BR112012012022B1 - pharmaceutical composition - Google Patents

Abstract

FIBRILLARY PROTEIN, COMPOSITION, METASTATIC CANCER CELLS, METHOD AND KIT TO SUPPRESS CANCER METASTASIS, AND, METHOD FOR MANUFACTURING A COMPOSITION. Method are provided to suppress cancer metastasis. Cancer metastasis is the most common cause of treatment failure and death in cancer patients. Tumor cell invasion and / or migration can be significantly inhibited after treatment of fibrillar proteins (r-VPl, F-HSA, and F-BSA) in vitro. In addition, rVPl can significantly suppress metastasis of murine and human breast cancer and metastasis of prostate and ovarian cancer of human in vivo while F-HSA can significantly suppress metastasis of murine breast cancer. Compositions of fibrillar proteins as therapeutic anti-cancer metastasis products and their methods of use are provided herein.

Description

INTRODUCTION

[001] Cancer metastasis is a process that involves a series of sequential steps and requires a cascade of host-tumor cell interactions (Steeg PS et al. (2007) Nature 449: 6713). These steps include the detachment of the primary tumor, invasion into and arrest of circulatory systems, the leakage into the parenchyma of organs; and proliferation in association with angiogenesis (Sawyer TK et al. (2004) Expert Opin Investig Drugs 13: 119). There are growing interests in investigating the mechanisms of migration and invasion, which are revealed to be a critical step in the metastatic process. Interference with any of these steps to block the metastatic cascade represents an attractive method to prevent the formation of metastatic tumor growth.

[002] Our previous data showed that recombinant capsid protein VP1 (rVPl) from foot-and-mouth disease virus (FMDV), induced apoptosis in several types of cancer cells via the integrin signaling pathway (Peng JM et al. (2004) J. Biol, Chem. 279: 52168-74). It was found that using the same process for folding rVPl in water-soluble form, globular bovine serum albumin (G-BSA) can be converted into fibrillar BS A (F-BSA), which, like rVPl, induced apoptosis of tumor cell via the integrin / FAK / Akt signaling pathway (Huang et al. (2009) BMC Biotechnol. 9: 2).

SUMMARY OF THE INVENTION

[003] Methods are provided to suppress cancer metastasis. Cancer metastasis is the most common cause of treatment failure and death in cancer patients. Tumor cell invasion and / or migration is significantly inhibited after contacting tumor cells with fibrillar proteins, which include, without limitation, rVPl, F-HSA, and F-BSA. The tumor cells of interest include carcinomas, for example breast carcinoma, ovarian epithelial adenocarcinoma, prostate adenocarcinoma, etc.

[004] In one aspect, the invention relates to a composition comprising a therapeutically effective amount of fibrillar human serum albumin and a pharmaceutically acceptable carrier for use in treating a mammal having cancer, for example, to suppress cancer cells, suppress metastasis, etc.

[005] In some embodiments of the invention, tumor cells are contacted in vivo with fibrillar proteins, which contact can be local, for example, intratumoral introduction or injection, or systemic. For example rVPl is shown here to significantly suppress metastasis of murine and human and human and prostate breast cancer and metastasis of ovarian cancer in vivo. FHSA is shown to significantly suppress breast cancer metastasis. In one embodiment, the invention relates to a composition as previously mentioned for use in the treatment of cancer in a mammal, wherein the cancer is at least one selected from breast cancer, ovarian cancer, cervical cancer, prostate cancer , and lung cancer.

[006] In some embodiments of the invention, fibrillar protein compositions as cancer antimetastasis therapeutic products are provided, where the composition provides a pharmaceutical formulation in a dose effective to inhibit metastasis. In another aspect, the invention relates to a method which comprises making a composition for use in treating a patient having cancer. The method comprises making fibrillary human serum albumin, and mixing fibrillary human serum albumin with a pharmaceutically acceptable carrier. In another aspect, the invention relates to a method which comprises dissolving HSA in an SDS solution; applying the dissolved HSA through a gel filtration column with a pore size to separate proteins of molecular weight 70 kDa and above; eluting the HSA from the column; and dialysing the solution against phosphate-buffered saline to remove the SDS.

BRIEF DESCRIPTION OF THE DRAWINGS

[007] Figure 1. The effects of rVPl on cell invasion / migration and cytotoxicity in MDAMB-231 cells, PC-3 cell, and 22Rvl cell. (A&B) Invasion / migration of MDA-MB-231 cell, PC-3 cell, and 22Rvl cells with rVPl treatment for 24 hours was measured using Boyden chamber assay. rVPl significantly suppressed tumor cell invasion / migration (C). The cytotoxicity of MDA-MB-231 cells, PC-3 cell, and 22Rvl cells with rVPl treatment for 24 hours was measured using the MTT assay. 0.1 pM and 0.2 pM rVPl did not affect tumor cell viability. The data represent means ± S.D. (n = 3). *: P <0.05, **: P <0.01 and ***: P <0.001 were relative to the control (0 pM rVPl treatment).

[008] Figure 2. The effects of rVPl on cell invasion and cytotoxicity in SK-OV-3 cells and CaSki cells. (A) Cell invasion of SK-OV-3 cells and CaSki cells with rVPl treatment for 24 hours was measured using the Boyden chamber assay. rVPl significantly suppressed tumor cell invasion (B) The cellular cytotoxicity of SK-OV-3 and CaSki cells treated with rVPl for 24 hours was measured using the MTT assay. 0.2 pM to 0.4 pM rVPl in SKOV-3 cells and 0.2 pM to 0.6 pM rVPl in CaSki cells did not affect cell viability. White bars, SKOV-3 cells; black bar, CaSki cells. The data represent means ± S.D. (n = 3). *: P <0.05; **: P <0.01; ***: P <0.001.

[009] Figure 3. The effects of rVPl on cell invasion and cytotoxicity in SK-OV-3ip.l cells. (A) SK-OV-3ip.l cells were plated in the upper chamber for 1 hour followed by treatment with rVPl for 24 h. After incubation, cells on the lower membrane surface were dissociated and counted. (B) SK-OV-3ip.l cells were treated with rVPl for 24 h after WST-1 assays were performed. Cell survival rates were determined by measuring absorbance at 450 nm (690 nm as a reference). The percentage of cell survival was calculated as (O.Dlralamcnlo / O.Dconlroie) x 100%. The data represent means ± S.D. (n = 3). *, P <0.05 and **, P <0.01 were related to the control. [0010] Figure 4. The metastatic capacity of MDA-MB-231 cells was lost after treatment with rVPl in vivo. 0.1 pM and 0.2 pM of MDA-MB-231 cells treated with rVPl in vitro for 24 hours after harvested and intravenously injected into the mice through the tail vein. After 14 days, the sacrifice was made. The gross appearance (A) and histopathology examination (B and C) of the lung in three different groups of mice were measured. rVPl significantly inhibited the metastatic capacity of MDA-MB-231 cells. ***: P <0.001.

[0011] Figure 5. rVPl suppressed PC-3 cells metastasized to the lymph nodes and inhibited osteolysis in pelvic bones. (A) Lymph nodes from normal mice that carry tumor with or without rVPl treatment. (B) The pelvic bones of normal mice that carry a tumor with or without rVPl treatment (arrow).

[0012] Figure 6. Histological aspect of SK-OV-3 liver tumor metastasis implanted in nude mice was prevented by rVPl. The representative liver sections with the H&E strain of rVPl treated and untreated mice. BALB / cAnN-Foxnl female nude mice were implanted with 5 x 106 SK-OV-3 cancer cells per mouse by intraperitoneal injection, (ab) After 60 days, the liver of PBS-treated mice carrying SK-OV-3 was surrounded by tumor cells, (cd) 340 days after implantation of SK-OV-3, the liver of mice treated with rVPl showed no evident invasion, (ef) The normal mouse liver (that is, without implantation of SK -OV-3).

[0013] Figure 7. The effects of F-HSA on cell migration, invasion and cytotoxicity in MDA-MB-231 cells, PC-3 cells, 22Rvl cells and CaSki cells. The in vitro migration and invasion assay, F-HSA significantly inhibited cell invasion (black bar) and migration (white bar) in MDA-MB-231 (A) cells, PC-3 cells (C), 22Rvl cells (E ), and CaSki cells (G) using the Boyden chamber assay. The effects of F-HSA on cytotoxicity in MDA-MB-231 cells, PC-3 cells, 22Rvl cells and CaSki cells using the MTT assay. (B, D, F, and H). Migration and invasion were represented as a percentage of the number of cells treated for that of untreated cells. The means ± S.D. of the three independent experiments with three replicates in each. P <0.01 and ***: P <0.001 were related to non-ratified cells.

[0014] Figure 8. F-HSA suppressed the TS / A cell from tumor metastasis in vivo. (A) Gross appearance and histopathological examination of the lungs of mice with or without treatment with F-HSA. (B & C) The weight of the related lung and the number of tumor foci in the lung of mice with or without F-HSA treatment. ***: P <0.001.

[0015] Figure 9. F-HSA suppressed the MDA-MB-231 cell from tumor metastasis in vivo. (A) Gross appearance and histopathological examination of the lungs of mice with or without FHSA treatment. (B & C) The weight of the related lung and the number of tumor foci in the lung with or without F-HSA treatment. ***: P <0.001.

[0016] Figure 10. The effects of F-BSA on cell invasion and cytotoxicity in CaSki cells. (A) The cellular invasion of CaSki cells treated with F-BSA for 24 hours was measured by a Boyden chamber assay. F-HSA significantly suppressed tumor cell invasion. (B) The cytotoxicity of CaSki cells treated with F-BSA for 24 hours was measured by MTT assay. F-HSA at 0.1 pM or 0.2 pM concentration had no effect on the cell viability of CaSki cells. The data represent means ± S.D. (n = 3). *: P <0.05; **: P <0.01.

