmaceutically acceptable salts thereof are administered orally, being optionally formulated for delayed release. In some embodiments, the cysteamine, cystamine or pharmaceutically acceptable salt thereof is formulated in an enterically coated tablet or capsule.

The delayed release formulation, in some embodiments, comprises an enteric coating that releases the cysteamine or cystamine when the formulation reaches the small intestine or a region of the gastrointestinal tract of a subject in which the pH is higher that approximately pH 4.5.

In some embodiments, the cysteamine, cystamine or pharmaceutically acceptable salts thereof is administered less than four times a day (eg, three times, twice or once a day). In some embodiments, the cysteamine, cystamine or pharmaceutically acceptable salts thereof are administered twice a day.

In various embodiments of the invention, the cysteamine, cystamine or pharmaceutically acceptable salt thereof is administered in a dose of about 0.01 mg to 1000 mg per kg / mg / k) of body weight per day. In some embodiments, the cysteamine, cystamine or pharmaceutically acceptable salt thereof is administered at a daily dose ranging from about 10 mg / kg to about 250 mg / kg or from about 1 00 mg / kg to about 250 mg / kg or from about 60 mg / kg or up to about 100 mg / kg or from about 50 mg / kg to about 90 mg / kg, or from about 30 mg / kg or up to about 80 mg / kg, or from about 20 mg / kg to about 60 mg / kg, or from about 10 mg / kg to about 50 mg / kg. In addition, the effective dose may be 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90 mg / kg, 1 00 mg / kg, 125 mg / kg, 1 50 m (kg, 175 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg , 500 mg / kg, and can increase by Increases of 25 mg / kg up to 1000 mg / kg, or can vary between any of two of the above values. In some embodiments, the cysteamine product is administered in a total daily dose of from about 0.25 g / m2 to 4.0 g / m2 body surface area, approximately 0.5-2.0 g / m2 body surface area, or 1-1.5 g / m2 of body surface area, or 1-1.95 g / m2 of body surface area, or 0.5-1 g / m2 of body surface area, or approximately 0.7-0.8 g / m2 of body surface area, or approximately 1.35 g / m2 of body surface area, or about 1.3 to about 1.95 grams / m2 / day, or about 0.5 to about 1.5 grams / m2 / day, or about 0.5 to about 1.0 grams / m2 / day, for example, at less approximately 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0., 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8.1.9 or 2 g / m2, or up to approximately 0.8, 0.9, 1.0 , 1.1, 1.2, 1.3, 11.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 3.25, 3.5 or 3.75 g / m2, or may vary between any two of the previous values.

In some embodiments, administration results in increased thiol levels compared to levels prior to the administration of cysteamine, cystamine or pharmaceutically acceptable salts thereof.

In some embodiments, the administration modulates the enzymatic activity of a matrix metalloproteinase (MMP, e.g., MMP-1, MMP-2, MMP-3, MMP-4, MMP-5, MMP-6, MMP-6, M P-8, MMP-9, MP-10, MMP-11, MP-12, MMP-13 and / or MMP-14) in a cancer cell of the tumor. In some embodiments, the enzymatic activity of MMP is decreased in a primary tumor.

In some embodiments, administration decreases metastatic nodules and / or ascites in the subject.

The methods described herein are useful for the inhibition or suppression of metastases from any cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, melanoma, prostate cancer, pancreatic cancer, head and neck cancer, lung cancer, non-small cell lung carcinoma, kidney cancer, colorectal cancer and gastric cancer.

The aspects of the invention which are described herein as methods (especially methods which involve treatment) may alternatively be described as (medical) uses of cysteamine, cystamine or pharmaceutically salts thereof. For example, in one variation, the use of cysteamine, cystamine or pharmaceutically acceptable salts thereof to inhibit or suppress tumor metastasis is described herein. In another variation, the use of cysteamine, cystamine or pharmaceutically acceptable salts thereof for treating pancreatic cancer is described herein.

The agents and compositions described herein for use in treatment are themselves aspects of the invention as well, for example, as compositions of matter.

In the methods (or uses) of treatment described herein, the methods optionally comprise administering a therapy of cancer auxiliary to the subject in combination with cysteamine, cystamine or pharmaceutically acceptable salts thereof. In some modalities, the auxiliary cancer therapy is selected from the group consisting of chemotherapy, surgery, radiotherapy, cancer vaccines, immunotherapy, gene therapy, thermotherapy, and laser therapy.

In some embodiments, the methods (or uses) described herein further comprise administering an additional therapeutic agent selected from the group consisting of an MMP inhibitor, a chemotherapeutic agent, a growth inhibitory agent, and a cytokine.

With respect to any combination treatment described herein, the cysteamine, cystamine or pharmaceutically acceptable salts thereof may be administered simultaneously with the other active agents, which may be in admixture with the agent or may be in a separate composition. Each composition preferably includes a pharmaceutically acceptable diluent, auxiliary or carrier. When the agents are administered separately, they can be administered in any order.

In another aspect, a method for decreasing matrix metalloproteinase (MMP) enzyme activity in a cancer cell comprising contacting the cell with cysteamine, cystamine or pharmaceutically acceptable salts thereof in an effective amount to decrease activity is described herein. Enzymatic of MMP in the cancer cell. In various embodiments, the MMP is selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-4, MP-5, MMP-6, MMP-6, MMP-8, MP-9, P-1 0, MP-1 1, MP-1 2, MMP-1 3 and MMP-14. In several embodiments, the MP is selected from the group consisting of MMP-9, MMP-1 2 and MMP-14. In several modalities, the MMP is MMP-9.

The preceding summary is not intended to define each aspect of the invention, and additional aspects are described in other sections, such as the Detailed Description. The entire document is intended to be related as a unified description, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. .

In addition to the foregoing, the invention includes, as a further aspect, all embodiments of the invention narrower in scope in any manner than the variations defined by prior specific paragraphs. For example, certain aspects of the invention that are described as a genre, and it should be understood that each member of a genus is, individually, an aspect of the invention. In addition, aspects described as a genre or selecting a member of a genre, it should be understood that combinations of two or more members of the genre are encompassed. Although the applicant (s) invented the full scope of the invention described herein, the applicants do not intend to divide the subject matter described in the prior art work of others into paragraphs. Therefore, in the event that prior statutory technique within the scope of a paragraph causes the attention of the applicant (s) by a Patent Office or another entity or individual, the applicant (s) reserve the right to exercise rights of amendment under the applicable patent laws to redefine the subject matter of that paragraph to specifically exclude such prior statutory technique or obvious variations of statutory technique from the scope of such paragraph. The variations of the invention defined by such amended paragraphs are also intended as aspects of the invention.

Brief description of the figures Figures 1 A and 1 B provide survival curves of Kaplan-Meier of mice harboring tumors of HS766T (Figure 1 A) and MIA-PaCa2 (Figure 1 B).

Detailed description The present disclosure relates, in general, to the discovery of the anti-invasive and / or anti-metastatic effects of cysteamine in human cancers.

Definitions As used herein and in the appended claims, in the singular forms "a", "an", and "the", "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the reference to "a derivative" includes a plurality of such derivatives and reference to "a patient" includes reference to one or more patients and so on.

Also, the use of "o" means "and / or" unless stated otherwise. Similarly, "understand", "understand", "understand", "include", "include" and "including" are interchangeable and are not intended to be limiting.

It will be further understood that where descriptions of various embodiments use the term "comprising", those skilled in the art would understand that in some specific cases, a modality may alternatively be described using the word "consisting essentially of" or "consisting of".

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood to one of ordinary skill in the art to which this description pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the methods and products described, exemplary methods, devices and materials are described herein.

The documents discussed before and throughout the text are provided only with their description before the date of presentation of the present application. Nothing here is to be construed as an admission that the inventors have no right to precede such a description by virtue of the foregoing description. Each document is incorporated by reference in its entirety with particular attention to the description to which it is cited.

The following references provide someone of skill with a general definition of many of the terms used in this description: Singleton, et al. , DICTIONARY OF MICROBIOLOGY AND MOLEUCLAR BIOLOGY (Dictionary of microbiology and molecular biology) (2nd ed., 1994); THE CAMBRI DGE DICTIONARY OF SCI ENCE AND TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS (The genetics glossary) 5th ed. , R. Rieger et al. (eds.), Springer Verlag (1991); and Hale and Marham, THE HARPER COLLI NS DICTIONARY OF BIOLOGY (1 991): As used herein, a "therapeutically effective amount" or "effective amount" refers to the amount of a cysteamine product, for example, cysteamine, cystamine or a pharmaceutically acceptable salt thereof, sufficient to result in improvement of symptoms, for example, treatment, cure, prevention or improvement of the relevant medical condition, or an increase in the speed of treatment, cure, prevention or improvement of such conditions, usually providing a statistically significant improvement in the population of patients treated. When referring to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When referring to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, either administered in combination, including serially or simultaneously. In several modalities, a quantity therapeutically Effectiveness of the cysteamine product improves symptoms associated with various cancers, including but not limited to, loss of appetite, pain, oral pain, upper abdominal pain, fatigue, abdominal swelling, persistent pain, bone pain, nausea, vomiting, constipation, loss of weight, headaches, rectal bleeding, night sweats, digestive discomfort and painful urination.