[0017] Figure 11. A table of the tumor implant with treatment with rVPl.

[0018] Figure 12 is an implementation of the experimental data showing a comparison of the fluorescence level of increasing the concentrations of F-HSA, HSA, and A β (1-42) then incubating with 20 pM of specific amyloid pigment ThT for 1 h.

[0019] Figure 13 is an implementation of the experimental data showing the cytotoxic effects of F-HSA on breast cancer cells.

[0020] Figure 14 is an implementation of the experimental data showing the cytotoxic effects of F-HSA on breast cancer cells.

[0021] Figure 15 is an implementation of the experimental data showing the cytotoxic effects of F-HSA on ovarian cells.

[0022] Figure 16 is an implementation of the experimental data showing the cytotoxic effects of F-HSA on cervical cancer cells.

[0023] Figure 17 is an implementation of the experimental data showing the cytotoxic effects of F-HSA on prostate cancer cells.

[0024] Figure 18 is an implementation of the experimental data showing the cytotoxic effects of F-HSA on prostate cancer cells.

[0025] Figure 19 is an implementation of the experimental data showing the cytotoxic effects of F-HSA on lung cancer cells.

[0026] Figures 20A to 20H are implementations of experimental data that show the effect of F-HSA in reducing migration and tumor cell invasion without affecting the viability of normal cells.

[0027] Figures 21A to 21C are implementations of experimental data showing the effect of F-HSA on suppression of TS / A cell metastasis from mouse breast cancer to lung.

[0028] Figure 22A-22C are implementations of experimental data showing the effect of F-HSA on suppressing mouse breast tumor MDA-MB-231 cell metastasis in the lung.

DEFINITIONS

[0029] In the following description, several terms conventionally used in the field of cell culture are used. In order to provide a clear and consistent understanding of the specification and the claims and the scope to be given to such terms, the following definitions are provided.

[0030] The terms "individual", "individual", and "patient" are used interchangeably here to refer to a mammal being evaluated for treatment and / or being. In one embodiment, the mammal is a human being. The terms "individual", "individual" and "patient" in this way include individuals having cancer (for example, colorectal cancer, ovarian or prostate adenocarcinoma, breast carcinoma, etc.), including those who have suffered or are candidates for resection (surgery) for the removal of cancerous tissue (for example, colorectal tissue with cancer). The individuals can be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, for example, mouse, rat, etc.

[0031] As used herein, the terms "treatment", "treat" and others, refer to administering an agent or performing a procedure (for example, radiation, a surgical procedure, etc.), for the purposes of obtaining an effect. The effect can be prophylactic in terms of complete or partial prevention of a disease or symptom thereof and / or it can be therapeutic in terms of achieving a partial or complete cure of a disease and / or symptoms of the disease. "Treatment", as used here, covers any treatment of any metastatic tumor in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in an individual who may be predisposed to the disease but has not yet been diagnosed as having it (for example, including diseases that may be associated with or caused by a primary disease; (b) inhibition of the disease, that is, preventing its development and (c) relieving the disease , that is, causing regression of the disease. In tumor treatment (for example, cancer), a therapeutic agent can directly decrease the metastasis of tumor cells.

[0032] The term "cell culture" or "culture" means of maintaining cells in an artificial in vitro environment. However, it should be understood that the term “cell culture” is a generic term and can be used to cover the cultivation of individual cells only, but also of tissues or organs.

[0033] The term "tumor", as used here, refers to the development and proliferation of all neoplastic, malignant or benign cells and all precancerous and cancerous cells or tissues.

[0034] The terms "cancer", "neoplasm" and "tumor" are used interchangeably here to refer to cells that exhibit autonomous unregulated development, such that they present an aberrant development phenotype characterized by significant loss of control in cell proliferation. In general, cells of interest for detection, analysis, classification or treatment in the present application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic and non-metastatic cells. Examples of cancer include, but are not limited to, breast cancer, colonic cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer and brain cancer.

[0035] Depending on the nature of the cancer, an appropriate patient sample is obtained. As used here, the phrase "cancerous tissue sample" refers to any cells obtained from a cancerous tumor. In the case of solid tumors that have not metastasized, a sample of tissue from the surgically removed tumor will typically be obtained and prepared for testing by conventional techniques. Alternatively, a sample of body fluid, such as a lymph, blood or serum sample, or a sample of exuding fluid, such as exudate from the cancerous organ (for example, breast exudation) can be collected and used as the sample to be analyzed. In the case of leukemias, lymphocytes or leukemic cells will be obtained and properly prepared. Similarly, in the case of any cancer that has metastasized, the cells can be removed from the body fluid, such as lymphatic fluid, blood, serum or a distally infected organ or its exudation.

[0036] As used here, the term "metastasis" refers to the development of a cancerous tumor in an organ or body part, which is not directly connected to the organ of the original cancerous tumor. Metastasis will be understood to include micrometastasis, which is the presence of an undetectable amount of cancer cells in an organ or body part that is not directly connected to the original cancer tumor organ. Metastasis can also be defined as several steps in a process, such as the removal of cancer cells from an original tumor site and the migration and / or invasion of cancer cells to other parts of the body. Therefore, the present invention considers a method of determining the risk of further development of one or more cancerous tumors in an organ or body part that is not directly connected to the original cancerous tumor organ and / or any steps in a process leading to that development.

[0037] The "pathology" of cancer includes all phenomena that comprise the patient's well-being. This includes, without limitation, abnormal or uncontrollable cell development, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretion products at abnormal levels, suppression or worsening of the inflammatory or immune response, neoplasia, pre-malignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.

[0038] As used here, the terms "cancer recurrence" and "tumor recurrence" and their grammatical variants, refer to the further development of neoplastic or cancer cells after the diagnosis of cancer. In particular, recurrence can occur when cancer cell development in cancer tissue still occurs. “Tumor spread”, similarly, occurs when the cells of a tumor spread in the local or distant tissues and organs; therefore, tumor spread encompasses tumor metastasis.

[0039] The term "diagnosis" is used here to refer to the identification of a molecular or pathological state, disease or condition, such as the identification of a molecular subtype of breast cancer, prostate cancer or other type of cancer.

[0040] The term "prognosis" is used here to refer to the prediction of the probability of death attributable to cancer or progression, including recurrence, metastatic spread and drug resistance, of a neoplastic disease, such as lung, colon, skin or esophageal. The term "prediction" is used here to refer to the act of prediction or estimation, based on specific observation, experience or reasoning. In one example, a doctor can predict the likelihood that a patient will survive, following surgical removal of a primary tumor and / or chemotherapy for a certain period of time without recurrence of cancer.

[0041] As used here, the phrase “disease-free survival”, refers to the loss of such recurrence and / or tumor spread and the fact that a patient after diagnosis, regarding the effects of cancer on the total life of the patient. The phrase "total survival" refers to the fact of the patient after diagnosis, despite the possibility that the cause of death in a patient does not occur directly due to the effects of cancer. The phrases, “disease-free survival probability”, “risk of recurrence” and variants thereof, refer to the likelihood of tumor recurrence or spread in a patient subsequent to the diagnosis of cancer, where the probability is determined according to the invention process.

[0042] As used here, the term "correlates", or "correlates with", and other terms, refer to a statistical association between the examples of two events, where the events include the numbers, series of data and others. For example, when events involve numbers, a positive correlation (also referred to here as a “direct correlation”) means that when one increases, the other also increases. A negative correlation (also referred to here as an “inverse correlation”) means that when one increases, the other decreases.

[0043] The term "isolated" is intended to mean that a compound is separated from all or some of the components that accompany it in its natural state. "Isolated" also refers to the state of a compound (for example, protein) separated from all or some of the components that accompany it during manufacture (for example, chemical synthesis, recombinant expression, culture medium and others).

[0044] A "biological sample" covers a variety of sample types obtained from an individual. The definition covers blood and other liquid samples of biological origin, samples of solid tissue, such as a biopsy specimen or tissue cultures or cells derived from these to their progeny. The definition also includes samples that have been manipulated in any way after obtaining them, such as by treatment with reagents; washed or enriched for certain cell populations, such as cancer cells. The definition also includes the sample that has been enriched for particular types of molecules, for example, nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cultured cells, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and others. A “biological sample” includes a sample obtained from a patient's cancer cell, for example, a sample comprising polynucleotides and / or poliopeptides that are obtained from a patient's cancer cell (for example, a cell lysate or other cell extract comprising polynucleotides and / or polypeptides) and a sample comprising cancer cells from a patient. A biological sample that comprises a patient's cancer cell can also include non-cancer cells.

DESCRIPTION OF THE SPECIFIC ACHIEVEMENTS

[0045] The present description refers to a process of producing fibrillar proteins and methods of treatment using fibrillar proteins. The treatment method involves suppressing cancer metastasis.

Treatment method

[0046] A method is described here to inhibit cancer metastasis. The method involves administering a therapeutically effective amount of the fibrillar structure protein to a patient in need thereof. The method may further involve the steps of providing a protein and changing the protein into a fibrillar structure prior to the administration of the fibrillar protein. The administration of fibrillar protein inhibits tumor cell invasion and / or migration (for example, in the surrounding tissues).

[0047] The present method finds use in a variety of cancer therapies (including cancer prevention and post-diagnostic cancer therapy) in a mammalian individual, particularly a human. Individuals having, suspected of having, or at risk of developing a tumor are considered for the therapy described here.

[0048] Anti-cancer therapies according to the individual method can be particularly targeted at cancer cells that are metastatic or are at risk of becoming metastatic. As such, fibrillar protein can be used in a therapeutic manner to carry out / prevent the adhesion and invasion of cancer cells in other tissues.

[0049] For example, cancer metastases that can be inhibited by the method of the present description include, but are not limited to, carcinomas, including adenocarcinomas, and particularly breast carcinomas, prostate adenocarcinoma and ovarian adenocarcinoma. Other metastases that can be treated include those that arise from developing cancer in the kidney, lung, liver, skin (eg, melanoma), colon, pancreas, or cervix.

[0050] The fibrillar structure protein used for the treatment of cancer can be an albumin, fibronectin, rVPl, rVP2, rVP3, Pl, or chimeric protein comprising parts of VP1, VP2, VP3 and / or VP4. Albumin proteins can be obtained from any animal of interest, for example, human serum albumin, bovine serum albumin, etc. In certain embodiments, the fibrillar protein additionally induces cancer cell apoptosis by modulating the Akt signaling pathway. In some examples, the fibrillar protein modulates α5β 1 integrin or v β 3 which leads to Akt deactivation. In other examples, fibrillar albumin binds fibrillar albumin to integrin and causes cell apoptosis mainly via the integrin / FAK / Akt / GSK-3β / caspase-3 pathway.

[0051] As noted above, the present method involves the administration of fibrillar proteins to an individual (eg, a human patient) to, for example, suppress invasion and migration of cancer cells. This can be accomplished by administering a fibrillar protein to the individual as described here in order to provide a decrease in cancer metastasis compared to individuals who are not administered the fibrillar protein. Therapies according to individual methods can also be useful in preventing relapse, reducing migration of cancer cells, reducing tumor size, reducing tumor burden and / or improving the clinical response in patients.

Types of cancer

[0052] The methods with respect to cancer considered here include, for example, the use of fibrillar protein therapy as an anti-cancer metastasis therapy. The methods are useful in the context of treating or preventing a wide variety of cancers, such as cancers that can metastasize (for example, carcinomas and sarcomas).

[0053] Carcinomas that may be sensitive to therapy by a method described here include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues ), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung , adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary carcinoma, renal cell, carcinoma, medullar ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilm's tumor, carcass cervical inoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma and nasopharyngeal carcinoma.

[0054] Sarcomas that may be sensitive to therapy by a method described here include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiossarcoma, endotheliosarcoma, lymphangiosarcoma, synangiosarcoma, lymph node , Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma and other soft tissue sarcomas.

[0055] Other solid tumors that may be sensitive to therapy by a method described here include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and neuroblastoma retinoblastoma.

[0056] Other cancers that may be sensitive to treatment according to the methods described here include atypical meningioma (brain), islet cell carcinoma (pancreas), medullary carcinoma (thyroid), mesenchyma (intestine), hepatocellular carcinoma (liver) , hepatoblastoma (liver), clear cell carcinoma (renal) and mediastinal neurofibroma.

[0057] Additional exemplary cancers that may be sensitive to treatment using the methods described here include, but are not limited to, cancers of neuroectodermal and epithelial origin. Examples of cancers of neuroectodermal origin include, but are not limited to, Ewings sarcoma, spinal tumors, brain tumors, early childhood neuroectodermal tumors, tubulocystic carcinoma, mucinous and cell bundle carcinoma, kidney tumors, mediastinal tumors, neurogliomas, neuroblastomas and sarcomas in adolescents and young adults. Examples of epithelial origin include, but are not limited to, small cell lung cancer, breast cancers, eye lenses, colon, pancreas, kidney, liver, ovary and bronchial epithelium.

Combinations with other cancer therapies

[0058] Therapeutic administration of fibrillar protein may include administration as a part of a therapeutic regimen that may or may not be in conjunction with additional standard anti-cancer therapeutic products, which include but are not limited to immunotherapy, agents chemotherapy and surgery (for example, as described below).

[0059] Furthermore, the therapeutic administration of fibrillar protein can also be the post-therapeutic treatment of the individual with an anti-cancer therapy, where the anti-cancer therapy can be, for example, surgery, radiation therapy, agent administration chemotherapeutic, and others. Cancer therapy using fibrillar proteins of the present description can also be used in combination with immunotherapy. In other examples, fibrillar proteins can be administered in combination with one or more chemotherapeutic agents (for example, cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP)), and / or in combination with radiation treatment and / or in combination with surgical intervention (for example, pre- or post-surgery to remove a tumor). When fibrillar proteins are used in conception with surgical intervention, fibrillar protein can be administered before, during or after surgery to remove cancer cells and can be administered systemically or locally to the surgical site. The fibrillar protein alone or in the combinations described above can be administered systemically (for example, by parenteral administration, for example, by an intravenous route) or locally (for example, in a tumor site, for example, by intratumoral administration ( for example, in a solid tumor, in a lymph node involved in a lymphoma or leukemia), administration in a blood vessel providing a solid tumor, etc.).

[0060] Any of a variety of cancer therapies can be used in combination with the fibrillar protein therapies described here. Such cancer therapies include surgery (for example, surgical removal of cancerous tissue), radiation therapy, bone marrow transplantation, chemotherapeutic treatment, biological response modifying treatment and certain combinations of the foregoing.

[0061] Radiation therapy includes, but is not limited to, X-rays or gamma rays that are released from an extremly applied source such as a beam or by implementing small radioactive sources.

[0062] Chemotherapeutic agents are non-peptide compounds (ie, non-proteinaceous) that reduce the proliferation of cancer cells and include cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, (vinca) plant alkaloids and steroid hormones.

[0063] Agents that act to reduce cell proliferation are known in the art and widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates and triazenes, which include, but are not limited to, meclorethamine, cyclophosphamide (CYTOXAN ™), melphalan (L-sarcolysin), carmustine ( BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chloro-zotocin, uracil mustard, chlormetin, ifosfamide, chlorambucil, pipobroman, triethylenomelaamine, triethylene thiophosphoramine, busulfan, dacaromide and dacarolazide.

[0064] Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors, which include, but are not limited to, cytarabine (CYTOSSAR-U), arabinoside cytosine, fluorouracil (5-FU) , floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), acid 5, 8- dideazatetrahydrofolic (DDATHF), leucovorin, fludarabine phosphate, pentostatin and gemcitabine.

[0065] Suitable natural products and their derivatives, (for example, crease alkaloids, anti-tumor antibiotics, enzymes, lymphokines and epipodophyllotoxins), include but are not limited to, Ara-C, paclitaxel (TAXOL®), docetaxel (TAXOTERE® ), deoxychoformicin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, for example, vincristine, vinblastine, vinorelbine, vindesine, etc .; podophyllotoxins, for example, etoposide, teniposide, etc .; antibiotics, for example, anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidin), idarubicin, doxorubicin, epirubicin and morpholino derivatives etc .; phenoxizone bisciclopeptides, for example, dactinomycin; basic glycopeptides, for example, bleomycin anthraquinone glycosides, for example, plicamycin (mitramycin); anthracenodiones, for example, mitoxantrone; azoleinopyrrole indolediones, for example, mitomycin; macrocyclic immunosuppressants, for example, cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc; and others.

[0066] Other anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide and droloxafma.

[0067] Agents that affect microtubules that have antiproliferative activity are also suitable for use and include, but are not limited to, alocolchicine (NSC 406042), Halicondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives ( for example, NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (TAXOL®), TAXOL® derivatives, docetaxel (TAXOTERE®), thiocolchicine (NSC 361792), tritil cisterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epitolones that include, but are not limited to, eptilone A, epothilone B, discodermolide; estramustine, nocodazole, and others.

[0068] Hormonal modulators and steroids (including synthetic analogs) that are suitable for use include, but are not limited to, adrenocorticosteroids, for example, prednisone, dexamethasone, etc .; estrogens and pregestins, for example, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc and adrenocortical suppressors, for example, aminoglutetimide; 17α-ethinylestradiol; dietilstilbestrol, testosterone, fhioximesterona, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutetimida, estramustina, medroxiprogidaida (Flutamide), Flutamide (Flour); Estrogens stimulate proliferation and differentiation, so compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0069] Other chemotherapeutic agents include metal complexes, for example, cisplatin (cisDDP), carboplatin, etc .; ureas, for example, hydroxyurea and hydrazines, for example, N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other anti-proliferative agents of interest include immunosuppressants, for example, mycophenolic acid, thalidomide, deoxyspergualin, azasporine, leflunomide, mizoribine, azaspiran (SKF 105685); IRESSA® (ZD 1839, 4- (3-chloro-4-fluorophenylamino) -7-methoxy-6- (3 - (4-morpholinyl) propoxy) quinazoline); etc.

[0070] "Taxanes" include paclitaxel, as well as any active taxane derivative or prodrug. “Paclitaxel” (which should be understood here to include analogs, formulations and derivatives, such as, for example, docetaxel, TAXOL, TAXOTERE (a formulation of docetaxel), paclitaxel 10-deacetyl analogs and 3'N-desbenzoyl- Paclitaxel 3'Nt-butoxycarbonyl) can be easily prepared using techniques known to those skilled in the art.

[0071] Paclitaxel should be understood to refer not only to the common chemically available form of paclitaxel, but analogs and derivatives (eg, TAXOTERE ™ docetaxel, as noted above) and paclitaxel conjugates (eg, paclitaxel-PEG, paclitaxel -dextran or paclitaxel-xylose).

[0072] In the treatment of some individuals according to the method of the present description, it may be desirable to use a high dose regimen in conjunction with a rescue agent for non-malignant cells. In such a treatment, any agent capable of rescuing non-malignant cells can be used, such as the citrovorum factor, folate derivatives or leucovorin. Such rescue agents are well known to those of ordinary skill in the art. Rescue agents include those that do not interfere with the ability of the present inventive compounds to modulate cellular function.