The "treatment" refers to prophylactic treatment or therapeutic treatment. In certain embodiments, "treatment" refers to the administration of a compound or composition to a subject for therapeutic or prophylactic purposes.

A "therapeutic" treatment is a treatment administered to a subject exhibiting signs or symptoms of pathology for the purpose of diminishing or eliminating those signs or symptoms. The signs or symptoms can be biochemical, cellular, histological, functional or physical, subjective or objective.

A "prophylactic" treatment is a treatment administered to a subject that does not exhibit signs of disease or exhibits only the early signs of the disease, for the purpose of decreasing the risk of developing pathology. The compounds or compositions of the disclosure may be given as a prophylactic treatment to reduce the likelihood of developing a pathology or to minimize the severity of the condition, if developed.

"Diagnosis" means identifying the presence, degree and / or nature of a pathological condition. The diagnostic methods differ in their specificity and selectivity. Although a method Particular diagnosis may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that will aid in the diagnosis.

"Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal subject, including humans and mammals. A pharmaceutical composition comprises a therapeutically effective amount of a cysteamine product, optionally another biologically active agent, and optionally a pharmaceutically acceptable excipient, carrier or diluent. In one embodiment, a pharmaceutical composition comprises a composition comprising the active ingredient (s), and the inert ingredients that form the carrier, as well as any product that results, directly or indirectly, from the combination, complex formation or aggregation of any two or more of the ingredients, or of the dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present disclosure encompass any composition made by mixing a compound of the invention and a pharmaceutically acceptable excipient, carrier or diluent.

"Pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, buffer, and the like, such as phosphate buffered saline, 5% dextrose aqueous solution, and emulsions (eg, oil / water or water / oil emulsion). Non-limiting examples of containers include auxiliaries, binders, fillers, diluents, disintegrants, emulsifying agents, wetting agents, lubricants, glidants, sweetening agents, flavoring agents and coloring agents. Suitable pharmaceutical carriers, excipients and diluents are described in Remington's Pharmaceutical Sciences, 1 9 ed. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend on the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous or intraperitoneal injection, or topical, transdermal or transmucosal administration).

A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use, including but not limited to metal salts (eg, sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines. . Examples of cysteamine derivatives include hydrochloride, bitartrate and phosphocysteamine derivatives. Cystamine and cystamine derivatives include sulfated cystamine.

As used herein, "pharmaceutically acceptable" or "pharmacologically acceptable" salt, ester or other derivative of an active agent comprises, for example, salts, esters or other derivatives refer to a material that is not biologically or otherwise undesirably, that is, the material can be administered to an individual without causing any undesirable biological effect or without interacting in a detrimental manner with any of the components of the composition in which it is contained or with any component present copper or in the body of the individual.

As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosages for human and animal subjects, each unit containing a predetermined amount of a compound of the description calculated in an amount sufficient to producing the desired effect, optionally in association with a pharmaceutically acceptable excipient, diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present disclosure depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

As used herein, the term "subject" encompasses mammalian subjects. Examples of mammalian animals include, but are not limited to, any member of the class of mammals: humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs and cats; laboratory animals including rodents, such as rats, mice and guinea pigs and the like. The term does not denote a particular age or gender. In several modalities, the subject is human.

Cancer The invention is contemplated as being useful with respect to any type of cancer. Examples of cancer include, but are not limited to, adrenocortical carcinoma, cancer related to AI DS, lymphoma related to AI DS, anal cancer, anorectal cancer, anal canal cancer, appendix cancer, infantile cerebellar astrocytoma, infantile cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma) , biliary cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brainstem glioma, astrocytoma cerebellar, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, primitive supratentorial neuroectodemal tumors, hypothalamic glioma and visual trajectory, breast cancer, bronchial adenomas / carcinoids, carcinoid tumor, gastrointestinal, nervous system cancer, nervous system lymphoma, cancer system central nervous system, central nervous system lymphoma, cancer cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary syndrome, endometrial cancer, esophageal cancer, cell tumor extracranial germinal, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, infraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer (stomach), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor , ovarian germ cell tumor, gestational trophoblastic tumor glioma, cancer of the head and neck, hepatocellular cancer (liver), Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet cell tumors (endocrine pancreas), Kaposi's sarcoma, kidney cancer, kidney cancer, kidney cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, oral cavity and lip cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma related with AIDS, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Waldenstram's macroglobulinemia, medulloblastoma, melanoma, intraocular melanoma (eye), merkel cell carcinoma, malignant mesothelioma, mesothelioma, metastatic squamous neck cancer, mouth cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer, Oropharyngeal cancer, ovarian cancer, cancer ovarian epithelial tumor, malignant low ovarian tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus cancer and nasal cavity, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor , multiple myeloma / plasma cell neoplasm, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and urethra, cancer transitional cells, retinoblastoma, rhabdomyosarcoma, cancer of salivary glands, ewing family of sarcoma tumors, Kaposi's sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), merkel cell skin carcinoma, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer (gastric), primitive supratentorial neuroectodermal tumors, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, cancer of thyroid, transitional cell cancer of the renal pelvis and urethra and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, uterine sarcoma, uterine body cancer, vaginal cancer, vulvar cancer and Wilm's tumor. In some modalities, the tumor is associated with a cancer selected from the group consisting of breast cancer, melanoma, prostate cancer, pancreatic cancer, head and neck cancer, lung cancer, non-small cell lung carcinoma, cancer kidney, colorectal cancer and gastric cancer. In some modalities, cancer is pancreatic cancer.

Matrix metaioproteinases (MMPs) In some embodiments, the cysteamine product inhibits the enzymatic activity of one or more matrix metaioproteinases (MMPs) in a cancer cell selected from the group consisting of MMP-1, MMP-2, MP-3, MMP-4, MP -5, MP-6, MP-7. MP-8, MP-9, MP-10, MMP-1 1, MMP-12, MP-1 3 and MMP-14. In some modalities, the product of cysteamine inhibits the enzymatic activity of MMP-2, MMP-9, MMP-12 and / or MMP-14 in a cancer cell. MMPs are a group of zinc-dependent endopeptidases involved in mammalian angiogenesis, wound healing and tissue remodeling (17). In cancer, MMPs play an important role in cell invasion and metastasis by controlling the degradation of the extracellular matrix (18). In particular, MMP-9 plays a central role in the invasion of pancreatic cancer, and its inhibition decreases liver metastasis of pancreatic cancer (1 5, 1 9). Hence, many MMP inhibitors of broad to narrow specificity have been investigated for their anti-cancer effects. Some MP inhibitors successfully suppress tumor growth and metastasis in animal models (20-22), but fail to show anti-cancer effects in clinical settings.

Cysteamine / cystamine Cysteamine plays a role in the formation of the glutathione protein precursor (GSH). In cystinosis, cysteamine acts by converting cystine to cystine and cysteine disulfide-cysteine, both of which are capable of leaving the lysosome through the cysteine and lysine transporters (Gahl et al., N Engl J Med 2002; 347 (2): 1 1 1 -21). Within the cytosol, the mixed disulfide can be reduced by its reaction with glutathione and the released cysteine can be used for additional GSH synthesis. GSH synthesis of cysteine is catalyzed by two enzymes, gamma-glutamylcysteine synthetase and GSH synthetase. This trajectory occurs in almost all types of cells, the liver being the largest producer and exporter of GSH. The mixed cysteine-cysteamine reduced disulfide will also release cysteamine, which, in theory, is then able to re-enter the lysosome, bind more cystine and repeat the process (Dohil et al., J Pediatr 2006; 148 (6): 764 -9). In a recent study in children with cystinosis, enteral administration of cysteamine resulted in increased plasma cysteamine levels, which subsequently caused prolonged efficacy in decreasing leukocyte cystine levels (Dohil et al., J Pediatr 2006; 148 ( 6): 764-9 This may have been due to "re-cycling" of cysteamine when adequate amounts of medication reached the lysosome.If cysteamine acts in this manner, then the production of GSH can also be significantly enhanced.

Cysteamine is a potent gastric acid secretagogue that has been used in laboratory animals to induce duodenal ulceration; Studies in humans and animals have shown that gastric acid hypersecretion induced by cysteamine is most likely mediated through hypergastrinemia. Cysteamine is currently approved by the FDA for use in the treatment of cystinosis, an intra-lysosomal cystine storage disorder. In previous studies in children with cystinosis who suffered upper gastrointestinal symptoms, a single oral dose of cysteamine (11-23 mg / kg) was shown to cause hypergastrinemia and a 2 to 3 fold increase in gastric acid hypersecretion, and a 50% increase in serum gastrin levels. Symptoms suffered by these individuals included abdominal pain, heartburn, nausea, vomiting and anorexy . The US patent application no. 1 1 / 990,869 and international publication published no. WO 2007/89670 (each of which is incorporated by reference herein in its entirety) showed that cysteamine-induced hypergastrinemia arises, in part, as a local effect on the antral-predominant gastric G cells in susceptible individuals. The data also suggest that this is also a systemic effect of gastrin release by cysteamine. Depending on the route of administration, plasma gastrin levels usually peak after intragastric delivery within 30 minutes, while plasma cysteamine levels peak thereafter.