Administration of fibrillar protein

[0073] The administration of fibrillar protein can be achieved through various methods to different parts of the body, including intratumoral, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, intraperitoneal , intraarterial and rectal. Other suitable routes include administration of the composition orally, buccal, nasal, nasopharyngeal, parenteral, enteric, gastric, topical, transdermal, subcutaneous, intramuscular, in tablet, solid, powder, liquid, aerosol, intralesional injection into the tumor, intralesional injection adjacent to the tumor, intravenous infusion and intra-arterial infusion. Administration can be carried out locally or systemically, with or without added excipients. Administration can also be performed through slow release to or around an individual's tumor sites.

[0074] A person skilled in the art will appreciate that a variety of suitable methods of administering a formulation of the present description to an individual or host, for example, an individual in need thereof, are available, and although more than one route can be used for the administration of a particular formulation, a particular route can provide a more effective and more immediate reaction than another route.

[0075] The phrase "therapeutically effective amount" refers to an amount that produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount can vary depending on such factors as the disease or condition being treated, the particular targeted constructions being administered, the size of the individual or the severity of the disease or condition. A person of ordinary skill in the art can empirically determine the effective amount of a particular compound without requiring undue experimentation.

[0076] According to exemplary implementations, the protein can be administered as part of a composition, which is described in more detail below. The composition can be in various forms including powders, creams, gels, ointments, ointments, solutions, tablets, capsules, sprays and plasters. Vehicles and carriers can be used to deliver the composition to the patient. Such carriers include solubilizing agents, diluents and dispersion media. These carriers are biocompatible, pharmaceutically acceptable and do not alter the treatment characteristics of the fibrillar protein. Excipients, adjuvants and other ingredients can also be included in the composition.

Dosage

[0077] In the methods, an effective amount of a fibrillar protein is administered to an individual in need of it. In particular, fibrillar proteins of specific interest are those that inhibit cancer metastasis in a host when fibrillar proteins are administered in an effective amount. The amount administered varies depending on the objective of the administration, the health and the individual physical condition to be treated, age, the taxonomic group of the individual to be treated (for example, human, non-human primate, primate, etc.), the degree of desired resolution, the formulation of the fibrillar protein composition, the physician's estimate of the treatment of the medical situation and other relevant factors. The quantity is expected to be in a relatively wide range that can be determined through routine tests. For example, the amount of fibrillar protein used to inhibit cancer metastasis is not much more than the amount that would otherwise be irreversibly toxic to the individual (ie, maximum tolerated dose). In other cases, the amount is around or well below the toxic threshold, but still in an immuno-effective concentration range or as low as the threshold dose.

[0078] Individual doses are typically not less than the amount required to produce a measurable effect on the individual and can be determined based on the pharmacokinetics and pharmacology for protein absorption, distribution, metabolism and excretion ("ADME") fibrillar of its by-products and, thus, based on the disposition of the composition within the individual. This includes consideration of the route of administration as well as the amount of dosage, which can be adjusted for topical applications (applied directly where the action is primarily desired for a local effect), enteric (applied through the digestive tract for local or systemic effects when retained in part of the digestive tract) or parenteral (applied by means other than the digestive tract for systemic or local effects). For example, the administration of fibrillar protein typically occurs through injection and often intravenous, intramuscular, intratumor or a combination of these.

[0079] Fibrillar protein can be administered by infusion or by local injection, for example, by infusion at a rate of about 50 mg / h to 400 mg / h, including about 75 mg / h to about 375 mg / h , about 100 mg / ha about 350 mg / h, about 150 mg / ha about 350 mg / h, about 200 mg / ha about 300 mg / h, about 225 mg / ha about 275 mg /H. Exemplary infusion rates can reach a therapeutic dose of, for example, about 0.5 mg / m2 / day to about 10 mg / m2 / day, including about 1 mg / m2 / day to about 9 mg / m2 / day, about 2 mg / m2 / day to about 8 mg / m2 / day, about 3 mg / m2 / day to about 7 mg / m2 / day, about 4 mg / m2 / day to about 6 mg / m2 / day, about 4.5 mg / m2 / day to about 5.5 mg / m2 / day. The administration (for example, by infusion) can be repeated in a desired period, for example, repeated in a period of about 1 day to about 5 days or once every several days, for example, about five days, about 1 month, about 2 months, etc. It can also be administered before, during, or after other therapeutic interventions, such as surgical intervention for the removal of cancer cells. Fibrillar protein can also be administered as part of a combination therapy, in which at least one of an immunotherapy, cancer chemotherapy or radiation therapy is administered to the individual (as described in more detail below).

[0080] The disposition of the fibrillar protein and its corresponding biological activity within an individual is typically calibrated against the fraction of fibrillar protein present in a target of interest. For example, a fibrillar protein once administered can accumulate with a glycoconjugate or other biological target that concentrates the material in cancer cells and cancerous tissue. In this way, dosing regimens in which fibrillar protein is administered in order to accumulate in a target of interest over time may be part of a strategy to allow for lower individual doses. This can also understand that, for example, the dose of fibrillar protein that is purified more slowly in vivo can be decreased with respect to the effective concentration calculated from in vitro assays (for example, the effective amount in vitro approaches the concentration mM versus less than mM concentrations in vivo).

[0081] As an example, the effective amount of a dosage or dosage regimen can be measured from the IC 50 of a given fibrillar protein for inhibiting cell migration. For “IC50” to be the concentration of a drug required by 50% inhibition in vitro. Alternatively, the effective amount can be measured from the EC50 of a given concentration of fibrillar protein. For "EC50" to be the concentration of plasma required to obtain 50% of the maximum effect in vivo. In the related embodiments, the dosage can also be determined on the basis of ED50 (effective dosage).

[0082] In general, with respect to the fibrillar protein of the present description, an effective amount is usually not more than 200X the calculated IC50. Typically, the amount of a fibrillar protein that is administered is less than about 200X, less than about 150X, less than about 100X and many embodiments less than about 75X, less than about 60X , 50X, 45X, 40X, 35X, 30X, 25X, 20X, 15X, 10X and even less than about 8X or 2X than the calculated IC50. In one embodiment, the effective amount is about IX to 50X of the calculated IC50, and sometimes about 2X to 40X, about 3X to 30X or about 4X to 20X of the calculated IC50. In other embodiments, the effective amount is the same as the calculated IC50, and in certain embodiments the effective amount is an amount that is more than the calculated IC50.

[0083] An effective amount may not be more than 100X the EC50 calculated. For example, the amount of fibrillar protein that is administered is less than about 100X, less than about 50X, less than about 40X, 35X, 30X, or 25X and many embodiments less than about 20X , less than about 15X and even less than about 10X, 9X, 9X, 7X, 6X, 5X, 4X, 3X, 2X or IX than the calculated EC50. The effective amount can be about IX to 30X of calculated EC50, and sometimes about IX to 20X, or about IX to 10X of calculated EC50. The effective amount can also be the same as the calculated EC50 or more than the calculated EC50. IC50 can be calculated by inhibiting cell migration / invasion in vitro. The procedure can be carried out by methods known in the art or as described in the examples below.

[0084] The effective dosage amount and / or dosing regimen can be readily determined empirically from trials, safety and escalation and dosage range trials, individual doctor-patient connections, as well as in vitro and in vitro trials. like those described in this one and illustrated in the experimental section, below. For example, a concentration used to perform the individual method in the mouse ranges from about 1 mg / kg to about 25 mg / kg based on the body weight of the mice. Based on these data, an example of a concentration of fibrillar protein that can be used in humans can vary from about 0.083 mg / kg to about 2.08 mg / kg. Another dosage can be determined from experiments on animal models using methods known in the art (Reagan-Shaw et al. (2007) The FASEB Journal 22: 659-661).

Pharmaceutical formulations

[0085] Also provided are pharmaceutical compositions containing the fibrillar protein used in the treatment methods described above. The term "fibrillary protein composition" is used herein as a matter of convenience to refer generically to compositions comprising a fibrillary protein of the present description, including conjugated fibrillary protein, or both. Compositions useful for suppressing the development and / or metastasis of cancer cells are described below.

[0086] Fibrillar protein compositions, for example, in the form of a pharmaceutically acceptable salt, can be formulated by oral, topical or parenteral administration, as described above. In certain embodiments, for example, where a fibrillar protein is administered as an injectable liquid (such as in these embodiments where they are administered intravenously or directly into a tissue), a fibrillar protein formulation is provided as a dosage form ready for use, or as a stable reconstitutable storage powder or liquid compound of pharmaceutically acceptable carriers or excipients.

[0087] Methods for producing the fibrillar protein suitable for administration to an individual (e.g., a human individual) are described below and also in U.S. Patent Publication No. 2008/0300186, the invention of which is incorporated by reference. An example method of formulating the fibrillar protein may involve a pharmaceutical composition that contains an effective amount of a fibrillar protein and a pharmaceutical excipient (e.g., saline). The pharmaceutical composition can optionally include other additives (for example, buffers, stabilizers, preservatives, and the like). An effective amount of fibrillar protein can be an effective amount to provide a decrease in cancer metastasis (for example, cancer migration and / or invasion). A therapeutic objective (for example, reduction in tumor burden and / or restriction of cancerous development) can be accompanied by single or multiple dosages under varying dosage regimens.

[0088] The concentration of fibrillar protein in pharmaceutical formulations can vary from less than about 0.1%, usually to or at least about 2% to as much as 20% to 50% or more by weight, and will be selected mainly by fluid volumes, viscosities, etc., according to the particular mode of administration selected and the patient's need. The resulting compositions can be in the form of a solution, suspension, tablet, pill, capsule, powder, gel, cream, lotion, ointment, aerosol or others. Current methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 18th ed., Mack Publishing Company, NY (1995).

[0089] According to another aspect of this invention, fibrillar HSA can be included in the pharmaceutical or nutraceutical composition along with additional active agents, carriers, vehicles, excipients, or auxiliary agents identifiable by a person skilled in the art in reading this description.

[0090] Pharmaceutical or nutraceutical compositions preferably comprise at least one pharmaceutically acceptable carrier. In such pharmaceutical compositions, the fibrillar HSA or equivalent fibrillar HSA forms the "active compound", also referred to as the "active agent." As used here, the “pharmaceutically acceptable carrier” language includes solvents, dispersion medium, coatings, anti-fungal and anti-bacterial agents, absorption and isotonic retarding agents, and others, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, for example, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium disulfide; chelating agents such as ethylene diaminetetraacetic acid; buffers such as acetates, citrates, or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be attached in ampoules, available syringes, or multiple dosing routes of glass or plastic.

[0091] Pharmaceutical compositions suitable for injectable use include aqueous solutions (where soluble in water) or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and the extent to which syringability easily exists must be fluid. It must be stable under the conditions of manufacture and storage and must be preserved against the action of contamination by microorganisms such as bacteria and fungus. The carrier can be a solvent or dispersion medium that contains, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and others. According to embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition are added. Prolonged absorption of the injectable compositions can be conducted at about including in the composition a retarding absorption agent, for example aluminum monostearate and gelatin.

[0092] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of the ingredients and listed above, as required, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other ingredients required from those listed above. In this case of sterile powders for the preparation of sterile injectable solutions, the preparation is prepared by vacuum drying or freeze drying, which produced a powder of the active ingredient plus any additional desired ingredient from a previously sterile solution thereof.

[0093] It is recognized that when administered orally, fibrillar protein must be protected from digestion. It is typically accompanied by the fibrillar protein complex with a composition to yield resistance to acidic and enzymatic hydrolysis or by packaging in an appropriately resistant carrier such as a liposome. The means of protecting a compound of interest from digestion are well known in the art.

[0094] Oral compositions include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules, for example, gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant like colloidal silicone dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint aroma, methyl salicylate, or strawberry, cherry, grape, lemon, or orange.

[0095] For administration by inhalation, the compounds are released in the form of an aerosol spray from the pressurized container or dispenser that contains a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.

[0096] In order to intensify the average serum life, the fibrillar protein preparations that are injectable can also be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, pegylated (Greenwald et al. (2003) Advanced Drug Delivery Rev 55: 217-250; Pasut et al. (2004) Expert Opin. Ther. Patents 14: 859-894) or other conventional techniques can be used that provide an extended serum half-life. A variety of methods are available for the preparation of liposomes, as described in, for example, Szoka et al. (1980) Ann. Rev. Biophys. Bioeng. 9: 467, U.S. Patent No. 4,235,871, 4,501,728 and 4,837,028. The preparations can also be provided in controlled release or low release forms and administration of the fibrillar protein compositions as a mixture or in a serial manner.

[0097] According to embodiments, intravitreal injection is accompanied using microparticles based on PLGA or nanoparticles (liposomes). PEG-based formulas can also be used. Consequently, the other methods for injectable pharmaceutical compositions are expressly considered for intravitreal injection.

[0098] Systemic administration can also be transmucosal or transdermal. For transmucosal or transdermal administration, the appropriate barrier penetrants will be permeable and are used in a formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be followed through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, ointments, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (for example, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal release.

[0099] In addition to the other forms of release, the compounds are released through eye drop or intraocular injection. With respect to eye drops, the compositions of the present description optionally comprise one or more excipients intended for topical application to the eye and nose. Excipients commonly used in pharmaceutical compositions intended for topical application to the eye, such as solutions or sprays, include, but are not limited to, tonicity agents, preservatives, chelating agents, buffering agents, surfactants and antioxidants. Suitable tonicity adjusting agents include mannitol, sodium chloride, glycerin, sorbitol and the like. Suitable preservatives include p-hydroxybenzoic acid ester, benzalkonium chloride, benzododecinium bromide, polyquaternium-1 and others. Suitable chelating agents include sodium edetate and the like. Suitable buffering agents include phosphates, borates, citrates, acetates and the like. Suitable surfactants include ionic or non-ionic surfactants, although nonionic surfactants are preferred, such as polysorbates, derivatives of polyethoxylated castor oil and tertiary octylphenol formaldehyde polymer (tiloxapol). Suitable antioxidants include sulfides, ascorbates, BHA and BHT. The compositions of the present description optionally comprise an additional active agent. With the exception of the optional preservative ingredient (e.g., polyquaternium-1), the compositions of the present description preferably do not contain any polymeric ingredient other than polyvinylpyrrolidone or polystyrene sulfonic acid.

[00100] When the compositions of the present description contain polyvinylpyrrolidone, the polyvinylpyrrolidone ingredient is preferably selected or processed to minimize the peroxide content. Freshly produced batches of polyvinylpyrrolidone are preferred in aged batches. In addition, particularly in cases where the composition will contain more than 0.5% polyvinylpyrrolidone, the polyvinylpyrrolidone ingredient must be heat treated (ie heated to a temperature above room temperature) before mixing with olopatadine in order to reduce a amount of peroxides in the polyvinylpyrrolidone ingredient and minimize the effect of peroxides on the chemical stability of olopatadine. While heat treatment of an aqueous solution of polyvinylpyrrolidone for prolonged periods will substantially reduce the amount of peroxides, it can lead to discoloration (yellow to yellowish brown) of the polyvinylpyrrolidone solution. In order to substantially reduce or eliminate peroxides without discoloring the polyvinylpyrrolidone solution, the pH of the aqueous polyvinylpyrrolidone solution must be adjusted to pH 11-13 before it is individually heated. Very short heating times are necessary to achieve significant reductions in peroxide levels if the pH of the polyvinylpyrrolidone solution is high.

[00101] A suitable method of heat treating the polyvinylpyrrolidone ingredient ingredient is as follows. First, dissolve the polyvinylpyrrolidone ingredient in the purified water to make a 4-6% solution, then increase the pH of the solution to pH 11-13, (an effective pH range is 11-11.5), then heated in a temperature in the range of 60- 121 ° C, preferably 65-80 ° C and more preferably 70-75 ° C. The elevated temperature should be maintained for approximately 30 120 minutes (preferably 30 minutes). After the heated solution cools at room temperature, add HCl to adjust the pH to 3.5-8, depending on the target pH for the olopatadine composition. administered as eye drops, the compositions preferably contain a tonicity adjusting agent in an amount sufficient to cause the final composition to have an ophthalmically acceptable osmolality (generally 150 to 450 mOsm, preferably 250 to 350 mOsm). The ophthalmic compositions of the present invention preferably have a pH of 4 to 8, preferably a pH of 6.5 to 7.5, and more preferably a pH of 6.8 to 7.2.

[00102] Particularly for the intended compositions to be administered as eye drops, the compositions preferably contain

[00103] The eye drop compositions of the present invention are preferably packaged in opaque plastic containers. A preferred container for an ophthalmic product is a low density polyethylene container that has been sterilized using ethylene oxide instead of gamma irradiation.

[00104] With respect to ophthalmic injectables, the pharmaceutical compositions of the invention are administered in the necessary areas of treatment by subconjunctival administration. A preferred method of subconjunctival administration of the eye is by injectable formulations comprising the pharmaceutical compositions described herein. Another preferred method of subconjunctival administration is by implantations comprising slow release compositions.

[00105] The compositions that are subconjunctivally released comprise, according to embodiments, an ophthalmic deposit formulation comprising an active agent for subconjunctival administration. According to embodiments, the ophthalmic deposit formulation comprises microparticles of the essentially pure active agent. The microparticles they comprise can be included in a pharmaceutically acceptable biocompatible polymer or a lipid encapsulating agent. The deposit formulations can be adapted to release all of substantially all of the active material over an extended period of time. The polymer or lipid matrix, if present, can be adapted to degrade sufficiently to be transported from the site of administration after the release of all or substantially all of the active agent. The deposit formulation can be a liquid formulation, which comprises an acceptable pharmaceutical polymer and an active agent dissolved or dispersed. In injection, the polymer forms a deposit at the injection site, for example, by precipitation or gel formation.

[00106] Solid articles suitable for implantation in the egg can be designed in such a way to understand the polymers and can be biocorrosible or non-biocorrosible. Biocorrosible polymers that can be used in the preparation of ocular implants carry out the compositions of the present verification include without restriction of aliphatic polyesters such as polymers and copolymers of poly (glycolide), poly (lactide), poly (c-caprolactone), poly (hydroxybutyrate) ) and poly (hydroxyvalerate), polyamino acids, polyorthoesters, polyanhydrides, aliphatic polycarbonates and lactone polyethers. Illustrative of suitable non-biocorrosible polymers are silicone elastomers.

[00107] According to embodiments, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biocompatible, biodegradable polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The methods for preparing such formulations will be apparent to the person skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. liposomal suspensions (including liposomes targeted by cells infected with monoclonal antibodies to specific antigens per cell) can also be used as pharmaceutically acceptable carriers.

[00108] The fibrillar protein composition can be administered as a simple pharmaceutical formulation. It can also be administered with an effective amount of another agent that includes other suitable compounds and carriers, and can also be used in combination with other active agents. The present invention, therefore, also includes pharmaceutical compositions that comprise pharmaceutically acceptable excipients.

[00109] Pharmaceutically acceptable excipients include, for example, any suitable vehicles, adjuvants, carriers or diluents, and are readily available to the public. The pharmaceutical compositions of the present invention can still contain other active agents as are well known in the art. Pharmaceutically acceptable excipients are also well known to those who are skilled in the art, and are readily available.