Patients with cystinosis are required to ingest oral cysteamine (CYSTAGON®) every 6 hours day and night. When taken regularly, cysteamine can suppress intracellular cystine by up to 90% (as measured in circulating white blood cells), and this has been shown to reduce the rate of progression of renal failure / transplantation and also obviate the need for replacement therapy. thyroid Due to the difficulty in taking CYSTAGON®, reducing the dosage required improves adherence to the therapeutic regimen. The international publication no. WO 2007/089670 demonstrates that the delivery of cysteamine to the small intestine reduces gastric distension and ulceration and increases AUC. The delivery of cysteamine in the small intestine is useful due to the improved absorption rates of the small intestine, and / or less cysteamine undergoing first pass hepatic clearance when absorbed through the intestine thin. A decrease in leukocyte cystine was observed within one hour of treatment.

In addition, sulfhydryl (SH) compounds, such as cysteamine, cystamine and glutathione are active intracellular antioxidants. Cysteamine protects animals against gastrointestinal radiation and bone marrow syndromes. The fundamental reason for the important anti-oxidant properties of SH compounds is further supported by observations in mitotic cells. These are more sensitive to radiation injury in terms of cellular reproductive death and it is noted that they have the lowest level of SH commissions. In contrast, the S phase cells, which are the most resistant to radiation injury using the same criteria, have demonstrated the highest levels of inherent SH compounds. In addition, when the mitotic cells were treated with cysteamine, they became very resistant to radiation. It has also been noted that cysteamine can directly protect cells against induced mutations. The protection is thought to result from purifying the free radicals, either directly or via protein-bound release of GSH. An enzyme that releases coenzyme A cysteamine has been reported in bird liver and pig kidney. Recently, studies have reported a protective effect of cysteamine against the hepatotoxic agents acetaminophen, bromobenzene and phalloidin.

Cystamine, in addition to its role as a radioprotector, has been found to alleviate tremors and prolong life in mice with the genetic mutation of Huntington's disease (HD). He medication can work to! increase the activity of proteins that protect nerve cells or neurons, from degeneration. Cystamine appears to inactivate an enzyme called transglutaminase and thus results in a reduction of huntingtin protein (Nature Medicine (2002) 8, 143-149). In addition, it was found that cystamine increases the levels of certain neuroprotective proteins. However, due to current methods and formulation of cystamine delivery, degradation and poor uptake require excessive dosing.

Cysteamine products In another aspect, the disclosure provides cysteamine products for use in the methods described herein.

A "cysteamine product" in the present disclosure generally refers to cysteamine, cystamine or a biologically active metabolite or derivative thereof, or combination of cysteamine and cystamine, and includes salts, esters, cysteamine amides or cystamine, alkylate, prodrugs, analogues, phosphorylated compounds, sulphated compounds, nitrosylated and glycosylated compounds or other chemically modified forms thereof (eg, chemically modified forms prepared by labeling with radionucleotides or enzymes and chemically modified forms prepared by linking polymers such as polyethylene glycol ). Thus, the cysteamine or cystamine can be administered in the form of a pharmacologically acceptable salt, ester, amide, prodrug or analog, or as a combination thereof.

In various embodiments, the cysteamine product includes cysteamine, cystamine or derivatives thereof. In any of the embodiments described herein, a cysteamine product may optionally exclude N-acetylcysteine.

The salts, esters, amides, prodrugs and analogs of the active agents can be prepared using standard procedures known to those skilled in the synthetic organic chemical art and described, for example, by J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure" (Advanced Organic Chemistry: Reactions, mechanisms and structure), 4th ed. (New York: Wiley-lnterscience, 1992). For example, basic addition salts are prepared from the neutral medicament using conventional means, involving the reaction of one or more of the free hydroxyl groups of active agent with a suitable base. In general, the neutral form of the medicament is dissolved in a polar organic solvent, such as methanol or ethanol and the base is added thereto. The resulting salt either precipitates or can be carried out by the addition of a solution of a less polar solvent. Suitable bases for forming basic addition salts include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like. The preparation of esters involves the functionalization of hydroxyl groups, which may be present within the molecular structure of the drug. The esters are usually acyl-substituted derivatives of free alcohol groups, i.e. portions, which are derived from carboxylic acids of the formula R-COOH, where R is alkyl, and is usually lower alkyl. The esters can be converted to the free acids, if desired, by using conventional hydrolysis or hydrogenolysis procedures. The preparation of amides and prodrugs can be carried out in an analogous manner. Other derivatives and analogs of the active agents can be prepared using standard techniques known to those skilled in the art of synthetic organic chemistry, or can be deduced by reference to the pertinent literature.

In various embodiments, the cysteamine product does not refer to nanoparticles (including, but not limited to, gold, silver, cadmium and iron nanoparticles) comprising cysteamine (e.g., Wu et al., Nanomedicine: Nanotechnology, Biology and Medicine ( Nanomedicine: Nanotechnology, biology and medicine), 8: 860,869, 201 1, Ghosh et al., Biomaterials, 34: 807-816, 2013, Unak et al., Surf N. Biointerfaces, 90: 217-226, 201 2, Petkova et al, Nanoscale Res. Lett., 7: 287, 2012, and U.S. Patent Publication No. 2010/0034735 or cysteamine incorporated into another active agent (e.g., Fridkin et al., J .. Comb. Chem. , 7: 977-986, 2005).

Pharmaceutical formulations The description provides cysteamine products useful in the treatment of cancer (e.g., to inhibit or suppress tumor metastasis or the treatment of pancreatic cancer). To administer cysteamine products to patients or test animals, it is preferable formulating the cysteamine products in a composition comprising one or more pharmaceutically acceptable carriers. The pharmaceutically or pharmacologically acceptable carriers or vehicles refer to molecular entities and compositions that do not produce allergic reactions, or other adverse reactions when administered using routes well known in the art, as described above, or are approved by the U.S. Food and Drug Administration or its counterpart of foreign regulatory authority as an acceptable additive for oral or parenterally administered pharmacists. Pharmaceutically acceptable carriers include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents and the like.

Pharmaceutical carriers include pharmaceutically acceptable salts, in particular where a basic group or acid is present in a compound. For example, when an acidic substituent, such as -COOH, is present, the ammonium, sodium, potassium, calcium salts and the like are contemplated for administration. Additionally, where an acid group is present, pharmaceutically acceptable esters of the compound (eg, methyl, tert-butyl, pivaloyloxymethyl, succinyl, and the like) are contemplated as preferred forms of the compounds, such esters being known in the art to modify the solubility and / or hydrolysis characteristics for use as promedication or sustained release formulations.

When a basic group (such as amino or heteroaryl radical) basic, such as pyridyl) is present, then an acid salt, such as hydrochloride, hydrobromide, acetate, maleate, pamoate, phosphate, methanesulfonate, p-toluenesulfonate and the like, is contemplated as a form for administration.

In addition, the compounds can form solvates with water or common organic solvents. Such solvates are contemplated as well.

The cysteamine products can be administered orally, parenterally, transocularly, intranasally, transdermally, transmucosally, by inhalation, vaginal, rectal or by intracranial injection. The term "parenteral" as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and / or surgical implant at a particular site is also contemplated. In general, compositions for administration by any of the above methods are essentially free of pyrogens, as well as other impurities that could be harmful to the recipient. In addition, the compositions compositions for parenteral administration are sterile.

The pharmaceutical compositions of the disclosure containing a cysteamine product as an active ingredient may contain pharmaceutically acceptable carriers or additives depending on the route of administration, examples of such carriers or additives include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like . The additives used are chosen from, but not limited to, the above or combinations thereof, as appropriate, depending on the dosage form of the description.

The formulation of the pharmaceutical composition will vary according to the selected route of administration (e.g., solution, emulsion). An appropriate composition comprising the cysteamine product to be administered can be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous solutions or alcoholic / aqueous suspensions or emulsions, including saline and buffered media. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, fixed or lactated Ringer's oils. Intravenous vehicles may include various additives, preservatives or replenishers of fluid, nutrient or electrolyte.

A variety of aqueous carriers, for example, water, buffered water, 0.4% saline, 0.3% glycine, or suspensions aqueous can contain the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum; Dispersing or wetting agents can be a naturally occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with aliphatic alcohols long chain, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example, ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

In some embodiments, the cysteamine product described herein can be lyophilized for storage and reconstituted in a suitable carrier before use. Any lyophilization and reconstitution technique can be used. Those skilled in the art appreciate that lyophilization and reconstitution can lead to varying degrees of activity loss and that levels of use may have to be adjusted to compensate.

Dispersible powders and granules suitable for the preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring and coloring agents, may also be present.

In one embodiment, the disclosure provides the use of an enteric coated cysteamine product composition. The enteric coatings prolong the release until the cysteamine product reaches the intestinal tract, usually the small intestine. Due to the enteric coatings, the delivery to the small intestine is improved, thereby improving the uptake of the active ingredient while the gastric side effects are reduced. Enterally-coated cysteamine products are described in international publication no. WO 2007/089670.

In some embodiments, the coating material is selected so that the therapeutically active agent is released when the dosage form reaches the small intestine or a region in which the pH is greater than pH 4.5. The coating can be a pH-sensitive material, which remains intact in the lower pH environment of the stomach, but which disintegrates or dissolves at the pH commonly found in the small intestine of the stomach. patient. For example, the enteric coating material begins to dissolve in an aqueous solution at pH between about 4.5 to about 5.5. For example, the pH sensitive materials will not experience significant dissolution until the dosage form has been emptied from the stomach. The pH of the small intestine gradually increases from about 4.5 to about 6.5 in the duodenal bulb to about 7.2 in the distal portions of the small intestine. In order to provide predictable dissolution corresponding to the transit time of the small intestine of about 3 hours (for example, 2-3 hours) and allow reproducible release therein, the coating should begin to dissolve within a pH range within. of the small intestine. Therefore, the amount of enteric polymer coating should be sufficient to dissolve substantially during the approximate transit time of three hours within the small intestine, such as the proximal and middle intestine.