[00110] For example, fibrillar protein compositions can be mixed with pharmaceutically acceptable carriers and conventional excipients (i.e. vehicles) and used in the form of aqueous solutions, tablets, capsules, elixirs, suspensions, syrups, wafers, plasters and others , but usually the fibrillar protein will be supplied as an injectable. The pharmaceutical compositions may contain common carriers or excipients, such as corn starch or gelatin, lactose, dextrose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid. Disintegrators commonly used in formulations include croscarmellose, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid. Preservatives and others can also be included. Each of these components are well known in the art. Such pharmaceutical compositions contain, in certain embodiments, from about 0.1 to about 90% by weight of the active compound, and more generally from about 1 to about 30% by weight of the active compound. See, for example, U.S. Patent No. 5,985,310, the description of which is incorporated into this by reference

[00111] Fibrillar protein compositions can be supplied in a pharmaceutically acceptable excipient, which can be a solution such as an aqueous solution, often a saline solution, or these can be supplied in powder form. The fibrillar protein compositions may contain other components, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonate, and others. The compositions may contain pharmaceutically acceptable auxiliary substances as required by the approximate physiological conditions such as pH adjustment and buffering agents, toxicity adjustment agents and others, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride , sodium lactate and others.

[00112] The choice of excipient will be determined in part by the particular compound, as well as the particular method used to administer the composition. Consequently, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following methods and excipients are merely exemplary and are by no means limiting.

[00113] A liquid composition will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in one of the suitable liquid carriers, for example, ethanol, glycerin, sorbitol, non-aqueous solvent such as polyethylene glycol, oils or water, with a suspending agent, preservative, surfactant, wetting agent, flavoring or coloring agent. Alternatively, a liquid formulation can be prepared from reconstitutable powder.

[00114] Formulations suitable for parenteral administration include sterile injectable aqueous and non-aqueous solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that yield an isotonic formulation with the blood of the intended container, and sterile aqueous and non-aqueous suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. The formulations can be presented in sealed single-dose or multiple-dose containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections. , immediately before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets from the group previously described.

[00115] The fibrillar proteins of the present invention and pharmaceutically acceptable salts thereof that are active when giving parenterally can be formulated by intramuscular, intrathecal, or intravenous administration. A typical composition for intramuscular or intrathecal administration will be a suspension or solution of the active ingredient in an oil, for example, peanut oil or sesame oil. A typical composition for intravenous or intrathecal administration will be a sterile isotonic aqueous solution that contains, for example, an active ingredient and dextrose or sodium chloride, or a mixture of dextrose and sodium chloride. Other examples are lactated Ringer injection, dextrose injection plus lactated Ringer, Normosol-M and dextrose, Isolyte E, acylated Ringer injection, and others. Optionally, a co-solvent, for example, polyethylene glycol, a chelating agent, for example, ethylenediamine tetracetic acid, and an anti-oxidant, for example, sodium metabisulfide can be included in a formulation. Alternatively, the solution can be freeze-dried and then reconstituted with a suitable solvent before use for administration.

[00116] The fibrillar proteins of the present invention and pharmaceutically acceptable salts thereof that are active in rectal administration can be formulated as suppositories. A typical suppository formulation will generally consist of the active ingredient with a lubricating and / or binding agent such as a gelatin or cocoa butter or other low-melt vegetables or synthetic wax or fat.

[00117] The fibrillar proteins of the present invention and pharmaceutically acceptable salts thereof that are active in topical administration can be formulated as transdermal compositions or transdermal delivery devices ("patches"). Such compositions include, for example, a support, active compound reservoir, a control membrane, contact and linear adhesive. Such transdermal patches can be used to provide continuous or batch infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the release of pharmaceutical agents is well known in the art. See, for example, U.S. Patent No. 5,023,252, incorporated herein by reference in its entirety. Such plasters can be constructed by continuous, pulsating, or in demand for the release of pharmaceutical agents.

[00118] The formulations of the present invention can be made in aerosol formulations to be administered by inhalation. These aerosol formulations can be placed on pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and others. These can also be formulated as pharmaceuticals for non-pressurized preparations such as for use in a nebulizer or atomizer.

[00119] Formulations suitable for topical administration may be presented as creams, gels, pastes, or foams, which contain, in addition to the active ingredient, such carriers as are known technique will be appropriate.

[00120] Suppository formulations also provided for mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as vaginal suppositories, tampons, creams, gels, pastes, foams.

[00121] The unit dosage form for oral or rectal administration such as syrups, elixir, and suspensions can be provided in which each dosage unit, for example, teaspoon, table spoon, tablet or suppository, contains a quantity predetermined composition that contains one or more fibrillar protein. Similarly, the unit dosage form for intravenous administration or injection can comprise the fibrillar protein in a composition such as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[00122] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for humans and animals, each unit containing a predetermined amount of the compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the new unit dosage form of the present invention depends on the particular compound and the effect to be achieved, and the pharmacodynamics associated with each of the compounds in the host.

[00123] One skilled in the art will readily appreciate that dosage levels may vary as a function of the specific fibrillar protein, the nature of the delivery vehicle, and others. Suitable dosages for a given compound are readily determined by that person skilled in the art by a variety of means.

[00124] The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, for the determination of LD5O (the lethal dosage to 50% of the population) and the ED50 (the therapeutically dosage) effective in 50% of the population). The dosage rate between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compounds that exhibit high therapeutic indexes are preferred. While compounds that exhibit toxic side effects can be used, care must be taken to design a delivery system that targets such compounds to the site of the affected tissue in order to minimize the potential damage to uninfected cells and therefore reduces side effects. .

[00125] The data obtained from tissue culture assays and animal studies can be used in the formulation of a dosage range for use in humans. The dosage of such compounds preferably sustains within the range of circulating concentrations that include the ED50 with no or little toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration used. For any compound used in the method of the invention, the therapeutically effective dosage can be initially estimated from cell culture assays. A dosage can be formulated in animal models to achieve a plasma circulating concentration range that includes the IC 50 (i.e., the concentration of the tested compound that achieves a mean-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful dosages in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[00126] The term "effective amount" refers to an amount of a biologically active molecule or conjugate or derivative thereof sufficient to exhibit a detectable therapeutic effect without undue side effects (such as toxicity, irritation and allergic response) providing a reason for reasonable risk / benefit used in the manner of the invention. The therapeutic effect may include, for example but not by way of limitation, cancer cells being substantially cytotoxic, but natural cells being less cytotoxic. The effective amount for an individual will depend on the type of individual, the size and health of the individuals, the nature and severity of the condition being treated, the method of administration, the duration of treatment, the nature of the concurrent therapy (if any), the specific formulations used, and others. Therefore, it is not possible to specify the effective amount of feed. However, the effective amount for one of the situation can be determined by a person with common skill in the technique using the experimentation routine based on the information provided in this.

Methods of making fibrillar protein

[00127] In accordance with the present invention, a method is described for treating cancer metastasis by administering a fibrillar protein. The fibrillar protein can be naturally occurring and, as such, can be isolated using methods known in the art for use in the treatment method described above. The fibrillar protein can also be made artificially by any method known in the art, for example, by changing a globular protein structure into a fibrillar protein structure. In one example, fibrillation of proteins can be induced by a process without the assistance of the fibril seed, as described in U.S. Patent No. 2008/0800186, the description of which is incorporated into this by reference. This process has the advantages that include control relief, homogeneity of production, and the possibility of classification. In addition, protein fibrillation can be induced by the process without the assistance of the fibril seed. Even a tiny amount of the protein would be applicable to this process. As used herein, the "protein" includes one or more proteins, protein fragments, polypeptides, or peptides. Proteins include both synthetic and naturally occurring proteins.

[00128] According to the method previously described in U.S. Patent Publication No. 2008/0800186, still a minuscule amount of protein would be applicable to this process. As used herein, "protein" includes one or more proteins, protein fragments, polypeptides or peptides. Proteins include both synthetic and naturally occurring proteins. The method can be used to convert natural proteins, with respect to their sequence, into fibrillar form in a simple and fast way. The method comprises the steps of dissolving a globular protein in a solution containing detergents and applying the solution to a molecular-sized column that can separate proteins of molecular weight 70 kDa or more, and eluting the protein with a solution containing detergent. In an exemplary implementation, the method comprises the steps of providing a globular protein, forming a solution containing the globular protein, adding a detergent to the solution containing the globular protein, and applying the solution to a molecular-sized column of a size of pore which may be the separate proteins of molecular weight 70 kDa and above, optionally in the presence of low detergent concentration.

[00129] Globular proteins, also known as spheroproteins, are one of the two main tertiary structure classes of proteins. Globular proteins are generally soluble and form spheroidal molecules in water. These have a secondary structure complex that comprises a mixture of secondary structure motifs, such as a-propellers, β-blades and arc structures. The other major class of tertiary structure of proteins are fibrillar proteins, or fibrous proteins. Fibrillar proteins are generally insoluble and have an elongated shape. These have a simpler secondary structure and are often based on only one type of secondary structure motif. In the examples of implementations, the globular protein is an albumin, for example human serum albumin, fibronectin, etc. recombinant unfolded proteins extracted from E. coli inclusion bodies with 8M urea can also be used, for example, recombinant capsid protein VP1 from the foot and mouth disease virus (rVPl), recombinant capsid protein VP2 from the virus foot and mouth disease (rVP2), recombinant capsid protein VP3 from the foot and mouth disease virus (rVP3), or PI protein precursor of VPÍ, VP2, VP3, and VP4. The protein can also be a chimeric protein comprising parts of VP1, VP2, VP3, and / or VP4, for example VP42, which comprises parts of both VP2 and VP4. Other globular proteins (eg, bovine serum albumin) can also be used, including both naturally occurring proteins and synthetic oligopeptides.