Enteric coatings have been used for many years to stop the release of the medicament from orally ingestible dosage forms. Depending on the composition and / or thickness, the enteric coatings are resistant to stomach acid for required periods before they begin to disintegrate and allow release of the drug into the lower stomach or upper part of the small intestine. Examples of some enteric coatings are described in U.S. Pat. 5,225,202, which is incorporated by reference fully herein. How I know set forth in U.S. Patent No. 5,225,202, some examples of coating previously employed are beeswax and glyceryl monostearate; beeswax, lacquer and cellulose; and cetyl alcohol, putty and lacquer, as well as lacquer and stearic acid (US Patent No. 2,809.91 8); polyvinyl acetate and ethyl cellulose (U.S. Patent No. 3,835,221); and neutral copolymer of polymethacrylic acid esters (Eudragit L30D) (F.W. Goodhar et al., Pham. Tech., pp. 64-71, April 1884); copolymers of methacrylic acid and methacrylic methylester (Eudragits), or a neutral copolymer of polymethacrylic acid esters containing metal stearates (Mehta et al., US Patent Nos. 4,728, 512 and 4,794,001). Such coatings comprise mixtures of fats and fatty acids, shellac and lacquer derivatives and cellulose acid phthalates, for example, those having a free carboxyl content. See, Remington on page 1 590 and Zeitova et al. (US Patent No. 4,432,966) for descriptions of suitable enteric coating compositions. Accordingly, increased adsorption in the small intestine due to enteric coatings of cysteamine product compositions may result in improved efficacy.

In general, the enteric coating comprises a polymeric material which prevents the release of cysteamine product in the low pH environment of the stomach but which ionizes at a slightly higher pH, usually a pH of 4 or 5, and thus dissolves sufficiently in the small intestine to gradually release the active agent in the I presented. Accordingly, among the effective enteric coating materials are polyacids having a pKa in the range of about 3 to 5. Suitable enteric coating materials include, but are not limited to, polymerized gelatin, shellac, methacrylic acid copolymer type CNF , cellulose phthalate butyrate, cellulose acid phthalate, cellulose propionate phthalate, polyvinyl acetate phthalate (PVAP), cellulose acetate phthalate (CAP), cellulose trimellitate acetate (CAT), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate, dioxypropyl methylcellulose succinate, carboxymethyl ethylcellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (H PMCAS) and acrylic acid polymers and copolymers, normally formed from methyl acrylate, ethyl acrylate, methyl methacrylate and / or methacrylate ethyl with copolymers of acrylic and methacrylic acid esters (Eudragit NE, Eudragit RL, Eudragit RS). In one embodiment, the cysteamine product composition is administered in an oral delivery vehicle, including but not limited to, a tablet or capsule form. The tablets are manufactured first by enterically coating the cysteamine product. One method for forming tablets herein is by direct compression of the powders containing the enteric coated cysteamine product, optionally in combination with diluents, binders, lubricants, disintegrants, colorants, stabilizers or the like. As an alternative to direct compression, compressed tablets can be prepared using wet granulation or dry granulation processes. The tablets can also be molded instead of compressed, starting with a wet material containing a suitable water soluble lubricant.

The preparation of delayed, controlled or sustained / extended release forms of pharmaceutical compositions with the desired pharmacokinetic characteristics is known in the art and can be accomplished by a variety of methods. For example, oral controlled delivery systems include controlled release by dissolution (e.g., encapsulation dissolution control or matrix dissolution control), controlled release by diffusion (reservoir devices or matrix devices), ion exchange resins, controlled release by osmosis or gastro-retentive systems. Controlled release by dissolution can be obtained, for example, by slowing the dissolution rate of a drug in the gastrointestinal tract, incorporating the drug in a soluble polymer, and coating medicament particles or granules with polymeric materials of varying thickness. The diffusion controlled release can be obtained, for example, by controlling the diffusion through a polymeric membrane or a polymeric matrix. The osmotically controlled release can be obtained, for example, by controlling the influx of solvent through a semipermeable membrane, which in turn carries the external drug through a laser-drilled orifice. Differences in osmotic and hydrostatic pressure on either side of the membrane govern fluid transport. The prolonged gastric retention can be achieved by, for example, altering the density of the formulations, the bioadhesion to the coating of the stomach, or increase the time of flotation in the stomach. For more detail, see the Handbook of Pharmaceutical Controlled Relay Technology (Handbook for controlled-release pharmaceutical technology), Wise, ed. , Marcel Dekker, I nc. , New York, NY (2000), incorporated by reference herein in its entirety, eg, Chapter 22 ("An Overview of Controlled Relay Systems").

The concentration of cysteamine product in these formulations can vary widely, for example, from less than about 0.5%, usually to at least about 1% up to at most 15 or 20% by weight and are selected primarily based on fluid volumes, manufacturing characteristics, viscosities, etc. , according to the particular management mode selected. Real methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed, Mack Publishing Company, Easton, Pa. (1 980).

Compositions useful for administration can be formulated with uptake or absorption enhancers to increase their efficiency. Such enhancers include, for example, salicylate, glycolate / linoleate, glycolate, aproptinin, bacitracin, SDS, caprate and the like. See, for example, Fix (J. Pharm.Scie., 85: 1282-1285, 1996) and Oliyai and Stella (Ann.Rev Pharmacol. Toxicol, 32: 521-544, 1993).

The enterically coated cysteamine product may comprise several excipients, as is well known in the pharmaceutical art, provided that such excipients do not exhibit a destabilizing effect on any component in the composition. Thus, excipients such as binders, bulking agents, diluents, disintegrants, lubricants, fillers, carriers and the like can be combined with the cysteamine product. Oral delivery vehicles contemplated for use herein include tablets, capsules, comprising the product. For solid compositions, diluents are usually necessary to increase the volume of a tablet or capsule so that a practical size is provided by compression. Suitable diluents include dicalcium phosphate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch and powdered sugar. The binders are used to impart cohesive qualities to an oral delivery vehicle formulation, and thus ensure that a tablet remains intact after compression. Suitable binder materials include, but are not limited to, starch (including corn starch and pregelatinized starch), gelatin, sugars (including sucrose, glucose, dextrose and lactose), polyethylene glycol, natural and synthetic waxes and gums, for example, acacia , sodium alginate, polyvinylpyrrolidone, cellulosic polymers (including hydroxypropyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, hydroxyethyl cellulose, hypromellose and the like) and Veegum. Lubricants are used to facilitate manufacture of oral delivery vehicles; Examples of suitable lubricants include, for example, magnesium stearate, calcium stearate, and stearic acid, and are usually present at no more than about 1 weight percent relative to tablet weight. The disintegrants are used to facilitate the oral delivery vehicle, (e.g., a tablet), disintegration or "decomposition" after administration, and are generally starches, clays, celluloses, algins, gums or cross-linked polymers. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffering agents and the like, for example, hate acetate, sorbitan monolaurate, triethanolamine acetate sodium, triethanolamine oleate and the like. If desired, flavoring agents, colorants and / or sweeteners can also be added. Other optional components for incorporation into an oral formulation include, but are not limited to, preservatives, suspending agents, thickening agents, and the like. The fillers include, for example, insoluble materials, such as silicon dioxide, titanium oxide, alumina, talc, kaolin, powdered cellulose, microcrystalline cellulose and the like, as well as soluble materials such as mannitol, urea, sucrose, lactose, dextrose. , sodium chloride, sorbitol and the like.

A pharmaceutical composition may also comprise a stabilizing agent, such as hydroxypropyl methylcellulose or polyvinylpyrrolidone, as described in U.S. Pat. 4,301, 146. Other stabilizing agents include, but are not limited to cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose trimellitate acetate, hydroxypropyl methyl cellulose phthalate, microcrystalline cellulose and sodium carboxymethyl cellulose; and vinyl polymers and copolymers, such as polyvinyl acetate, polyvinyl acetate phthalate, vinyl acetate, crotonic acid copolymer, and ethylene-vinyl acetate copolymers. The stabilizing agent is present in an amount effective to provide the desired stabilizing effect; in general, this means that the proportion of product of cysteamine to the stabilizing agent is at least about 1: 500 w / w, more commonly about 1: 99 w / w.

The tablet, capsule or other oral delivery system is manufactured by first encapsulating the cysteamine product. One method for forming tablets herein is by direct compression of the powders containing the enteric coated cysteamine product, optionally in combination with diluents, binders, lubricants, disintegrants, colorants, stabilizers or the like. As an alternative for direct compression, compressed tablets can be prepared using wet granulation or dry granulation processes. The tablets can also be molded instead of compressed, starting with a wet material containing a suitable water-soluble lubricant.

In various embodiments, the enteric coated cysteamine product is granulated and the granulation is compressed into a tablet or filled in a capsule. The capsule materials can be either hard or soft, and are normally sealed, such as gelatin bands or the like. Tablets and capsules for oral use will generally include one or more commonly used excipients as discussed herein.