[00130] Surfactants, also referred to here as detergents, are substances that decrease the surface tension of water and increase the solubility of organic compounds. Detergents can be ionic, which include cationic, anionic and zwitterionic detergents, as well as non-ionic. Detergents play a role in breaking non-covalent bonds in proteins, thereby denaturing proteins so that they lose their natural shape or conformation. In exemplary implementations, the detergent used is sodium dodecyl sulfate (SDS), obtained from Sigma. In other exemplary implementations, the detergent used is Zwittergent 3-14, obtained from Calbiochem.

[00131] Amyloids are fibrous β-crossed protein aggregates. Numerous proteins are capable of converting to amyloid-like fibrils with characteristics that include fibrillar morphology, protofilament substructure, β-crossing diffraction pattern, an increase in β structure, Congo Red binding, and ThT binding. In exemplary implementations, the globular protein is converted to form amyloid-like fibrils, which allows the converted protein to be identified by its amyloid-like properties.

[00132] The protein for the treatment of cancer can be selected based on the severity of the disease and the desired cytotoxicity to the cancer cells. In exemplary implementations, for greater cytotoxicity to cancer cells are selected. Fibrillar proteins can include fibrillar proteins derived from the same types of protein or more than one, more than two, more than three or more types. For example, the treatment methods described above can deliver fibrillar proteins rVPl and fibrillar BSA together in one dosage or separately.

[00133] According to implementations, chromatography can be used in the process to convert the globular protein structure into a fibrillar protein structure and separate them. In general, chromatography is performed using columns, although other methods, such as those used for thin layer chromatography, may also be possible. Chromatography techniques include size exclusion, affinity and ion exchange. Although a batch-type production of fibrillar proteins is possible, using a column process allows globular proteins to be converted into a fibrillar form in a fast, firm, efficient and continuous manner. The classification of this process is also possible with the use of columns.

[00134] According to the exemplary implementations, size exclusion chromatography with pearl pore size of at least about 70 kDa is used. The pore size of pearl that is used can vary depending on various characteristics of the globular protein, for example, its size. The pore size plays a role in allowing proteins to enter the pearl matrix, thereby leading to the mechanical forces that contribute to the fold / unfold and intensifies fibrillogenic assembly. In exemplary implementations, the dimensionally molecular column used is a Superdex 200. In other exemplary implementations, the molecular dimensioning column used is an HW55S.

[00135] For column chromatography, a buffer solution containing low concentrations of detergent can be used to elute the column. In exemplary implementations, the molecular sizing column is eluted with a buffer solution containing 25 mM Tris-HCI (pH 8.0), 1 mM EDTA, 0.1 M NaCl and 0.05% SDS. In other exemplary implementations, the molecular sizing column is eluted with a buffer solution containing 25 mM Tris-HCI, pH 8.0.1 mM EDTA, 0.1 M NaCl and 0.05% Zwittergent 3-14 . The eluent can be collected as fractions and the fractions containing the subsequently joined fibrillar protein. The joined fraction can then be further filtered to purify and isolate the fibrillar protein, for example, dialysis against PBS to remove SDS or Zwittergent 3-14.

[00136] According to the implementations, human serum albumin (HSA) can be made in fibrillar human serum albumin by the processes described here to create fibrillar proteins. According to implementations, human serum albumin has been confirmed to convert to fibrillar form by the processes described here. With regard to the creation of fibrillar proteins, U.S. Patent No. 7,488,800 is incorporated by reference.

[00137] Fibrillar HSA (F-HSA) has unexpectedly been found to be at least as potent as the recombinant capsid protein from foot and mouth disease virus (rVPl) causing apoptosis in a variety of cancer cells. Among the advantages of using F-HSA instead of rVPl as a cancer therapy is that HSA is an endogenous human protein. Therefore, HSA or its derivatives with similar sequence and composition must be less likely than foreign proteins, such as rVPl, to induce immunogenicity and neutralization of antibodies during clinical applications.

[00138] According to the implementations, the F-HSA was generated by dissolving HSA in a 1% SDS solution, passing through a Superdex-200 gel filtration column and eluting with a buffer solution containing 25 mM of Tris-HCI (pH 8.0), 1 mM EDTA, 0.1 M NaCl, and 0.05% SDS (Fig. 1). After analysis against PBS to remove the SDS, it was observed that different HSA, the F-HSA eluted from the Superdex-200 column showed the enhanced fluorescence level of the specific amyloid pigment ThT in a dose dependent manner.

[00139] It was then determined that the cytotoxicity induced by F-HSA in cancer cells. As the fibrillar serum albumin bound to receptors, such as integrins on the cell surface while serum albumin may not be believed, the change in the structure of serum albumin from the globular to the fibrillar form allowed the proteins to selectively target cancer cells that express more integrin α5βl than normal cells. F-HSA inhibited the dose of breast cancer cell development including dependent cells TS / A (murine breast adenocarcinoma) and MDA-MB231 (human breast adenocarcinoma) with an IC50 of 0.15 (Fig. 13) and 0.48 pM (Fig. 14), respectively. F-HSA inhibited the development of an SKOV3 ovarian cancer cell with an IC50 of 0.6 pM (Fig. 15) and the development of a CaSki cervical cancer cell with an IC50 of 1.1 pM (Fig. 16). F-HSA also induced cytotoxicity in prostate cancer cells PC-3 and 22Rvl cells with an IC50 of 0.35 (Fig. 17) and 0.2 pM (Fig. 18), respectively. In addition, F-HSA induces cytotoxicity in several lung cancer cell lines (Fig. 19).

[00140] According to the implementations, therefore, a method for the treatment of cancer is described. The method comprises the steps of providing HSA, changing the HSA into a fibrillar structure and administering a therapeutically effective amount of F-HSA to a patient in need. The conversion of HSA to fibrillar form increases its cytotoxic effects on target cells.

[00141] In exemplary implementations, cancer is kidney, breast, lung, prostate, liver, cervical or ovarian cancer. In exemplary implementations, fibrillar HSA plays a role in inducing cancer cell apoptosis by modulating the Akt signaling pathway. In some instances, fibrillar HSA modulates α5βl or avβ3 integrin which leads to Akt deactivation. In other examples, fibrillar HSA binds to integrin and causes cell apoptosis mainly via the integrin / FAK / Akt / GSK-36 / caspase-3 pathway.

[00142] The fibrillar HSA protein, derivative, orthologist or other protein having substantial identity to HSA for the treatment of cancer can be selected based on the severity of the disease and the desired cytotoxicity to the cancer cells. In exemplary implementations, for greater cytotoxicity to cancer cells, a protein with an RGD motif or higher molecular weight is selected. The RGD motif is a ligand for integrins. It was shown that fibrillar proteins induced cell death by modulating the integrin / Akt signaling pathway. It was observed that fibrillar proteins with RGD motifs, such as rVPl-5200 and FN-5200, were more cytotoxic than those without RGD motifs, such as BSA-5200 and rVP3-5200.

[00143] "Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. A typical variant of a polynucleotide differs from the nucleotide sequence of another reference polynucleotide. Changes in the variant's nucleotide sequence may or may not change the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed here.

[00144] A typical variant of a polypeptide differs from the amino acid sequence of another reference polypeptide. In general, the differences are limited so that the sequences of the reference polypeptide and the variant are almost all similar and, in many regions, identical. A variant and the reference polypeptide may differ from the amino acid sequence by one or more substitutions, additions and deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide can be a naturally occurring one such as an allelic variant or it can be a variant that is known to occur naturally. Variants of non-naturally occurring polynucleotides and polypeptides can be made by mutagenesis techniques or by direct synthesis.

[00145] An "orthologist" indicates a polypeptide or polynucleotide obtained from another species that is the functional counterpart of a polypeptide or polynucleotide of a different species. The sequence differences between orthologists are the result of specialization.

[00146] As used in this, the twenty conventional amino acids and their abbreviations follow conventional use. See Immunology — A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Stereoisomers (eg, D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids can also be suitable components for the polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, y-carboxyglutamate, eN, N, N-trimethylisine, ε-N-acetylisin, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylstidine, 5-hydroxylisine, σ-methylilarginine and other similar amino acids and imino acids (eg, 4-hydroxyproline). In the polypeptide notation used in this, the left direction is the direction of the amino terminal and the right direction is the direction of the carboxy terminal, according to standard usage and convention.

[00147] When applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optionally aligned, such as by the GAP or BESTFIT programs using default slit weights, share at least 80 percent sequence identity , preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity and most preferably at least 99 percent sequence identity. Preferably, residue positions that are identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar secondary chains. For example, a group of amino acids having aliphatic secondary chains is glycine, alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic hydroxyl secondary chains is serine and threonine; a group of amino acids having secondary chains containing amide is asparagine and glutamine; a group of amino acids having secondary aromatic chains is phenylalanine, tyrosine and tryptophan; a group of amino acids having basic secondary chains is lysine, arginine and histidine and a group of amino acids having secondary chains containing sulfur is cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic and asparagine-glutamine.