In a further embodiment, the cysteamine product is formulated as a capsule. In one embodiment, the capsule comprises the cysteamine product and the capsule is then enterically coated. Capsule formulations are prepared using techniques known in the art.

A suitable pH-sensitive polymer is one that will dissolve in the intestinal environment at a higher pH level (pH greater than 4.5), such as within the small intestine and thereby allow the release of the pharmacologically active substance in the bowel regions. thin and not in the upper portion of the Gl tract, such as the stomach.

For administration of the dosage form, i.e., the tablet or capsule comprising the enteric coated cysteamine product, a total weight in the range of about 1000 mg to 1000 mg is used. The dosage form is administered orally to a subject in need thereof.

In addition, several prodrugs can be "activated" by using enteric coated cysteamine. The promedicamentos are pharmacologically inert, they by themselves do not work in the body, but once they have been absorbed, the promedicamento is decomposed. The promedication approach has been used successfully in a variety of therapeutic areas including antibiotics, antihistamines and ulcer treatments. The advantage of using prodrugs is that the active agent is chemically camouflaged and no active agent is released until the drug has left the intestine and into the cells of the body. For example, a variety of promedications use S-S links. Weak reducing agents, such as cysteamine, reduce these bonds and release the medication. Accordingly, the compositions of the disclosure are useful in combination with prodrugs for the synchronized release of the medicament. In this aspect, a prodrug can be administered followed by administration of an enteric-coated cysteamine composition of the description (at a desired time) to activate the prodrug.

Dosage and administration The cysteamine product is administered in a therapeutically effective amount; normally, the composition is in unit dosage form. The amount of cysteamine product administered is, of course, dependent on the age, weight and general condition of the patient, the seriousness of the condition being treated, and the judgment of the attending physician. Suitable therapeutic amounts will be known to those skilled in the art and / or are described in the relevant reference texts and literature. The current non-enteric coated doses are approximately 1.35 g / m2 of body surface area and are administered 4-5 times per day (Levtchenko et al. to the. , Pediatr Nephrol. 21: 1 1 0-1 13, 2006). In gn aspect, the dose is administered either once a day or multiple times per day. The cysteamine product can be administered less than four times per day, for example, once, twice or three times per day. In some embodiments, an effective dosage of cysteamine product may be within the range of 0.01 mg to 1000 mg per kg (mg / kg) of body weight per day. In some embodiments, the cysteamine, cystamine or pharmaceutically acceptable salt thereof is administered at a daily dose ranging from about 10 mg / kg to about 250 mg / kg, or from about 100 mg / kg to about 250 mg / kg. , or from about 60 mg / kg to about 100 mg / kg or from about 50 mg / kg to about 90 mg / kg, or from about 30 mg / kg to about 80 mg / kg, or from about 20 mg / kg to about 60 mg / kg, or from about 10 mg / kg to about 50 mg / kg. In addition, the effective dose can be 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 1 50 mg / kg, 1 75 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, and may increase by 25 mg / kg in increments up to 1000 mg / kg, or may vary between any two of the above values. In some embodiments, the cysteamine product is administered in a total daily dose from - about 0.25 g / m2 to 4.0 g / m2 surface area body, for example, at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9 or 2 g / m2, or up to about 0.8, 0.9, 1 .0, 1. 1, 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.2, 2.5, 2.7, 3.0, 3.25, 3.5 or 3.75 g / m2 or may vary between any of the above values. In some embodiments, the cysteamine product can be administered at a total daily dose of approximately 0.5-2.0 g / m2 body surface area, or approximately 0.7-0.8 g / m2 body surface area, or approximately 1.35 g. / m2 of body surface area, or about 1.3 to about 1.95 grams / m2 / day, or about 0.5 to about 1.5 grams / m2 / day, or about 0.5 to about 1.0 grams / m2 / day, preferably at a frequency of less than four times a day, for example, three, two or once a day. The salts or esters of the same active ingredient can vary in molecular weight depending on the type and weight of the salt or ester portion. For administration of enteric dosage form, for example, a tablet or capsule or other oral dosage form comprising the enteric coated cystamine product, a total weight in the range of about 100 mg to 1000 mg is used.

Administration can continue for at least 3 months, 6 months, 9 months, 1 year, 2 years, or more.

Combination therapy The therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with auxiliary cancer therapy, such as surgery, chemotherapy, radiotherapy, thermotherapy and laser therapy, and may provide a beneficial effect, for example, reducing tumor size, slowing tumor growth rate, inhibit metastasis or otherwise improve the overall clinical condition, without necessarily eradicating cancer. The cytostatic and cytotoxic agents that target cancer cells are contemplated specifically for combination therapy. Likewise, agents that focus on angiogenesis or lymphangiogenesis are contemplated specifically for combination therapy.

As used herein, a "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulphonates such as busulfan, improsulphan and poiposulphan; aziridines, such as benzodopa, carbocuone, meturedopa and uredopa; ethylene imines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and thiimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic topotecan analog); Bryostatin; Callistatin; CC-1 065 (including its synthetic analogues of adozelesin, carzelesin and bizelesin); cryptophycins (in particular cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM 1); eleutherobin; pancratistatin; a sarcodictiin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterin, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; vinca alkaloids; epidophyllotoxins; antibiotics such as enediin antibiotics (eg, calicheamicin, especially gammali calicheamicin and omegalic calicheamicin; L-asparaginase; anthracenedione-substituted urea; methylhydrazine derivatives; dinemicin, including dynemycin A; bisphosphonates, such as clodronate; a esperamycin; as chromophore of neocarzinostatin and chromophores of enediin antibiotics related to chromoprotein), aclacinomisins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L -norleucine, doxorubicin ADRIAMYCIN® (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxidoxorubicin), epirubicin, esububicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin , puromycin, chelamicin, rodoubicin, streptonigrin, streptozocin, tubericidin, ubenimex, zinostatin, zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, tiamiprin, thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocythabin, floxuridine; androgens, such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal, such as aminoglutethimide, mitotane, triolestane; Folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabuchil; bisantrene; edatraxate; defofamin; demecolcine; diazicuone; elfornitin; eliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitiaerin; pentostatin; fenamet; pirarubicin; losoxanthione; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofirano; spirogermanium; tenuazonic acid; triazicuone; 2,2,2"-trichlorothiiethylamine, trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine), urethane, vindesine, dacrbazine, manomustine, mitobronitol, mitalactol, pipobroman, gacitosin, arabinoside (" Ara-C ") cyclophosphamide, thiotepa, taxoids, for example, paclitaxel TAXOL® (Bristol-Myers Squibb Oncology, Princeton, NJ), paclitaxel nanoparticle formulation designed with albumin, free of Cremophor ABRAXANEMR (American Pharmaceutical Partners, Schaumberg, Illinois) and docetaxel TAXOTERE ® (Rhóne Poulenc Rorer, Antony, France), chlorambucil, gemicitabine GEMZAR®, 6-thioguanine, mercaptopurine, methotrexate, platinum coordination complexes, such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine NAVELBI NE®; novantrone; teniposide; edatrexate; Daunomycin; aminopterin; xeloda; ibandronate; irinotecano (for example, CPT-1 1); Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids, such as retinoic acid; capecitabine; leucovorin (LV); irenotecano; adrenocortical suppressor; adrenocorticosteroids; progestins; estrogen; androgens; analogs of hormones released from gonadotropin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in this definition are anti-hormonal agents that act to regulate or inhibit the action of hormones on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen NOLVADEX®). ), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY1 1 701 8, onapristone and FARESTON-toremifene; aromatase inhibitors that inhibit the aromatase enzyme, which regulates the production of estrogen in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, megestrol acetate MEGASE®, AROMASL® exemestane, formestanie, fadrozole, vorozole RIVISOR®, letrozole FEMARA® and anastrozole ARTMI DEX®; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as traoxacitambin (a cytosine analog of 1,3-dioxolane nucleoside); antisense oligonucleotides, in particular those which inhibit expression of genes in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes, such as VEGF-A expression inhibitor (e.g., ANGIOZYME® ribozyme) and an HER2 expression inhibitor; vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTI N® vaccine and VAXID® vaccine, rJL-2 PROLEUCI®; Topoisomerase 1 inhibitor LURTOTECAN®; rmRH ABARELLX®; and salts, pharmaceutically acceptable acids or derivatives of any of the foregoing.

A "growth inhibitory agent" as used herein refers to a compound or composition which inhibits growth of a cell in vitro and / or in vivo. In this manner, the growth inhibitory agent can be one that significantly reduces the percentage of S-phase cells. Examples of growth inhibitory agents include agents that block cell cycle progression (at a different site of S phase), such as agents inducing G 1 arrest and phase M arrest. Classical M-phase blockers include vinca (vincristine and vinblastine); TAXOL® and topo I I inhibitors, such as doxorubicin, epirubicin, daunarubicin, etopósito and bleomycin. Those agents that stop G1 also overflow into phase S arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C.

Cytokines that are effective in inhibiting tumor metastasis are also contemplated for use in combination therapy. Such cytokines, lymphokines or other hematopoietic factors, include, but are not limited to, M-CSF, GM-CSF, TNF, I L-1, I L-2, I L-3, IL-4, I L-5 , IL- 6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12, IL-13, IL-14, IL-1 5, IL-16, IL-17, IL- 18, I FN, TNFa, TNF 1, TNF 2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, cell factor, adre and erythropoietin.

In some embodiments, the methods described herein comprise administering an MMP inhibitor (eg, an MMP-2 inhibitor, an MMP-12 inhibitor and / or an MMP-9 inhibitor) to the subject. As used herein, "MMP inhibitor" is an agent that directly or indirectly inhibits the activity of MMP. This includes an agent that blocks the activity of MMP or an agent that blocks an MMP production path. The agent causes a reduction in MMP activity in a cancer cell (or cancerous tissue) notwithstanding the mechanism of its action. Representative examples of MMP inhibitors include tissue inhibitors of metalloproteinases (TIMPs) (e.g., TI MP-1, TIMP-2, TIMP-3 or TI MP-4), 02-macroglobulin, tetracyclines (e.g., tetracycline, minocycline). and doxycycline), hydroxamates (for example, BATIMASTAT, MARIMISTAT and TROCADE); chelators (e.g., EDTA, cysteine, acetylcysteine, D-penicillamine and gold salts), synthetic MMP fragments, succinyl mercaptopurines, phosphonamidates and hydroxamic acids.

Broad spectrum inhibitors that inhibit more than one type of MMP are also contemplated. Exemplary broad spectrum MMP inhibitors include, but are not limited to, GM6001, batimastat, marimastat, prinomastat, BAY 12-9566, MMI270 (B), BMS-275291, and metastat. Inhibitors that are capable of inhibiting MMP2, MMP9 or both MMP2 and MP9 are specifically contemplated. A MMP-2 / MMP-9 exemplary inhibitor includes, but is not limited to, SB-3CT. For example, in one embodiment, combination therapy comprising administration of the cysteamine product and an MMP-2 / MMP-9 inhibitor is contemplated specifically.

Assays for measuring inhibition / suppression of MMP are readily known in the art, and include, for example, the following: Cawston T. E., Barrett A.J. , "A rapid and reproducible assay for collagenase using (14C) acetylated collagen" (A rapid and reproducible assay for collagenase using 14C acetylated collagen), Anal. Biochem. 35: 1 961 -1965 (1 963); Cawston T. E., Murphy G. "Mammalian collagenases" (Collagenases of mammalian), Methods in Enzymology 80:71 1 (1981); Koshy P.T.J. , Rowan A. D., Life P. F., Cawston TE, ("96-well píate assays for measuring collagenase activity using (3) H-acetylated collagen" (96-well plate assays to measure collagenase activity using collagen (3) H-acetylated), Anal Biochem 99: 340-345 (1979); Stack MS, Gray RD, "Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide" (Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide), J Biol. Chem. 264: 4277-4281 (989); and Knight CG, Willenbrock F., Murphy G, "A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases" (A novel peptide labeled with coumarin for assays continuous sensitive matrix metalloproteinases ", FEBS Lett 296: 263-266 (1992).

The methods of treatment described herein include optionally monitoring the effect of the therapeutic composition on the tumor. For example, the size of the tumor can be determined, as can the presence of metastasis. The measurement of the degree of metastasis is also contemplated, for example, by measuring the number of metastatic modules or by measuring ascites associated with metastasis.

The cysteamine product and other medicaments / therapies can be administered in combination either simultaneously in a single composition or in separate compositions. Alternatively, the administration is sequential. Simultaneous administration is achieved by administering a single pharmacological protein composition or formulation that includes both the cysteamine product and one or more other therapeutic agents. Alternatively, the one or more other therapeutic agents are taken separately at about the same time as a pharmacological formulation (eg, tablet, injection or beverage) of the cysteamine product.

In several alternatives, the administration of the cysteamine product can precede or follow the administration of the other therapeutic agent (s) at intervals ranging from minutes to hours. For example, in various embodiments, it is further contemplated that the agents are administered in a separate formulation and are administered concurrently, concurrently referring to agents administered within 30 minutes of each other.

In modalities where the or the other therapeutic agents and the cysteamine product are administered separately, one would generally ensure that the cysteamine product and the other therapeutic agent (s) are administered within an appropriate time of one another so that both the cysteamine product and the other therapeutic agent (s) can exert, synergistically or additively, a beneficial effect about the patient. For example, in various embodiments the cysteamine product is administered within about 0.5-6 hours (before or after) of the other therapeutic agent (s). In various embodiments, the cysteamine product is administered within about 1 hour (before or after) of the other therapeutic agent (s).

In another aspect, the second agent is administered prior to the administration of the cysteamine composition. The above administration refers to the administration of the second agent within the range of one week before treatment with cysteamine, up to 30 minutes before the administration of cysteamine. It is further contemplated that the second agent is administered subsequent to the administration of the cysteamine composition. Subsequent administration means describing administration from 30 minutes after the cysteamine treatment until one week after the administration of cysteamine.

Animal models The cysteamine products can be evaluated in animal models known in the art for the disease indications contemplated herein. Exemplary animal models for cancer metastatic diseases are described in Kim et al., (Biochem. Biophis, Res. Comm., 394: 443-447, 2010) (breast cancer); Bresalier et al., (J. Clin. Invest., 87: 1037-1045, 1991) (Colorectal cancer); Furikawa et al., Cancer Res., 53: 1204, 1993) (stomach cancer); Gingrich et al., (Cancer Res., 56: 4096, 1996) (Prostate cancer); Cranmer et al., (Melanoma Res., 15: 325-356, 2005) (melanoma); Zheng et al., Oncogene, 26: 6896-6904, 2007) (lung cancer); and Wang et al, (Neuropatol, Appl. Neurobiol, 37: 189-205, 2011) (brain cancer).

Kits The description also provides kits for performing the methods of the description. In various embodiments, the kit contains, for example, bottles, vials, ampoules, tubes, cartridges and / or syringes comprising a liquid formulation (e.g., sterile injectable) or a solid formulation (e.g., lyophilized). The kits may also contain pharmaceutically acceptable carriers or carriers (e.g., solvents, solutions and / or buffers) to reconstitute a solid formulation (e.g., lyophilized) in a solution or suspension for administration (e.g., by injection), including without limitation reconstitute a lyophilized formulation in a syringe for injection or to dilute concentrate to a lower concentration. Additionally, extemporaneous injection solutions and suspensions may be prepared from, for example, sterile powder, granules or tablets comprising a composition containing cysteamine product. Kits can also include devices dispensers, such as injection or aerosol dispensing devices, pen injectors, autoinjectors, needleless injectors, syringes and / or needles. In various embodiments, the kit also provides an oral dosage form, e.g., a tablet or capsule or other oral formulation described herein, of the cysteamine product for use in the method. The kit also provides instructions for use.

Although the description has been described in conjunction with specific embodiments thereof, the foregoing description as well as the examples that follow, are intended to illustrate and not limit the scope of the description. Other aspects, advantages and modifications within the scope of the description will be apparent to those skilled in the art.

Examples Materials and methods Cell culture and reagents: Pancreatic cancer cell lines (HS766T, MIA-PaCa2, Panc-1, ASPC-1, PK-1, Mpanc96, BxPC-3, KLM, HPAF-I I and SW1 990) were obtained from American Type Culture Collection (Manassas, VA). Cysteamine hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in distilled water.

Cell migration assay: The cell migration assay is a classical wound healing assay (25). For this assay, the cells were cultured in 1 cm petri dishes until they were completely confluent. The cell monolayers were scraped with a handful of sterile yellow micropipettes and washed with PBS three times: the cells were then cultured in medium containing 9-5 mM cysteamine for 24 hours. Several random fields were selected and photographs were taken using an inverted microscope. The average area (mm2) of the opening between the cell layers was calculated using I PLab imaging software (BD Biosciences-Bioimag, Rockville, MD).

Matrigel Invasion Assay: Cell invasion was assayed in BD BioCoat Matrigel invasion chambers (BD Biosciences, 24 cavities, pore size 8 p.m.) as previously described (26). Briefly, the cells were incubated with different concentrations of cysteamine (9-5 mM) for 24 hours. Uninvaded cells were removed from the upper surface of the membrane with a cotton swab and the cells on the lower surface of the membrane were fixed and stained with H &E; Three random fields per camera were counted. The data were shown as mean ± S.D. of triplicate determinations.

Cellular viability assay: Cellular viability of pancreatic cancer cell lines was measured by mean number of viable cell numbers. Briefly, 3 x 1 05 cells were sealed per cavity in a 6-well plate with 2.0 m illiliter of complete medium and incubated overnight for plating. The cells were treated with different concentrations of cysteamine for 24-48 hours. After incubation, the cells were separated with trypsin, washed and stained with 0.4% trypan blue. The cell number was counted manually using a hemocytometer and presented as the number of viable cells per milliliter.

Measurement of MMP activity: The activity of MJPs was measured by fluorogenic peptide substrate Mca-KPLGL-Dpa-AR-NH2 (R & D Systems, Emeryville, CA). The total cellular protein of each pancreatic cancer cell line was harvested using lysis buffer (containing 50 mM Tris-HCl, 10 mM CaCl2, 0.05% Brij35 and 0.25% Triton-X) without an MP inhibitor. The substrate and 10 pg of total cell protein in the buffer were mixed in a 96-well black wall plate. After 1 hour of incubation, fluorescence units were determined using excitation / emission = 320/405 nm.

Zymographic substrate gel assay for MMP activity: Gelatin gel zymography was performed on H57666 cell phones and pancreatic cancer cell line MIA-PaCa2 with or without culture with different concentration of cysteamine essentially as previously described (27) . Briefly, 50 g of total protein was subjected to electrophoresis on 10% SDS-AGE containing 0.1% gelatin as substrate. After washing with 1% Triton X-100 in 50 mM Tris / HCl, pH 7.5 for 1 h to remove SDS, the gels were incubated overnight at 37 ° C in 50 mM Tris / HCl, pH 7.5, containing 150 mM NaCl, 10 mM CaCl2 and 0. 1% Triton X-100, before staining with blue safety stain simply. The gels were washed with deionized water at room temperature. The enzymes Gelatin degraders were identified by their ability to digest gelatin as demonstrated by clear zones of digested gelatin. Relevant band intensities were quantified by scanning densitometric analysis and normalized to cell number.

Quantitative Reverse Transcription PCR (qRT-PCR): qRT-PCR was performed as previously described using QuantiTect SyBR Green PCR kits (QIAGEN, Valencia, CA) (28). The gene-specific primers for human MMP-9, MMP-1 2, MMP-14 and 13-actin were either purchased from QIAGEN or synthesized at the CBER central facility. Gene expression was normalized to β-actin before the change of duplication in gene expression was determined.

Enzyme-Linked Immunosorbent Assay (ELISA): The NI MP-9 protein level was determined using the total human MMP-9 DuoSet kit (R & D Systems, Emeryville, CA) following the manufacturer's instruction. Ten micrograms of total protein were divided into the ices in each cavity and the concentration of MMP-9 was determined by the colorimetric method.

IC50 of cysteamine against MMPs activity: The IC50 of cysteamine and batimastat was determined using a fluorimetric MMP inhibitor profiling kit (Enzo Life Science, Farmingdale, NY), following the manufacturer's instruction. Briefly, each MMP enzyme was mixed with various concentrations of cysteamine and batimastat in 96-well black-walled plates and incubated for 30 minutes. After administration of fluorogenic substrate, measured a fluorescent rate of increase using excitation / emission = 320/405 nm.

Measurement of metastasis, survival and body weight of animals implanted with orthotopic pancreatic cancer in the mouse model: female hairless mice between 5 and 6 weeks of age were kept in a barrier installation in the HEPA filtered rail. All animal studies were conducted under the approved protocol # 2000-06 by the CBER I Institutional Animal Care and Use Committee in accordance with the principles and procedures outlined in the NIH Guideline for the Care and Use of Laboratory Animáis (NIH Guidelines for Care and use of laboratory animals). For injection of orthotopic tumor cells, the pancreas was carefully exposed and 2.0x1 06 of MIA-PaCa2 and H5766T cells were injected into the organ. The pancreas was then returned to the peritoneal cavity and the abdominal wall and skin were closed with skin fasteners (29, 30). Starting on day 4 after tumor implantation, increasing doses of cysteamine (0.25, 100 or 250 mg / kg / day) were injected subcutaneously twice a day until the end of the experiment. On day 30, mice were sacrificed and the number of metastatic nodules visible to the naked eye and whose sizes were > 5 mm in diameter were counted. The total weight of primary tumor and metastatic nodules were also measured. The images were taken immediately after sacrificing the animals. Additionally, mouse survival time was monitored in an independent experiment. The mice were sacrificed when they had ascites severe or cachexia. Mice in both tumor models were weighted on day 10 and day 25 after treatment with cysteamine.

Measurement of enzymes in mouse serum: The mouse blood was collected from the vein of the tail. Serum levels of alanine aminotransferase (ALt), creatine kinase (CK), aldolase and creatinine (Cr) were measured using different kits obtained from Pointe Scientific, Inc. (Canton, MI) and Caldon Biotech, INc. (Vista, Ca), following the instruction of the manufacturer.

MMP activity and expression of MMP-9 in primary tumors: When the mice were sacrificed on day 30, the primary tumors were collected. For total protein extraction, the tumor was soaked with lysis buffer and homogenized using TissueRuptor (QIAGEN); the supernatant was collected. Total RNA was extracted using the FastRNA pro green kit (MP Biomedicals, Solon, OH) following the manufacturer's instructions. The MMP activity, gelatin hydrolyzing MMP activity and the rnRNA and protein levels of MMP-9 were determined as mentioned above.

Statistical analysis: The data for enzymatic activity, ELISA and qRT-PCR were compared between each group by ANOVA. Survival cures were generated by the Kaplan-Meier method and compared using the logarithmic grade test.

Example 1 Cysteamine inhibits the migration and invasion of pancreatic cancer cells without being cytotoxic to cells The following example describes the effect of cysteamine on invasion and migration of pancreatic cancer cells.

The effect of cysteamine on invasion of pancreatic cancer cells was determined using a matrigel invasion assay. For this trial, ten different pancreatic cancer cell lines in a matrigel invasion chamber were used. The results showed that cysteamine inhibited cell invasion in a concentration-dependent manner. Even the lowest concentration of cysteamine (0.05 mM) significantly inhibited cell invasion in all pancreatic cancer cell lines.

The effect of cysteamine on the migration of the same pancreatic cancer cell lines in the invasion assay was also determined. For the migration assay, a wound healing assay (described above in Example 1) was used. The results indicated that cell migration was significantly inhibited at and above 0.05 mM cysteamine in all ten cell lines. Because cysteamine mediates similar effects in all ten cell lines used in these assays, two random cell lines were chosen for all other assays.

The cytotoxicity of cysteamine against two pancreatic cancer cell lines (H5766T and MIA-PaCa2) was also examined and the number of viable cells after 24 and 48 hours of treatment with cysteamine were counted. Cysteamine showed cellular toxicity at > 12.5 mM concentration in both cell lines tested, but no evidence of toxicity was observed at lower concentrations since the number of viable cells was similar to untreated cells. These results suggest that both migration and cell invasion were inhibited at a non-cytotoxic concentration of cysteamine.

Example 2 Cysteamine inhibits the enzymatic activity of MMP in pancreatic cell lines The following example investigated the mechanism of inhibition of migration and cellular invasion by cysteamine, by evaluating the effect of cysteamine on the enzymatic activity of MMP.

Cysteamine inhibited MMP activity in two representative pancreatic cancer cell lines tested in a concentration-dependent manner. The IC50 (the concentration of cysteamine at which 50% of MMP enzymatic activity (proteolysis) is inhibited) was calculated by ELISA (the results of which are given below in Table 1).

Table 1. MMP profiling Cysteamine directly inhibited each enzymatic activity of MMP, with an IC50 of 38-460 μ ?. Because MMP- plays a central role in pancreatic cancer invasion, the protein and mRNA levels of MMP-9 were examined in both cell lines. In contrast to the enzymatic activity, the protein and mRNA levels of MMP-9 were modestly increased at the highest concentration of cysteamine (5 mM). The mRNAs for two other MMPs (ie, MMP-12 and MMP-14) showed a modest increase similar to MMP-9 in response to treatment with cysteamine. In contrast, the results of zymographic gelatin hydrolyzing activity for MMP-9 differed significantly from mRNA expression and both pro and active MMP-9 activities decreased significantly in a dose-dependent manner.

Example 3 Cysteamine decreases metastasis and prolongs the survival of immunodeficient mice implanted orthotically with human pancreatic cancer The following example investigated the anti-metastasis effect of cysteamine in two mouse models using orthotic spikes using human pancreatic cancer cell lines.

Mice were treated twice daily with cysteamine subcutaneously from day 4 after tumor implantation until the end of the experiment. On day 30, the number and weight of primary metastatic and tumor nodules were measured. In both tumor models, the size and weight of the primary tumors showed no difference between the control and treated groups. However, cysteamine significantly decreased the number of metastatic nodules in a dose-dependent manner. At the highest dose (250 mg / kg / day), cysteamine significantly decreased the number of H5766T tumor metastases by -90% (from 34 to 3.6). The total weight of MIA-PaCa2 tumor metastatic nodules also decreased by ~ 90% at the highest dose. In addition, two of 5 mice in the H5766T tumor model and 4 of 5 mice in the MIA-PaCa2 tumor model developed ascites in their peritoneal cavity. However, no mouse developed ascites at doses of 100 and 250 mg / kg / day in any tumor model. The survival of mice between different treatment groups was also determined. The survival time of the mouse was significantly prolonged when treated with 100 mg / kg / day of cysteamine in both tumor models (Figures 1 A and 1 B).

The general condition and body weight of mice throughout the experimental period was also monitored. There was no difference significant in the overall appearance and body weight between four groups of animals in both tumor models. In a similar way, there was no alteration in serum enzymes representing liver function (ALT) or muscle damage (Aldolase) or evidence of skeletal muscle damage or kidney function (creatinine kinase and creatinine) in the groups treated with cysteamine. Moreover, no organ toxicity was detected in any vital organ, such as the liver, kidney, brain, heart and lung in mice treated with cysteamine when evaluated by histological examination.

Example 4 Cysteamine decreases MMP activity in primary orthotopic tumors The activity of MMP in primary orthotopic tumors was measured on day 30. Cysteamine decreased the activity of MMPs in both tumors (H5766T and MIA-PaCa2) at doses of 100 and 250 mg / kg of cysteamine (Figure 8A). In the same samples, the mRNA was measured by q-RT-PCR and the protein levels of MMP-9 by ELISA. In contrast to in vitro results, cysteamine did not affect MMP-9 mRNA and protein levels in primary tumors collected from mice. However, the zymography assay for MMP-9 showed a dose-dependent decrease in gelatinase activity.

Discussion These results demonstrate that cysteamine inhibits the migration and invasion of pancreatic cancer cells through the direct inhibition of MMP enzymatic activity in vitro. This newly discovered property of cysteamine resulted in the inhibition of metastasis of human pancreatic cancer cells implanted orthopeptically on the pancreas of immunodeficient mice; the effect was dependent on the dose of cysteamine. Cysteamine decreased not only the number of metastases in the peritoneal cavity, but also decreased the ascites generated by aggressive pancreatic tumor metastasis. In contrast, no significant change in the size or weight of the primary pancreatic tumor was observed. Consistent with this observation, cysteamine did not cause any effect on cell viability in pancreatic cancer lines until a concentration which caused significant inhibition of cell migration and invasion. Mice treated with cysteamine survived more compared to the control mice treated with excipient. Both in vitro and in primary tumors in vivo, the enzymatic activity of MMP decreased with cysteamine treatment while its expression at mRNA and protein levels did not change the zymographic results, confirming that cysteamine induced inhibition of MVP in vitro and in vivo. These observations indicate that the block of catalytic activities of MMPs by cysteamine plays a role in the inhibition of tumor metastasis in the animal model of pancreatic cancer.

The anti-metastatic effects of cysteamine were mediated without any visible signs of toxicity. Mice treated with even the Higher dose of cysteamine (250 mgkg / day) did not exhibit adverse effects related to overall appearance, body weight, muscle damage, serum enzymes and serum creatinine levels. In addition, the main organs of treated animals showed no evidence of histological damage. These observations are consistent with the known safety profile of cysteamine in humans (2,3). Cysteamine caused only gastrointestinal symptoms in subjects, since it increased the production of gastric acid and decreased gastrointestinal motility (31). However, these effects were controlled by concomitant use of proton pump inhibitor (32). It is worth noting that cysteamine sufficiently inhibited the migration and invasion of cancer cells in vitro at 50 μ ?, a concentration that can be achieved in vivo. Oral administration of cysteamine (given every 6 hours at 690 to 90 mg / kg of body weight per day) can increase the plasma level of cysteamine to ~ 50 μ? (3,33-35). In addition, 100 mg / kg / day of injection s.c. of cysteamine in animals is similar to the dosage used for cystinosis, and this dose produced a significant decrease in tumor metastasis and prolongation of survival. It is contemplated that cysteamine can be safely administered in the clinic for the control of pancreatic cancer metastasis.

Both mRNA and protein levels for M P-9 were moderately to moderately up-regulated in vitro at the highest dose of cysteamine. Similarly, mRNA from MMP-1 2 and 14 were also moderately over-regulated at the highest dose. This increase in MMP-9 was not observed in vivo, perhaps because the pancreatic cancer cells were incubated with cysteamine alone for 24 hours, while the tumors in vivo were continuously exposed to cysteamine for 27 days. Cysteamine levels in tumors in vivo were not measured because the intracellular metabolic fate of cysteamine in vivo is complex and difficult to measure since it binds to free thiol, in particular the cellular protein cysteines. Instead, the activity levels and cysteamine were extrapolated for the biological effects of cysteamine on the activities of MMP and tumor growth. However, the levels of MMP-9, MMP-1 2 and MMP-14 increased to the highest concentration in vitro may represent a temporary compensatory effect of cells due to a sudden decrease in MMP activity by cysteamine. In contrast, the catalytic activity of MMP-9 to hydrolyze gelatin in the zymographic assay showed no such up-regulation after treatment with higher dose of cysteamine from pancreatic cancer cell lines in vitro as well as in vivo orthotopic tumors. In fact, in the zymography assay for MMP-9, cysteamine caused a dose-dependent decrease in gelatinase activity. These results suggest that the enzymatic inhibition of MMP-9 by cysteamine can be involved in the decrease of invasion and metastasis of pancreatic cancer. MMP1, MMP2, MMP7 and MMP9 have also been shown to be expressed in pancreatic tumor (36, 37), but the effect of cysteamine on protein and mRNA levels for all these MMPs was not examined in the present. However, it is important to note that the cysteamine inhibited the enzymatic activity of MMPs, which includes all MMPs.

The anti-MPC activity of cysteamine was lower than that of specific MMP inhibitors, such as batimastat and marimastat (IC50 nM at low μ compared to the μ high range for cysteamine), but cysteamine can be better tolerated in vivo. In fact, batimastat had problems for poor oral bioavailability (24) and marimastat failed in the clinic since higher dosage produced high musculoskeletal toxicities and poor survival in patients with breast cancer metastases (38). In the present study, mice tolerated up to 250 mg / kg / day of cysteamine without any biochemical or histological evidence of visible toxicity or muscle damage.

In previous cancer treatment studies, cysteamine was used because of its anti-oxidant and radio-protective effects (39). During these studies, it was observed that cysteamine also had anti-carcinogenic and anti-proliferative activities in a variety of cancers. It is reported in the present and in Fujisawa et al (PlosONe, 7: e34437, 2012) that cysteamine exerts anti-MMP and anti-metastatic effects. However, in this study, cysteamine did not affect the size of the primary pancreatic tumor. Based on these insights, it is proposed that cysteamine may be useful as a mono-therapy prior to surgery to prevent metastasis, as an adjunct, or as a component of combination therapy for advanced-stage disease to prolong the survival of patients with pancreatic cancer.

All publications and patents mentioned herein are incorporated herein by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present request, including any definition herein, will be sent.

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Claims (20)

1. REVIEW DICAC IONS 1 . A method for inhibiting or suppressing metastasis of a tumor in a mammalian subject comprising administering cysteamine, cystamine or pharmaceutically acceptable salts thereof to the subject in an amount effective to inhibit tumor metastasis. 2. A method for treating pancreatic cancer in a mammalian subject comprising administering cysteamine, cystamine or pharmaceutically acceptable salts thereof to the subject in an amount effective to treat the cancer. 3. The method of claim 1 or claim 2, wherein the cysteamine, cystamine or pharmaceutically acceptable salts thereof are administered orally. 4. The method of any of the preceding claims, wherein the cysteamine, cystamine or pharmaceutically acceptable salts thereof are formulated for delayed release. 5. The method of claim 4, wherein the delayed release formulation comprises an enteric coating that releases the cysteamine or cystamine when the formulation reaches the small intestine or a region of the gastrointestinal tract of a subject in which the pH is greater than about pH 4.5. 6. The method of any of the preceding claims, wherein the cysteamine, cystamine or pharmaceutically acceptable salts thereof is administered less than four times a day. 7. The method of any of the preceding claims, wherein the cysteamine, cystamine or pharmaceutically acceptable salts thereof is administered twice a day. 8. The method of any of the preceding claims, wherein the administration results in increased thiol levels compared to the levels prior to administration of the cysteamine, cystamine or pharmaceutically acceptable salts thereof. 9. The method of any of the preceding claims, wherein the cysteamine, cystamine or pharmaceutically acceptable salts thereof are formulated in a tablet or capsule, which is enterically coated. 10. The method of any of claims 1 and 3-9, wherein the tumor is associated with a cancer selected from the group consisting of breast cancer, melanoma, prostate cancer, pancreatic cancer, head and neck cancer, lung cancer , non-small cell lung carcinoma, renal cancer, colorectal cancer and gastric cancer. eleven . The method of any of the preceding claims, further comprising administering to the subject auxiliary cancer therapy. 12. The method of claim 1, wherein the auxiliary cancer therapy is selected from the group consisting of chemotherapy, surgery, radiotherapy, thermotherapy, cancer vaccines, immunotherapy, gene therapy and laser therapy. 13. The method of any of the preceding claims, further comprising administering an additional therapeutic agent selected from the group consisting of an MMP inhibitor, a chemotherapeutic agent, a growth inhibitory agent, a cancer vaccine, a gene therapy product, an immunotherapy and cytokines. 14. The method of any of the preceding claims, wherein the cysteamine modulates the enzymatic activity of a matrix metalloproteinase (MMP). 15. The method of claim 14, wherein the enzymatic activity of MMP is decreased in a primary tumor. 16. The method of any of the preceding claims, wherein the cysteamine, cystamine or pharmaceutically acceptable salt thereof decreases metastatic nodules in the subject. 17. The method of any of the preceding claims, wherein the cysteamine, cystamine or pharmaceutically salt thereof decreases ascites in the subject. 18. The method of any of the preceding claims, wherein the cysteamine, cystamine or pharmaceutically acceptable salt thereof is administered in a dose of about 10 mg / kg to about 250 mg / kg. 19. A method to decrease the enzymatic activity of matrix metalloproteinase (MMP) in a cancer cell comprising contacting the cell with cysteamine, cystamine or pharmaceutically acceptable salts thereof in an amount effective to decrease enzymatic activity of MMP in the cancer cell. 20. The method of claim 20, wherein the MMP is selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-4, MMP-5, MMP-6, MMP-6, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13 and MMP-14.