[00148] As discussed here, minor variations in amino acid sequences of compositions having cytotoxic activities are considered to be covered by the present invention, provided that variations in the amino acid of the HSA sequence maintain at least 75%, at least 80%, at least at least 90%, at least 95%, or at least 99% identity. In particular, conservative amino acid substitutions are considered. Conservative substitutions are those that take place within a family of amino acids that are related within their secondary chains. Genetically encoded amino acids are generally divided into families: (1) acid = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, senna, threonine, tyrosine. The most preferred families are: serine and threonine are aliphatic-hydroxy family; asparagine and glutamine are a family containing amide; Alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine or similar replacement of an amino acid with a structurally related amino acid will not have a major effect on binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within a structure site. Whether an amino acid change results in a functional peptide can easily be determined by estimating the specific activity of the polypeptide derivative. The protein or peptide fragments or analogs of the present invention can be readily prepared by those of ordinary skill in the art. The preferred amino and carboxy termini of fragments or analogues occur near the boundaries of the functional domains. The structural and functional domains can be identified by comparing the nucleotide or amino acid sequence data. Preferably, computerized comparison methods are used to identify the predicted sequence motifs or protein conformation domains that occur in other proteins of known structure or function. Methods of identifying protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253: 164 (1991). Thus, the preceding examples demonstrate that those of skill in the art can recognize the sequence motifs and structural conformations that can be used to define the structural and functional domains according to the invention.

[00149] Effective amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change the binding affinity for the formation of protein complexes, (4) change the binding affinities and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include several mutations from a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domains that form intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the precursor sequence (for example, an amino acid substitution should not tend to break a helix that occurs in the precursor sequence or interrupt other types and secondary structure that characterize the precursor sequence). Examples of secondary and tertiary polypeptide structures recognized in technique are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Pubfishing, New York, NY (1991)), and Thornton et at Nature 354: 105 (1991), who are each incorporated here by reference.

[00150] The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino terminal or carboxy terminal deletion, but when the remaining amino acid sequence is identical to the corresponding positions in the deduced naturally occurring sequence, for example example, of a full-length cDNA sequence. The fragments are typically less than 5, 6, 8 or 10 amino acids in length, preferably at least 14 amino acids in length, more preferably at least 20 amino acids in length, usually at least 50 amino acids in length and even more preferably at least 70 amino acids of lenght.

[00151] In general, the method of making fibrillar proteins involves dissolving proteins in an SDS or other suitable detergent solution; applying the dissolved proteins through a gel filtration column with a pore size that can separate the proteins of 70 kDa of molecular weight and above; eluting the protein from the column and subjecting the eluate to dialysis against buffered saline to remove SDS or detergents.

[00152] In the first stage, the globular protein is generally dissolved in the form of a solution. In one example, the globular protein is dissolved in PBS with surfactants. Surfactants, also referred to here as detergents, are substances that decrease water surface tension and increase the solubility of organic compounds. Detergents can be ionic, cationic, anionic and zwitterionic detergents, as well as non-ionic. Detergents play a role in disrupting non-covalent bonds in proteins, thereby denaturing proteins, such that they lose their natural shape or shape or shape. In exemplary implementations, the detergent used is sodium dodecyl sulfate (SDS), obtained from Sigma. In other exemplary implementations, the detergent used is Zwittergent 3-14, obtained from Calbiochem.

[00153] In some aspects of the method, size exclusion chromatography with a pearl pore size of at least about 70 kDa is used. The pearl pore size used can vary depending on various characteristics of the globular protein, for example, its size. The pore size plays a role in allowing proteins to enter the pearl matrix, thereby leading to mechanical forces that contribute to protein folding / unfolding and intensify fibrillogenic assembly. In exemplary implementations, the molecular sizing column used is a Superdex 200. In other exemplary implementations, the molecular sizing column used is a HW55S. Further details of the method producing fibrillar proteins can be found in US Patent Publication No. 2008/0800186, the description of which is incorporated herein by reference.

[00154] The following examples are performed in order to provide those of ordinary skill in the art with a complete description and description of how to make and use the individual invention are not intended to limit the scope that is related to the invention.

EXPERIMENTAL Example 1 Methods

[00155] Lineages and cell cultures. SK-OV-3 cells (human ovarian carcinoma cell line; ATCC HTB-77) and SK-OV-3ip.l cells and CaSki cells (human cervical carcinoma cell line; ATCC CRL-1550) were maintained at 37 ° C in McCoy 5A and RPMI-1640 medium, respectively. MDA-MB-231 cells (human breast adenocarcinoma cell line; ATCC HTB-26) and TS / A cells (murine breast adenocarcinoma cell line) were maintained at 37 ° C in Dulbecco's modified Eagle medium (DMEM) / medium F12 and DMEM, respectively. PC-3 cells (human adenocarcinoma cell line; ATCC CRL-1435) and 22Rvl cells (human prostate carcinoma cell line; ATCC CRL-2505) were maintained at 37 ° C in RPMI-1640 medium. All media were supplemented with 10% fetal bovine serum (FBS), 2 mM Lglutamine, 100 units / ml penicillin and 100 pg / ml streptomycin.

[00156] Cytotoxicity Assay. Cell survival was determined by the Mtt assay or Wst-1 assay. Soon, Lx105 cells / Tumor cell reservoirs were seeded in the plates of 96 serum-free medium reservoirs and incubated for 1 hour. The treatment of cells with a series of concentrations of Rvpl, F-bsa, or F-hsa was performed in serum-free medium for 24 hours at 37 ° C. After the treatment, the Mtt solution was added to each reservoir (0.5 mg / ml), followed by 4 hours of incubation. The viable cell number is directly proportional to the production of Formazan, which, following solubilization with Isopropanol, can be spectrophotometrically measured at 570 Nm by an Elisa plate reader. Data expressed as a percentage of the non-treatment condition and presented as the mean ± S.D.

[00157] The WST-1 test was performed according to the manufacturer's instructions (Roche, Mannheim, Germany). Soon, 2 x 104 cells were added to 100 pl of medium per well in a 96 well plate and incubated at 37 ° C in 5% CO2 overnight in a humidified incubator. The cells attached to the reservoirs were incubated in serum-free medium and treated with serial dilutions of rVPl. After incubation at 37 ° C in 5% CO2 for 16 hours allowing the drug to take effect, 10 µl of WST-1 reagent was added to each well, and the plate was then mixed on a shaking table at 150 rpm for 1 hour. min. After incubation at 37 ° C in 5% CO2 for an additional 2 hours allowing the WST-1 reagent to be metabolized, the cell survival ratio was determined by the optical density (450 nm wavelength tested, 690 nm wavelength reference waves). The percentage of cell survival was calculated as (O.D.treatment / O.D.controie) x 100% while the percentage of development of inhibition was calculated as [1- (O.D.treatment / O.D.controie)] x 100%. IC50 is the concentration at which the reagent produces 50% inhibition of cell viability.

[00158] Cell migration and invasion test. Cell migration and invasion assays were performed following the Boyden chamber migration and invasion assay (Corning). The 8-pm pore membranes of the upper chambers were coated with 20 pg / ml of fibronectin by the cell migration assay or 40 pg / ml of Matrigel for the cell invasion assay and placed in a reservoir with 1 ml of PB S and incubated for 2 hours at 37 ° C. The cancer cells (1 x 105) were resuspended in serum-free medium and placed in the upper chamber for 1 hour. Various concentrations of fibrillar proteins such as rVPl were added in the upper chamber then the culture medium (10% FBS medium) was added in the lower chamber. The cells were incubated for 24 hours at 37 ° C. At the end of the incubation, the cells at the top of the membrane were removed by cleaning with a cotton swab and the cells migrated on the bottom membrane surface were dissociated using the cell dissociation solution (Sigma) and counted by flow cytometry (BD company).

[00159] Establishment of metastatic implantation in a murine model of TS / A mammary adenocarcinoma in BALB / c mice. The TS / A murine mammary adenocarcinoma cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin and 100 pg / ml streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37 ° C. After collection, TS / A cells (3 x 105/100 pl of PBS / mouse) were intravenously injected into the lateral tail vein of nine mice per group. Control medium or fibrillar proteins such as F-HSA were given i.v. once every two days for ten times. Finally, three mice per group were sacrificed and others were used for survival trials

[00160] In another experiment, TS / A murine mammary adenocarcinoma cells were first cultured in DMEM supplemented with 0% FBS, 2 mM L-glutamine, 100 U / ml penicillin and 100 pg / ml streptomycin or without fibrillar proteins such as rVPl treatment (0.1-0.2 pM) in a humidified atmosphere of 95% air and 5% CO2 at 37 ° C. After collection, TS / A cells pretreated with or without rVPl were intravenously injected (3 x 106/100 pl of PBS / mouse) into the lateral tail vein of nine mice per group, respectively. After 14 days, all nine mice were sacrificed and the lungs were collected.

[00161] Establishment of the implantation of metastatic xenograft in a murine model of human breast cancer. Human MDA-MB-231 breast adenocarcinoma cells were cultured in DMEM / F12 supplemented with 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin and 100 pg / ml streptomycin in a humidified atmosphere of 95 % air and 5% CO2 at 37 ° C. After collection, MDA-MB-231 cells (3 x 106/100 pl of PBS / mouse) were intravenously injected into the lateral tail vein of nine mice per group. One day after the tumor injection, fibrillar proteins such as F-HSA (1mg / Kg BW) were given i.v. once every two days for ten times while the control group were injected with only the medium. Three mice per group were sacrificed at this point in time and the rest of the mice were kept to measure survival rate.

[00162] The establishment of orthotropic xenograft implantation in a murine model of human prostate cancer. Human PC-3 prostate cancer cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin and 100 pg / ml streptomycin in a humidified atmosphere of 95 % air and 5% CO2 at 37 ° C. After collection, the PC-3 cells were washed and orthotopically injected into the prostate (1 x 106/20 pL of PBS / mouse). In one group, four weeks after PC-3 implantation, fibrillar proteins such as rVPl (25 mg / kg) were given i.v., while in the other group rVPl was given 10 weeks after cancer implantation. The same amount of rVPl was administered three times a week for 6 weeks and at the end of the rVPl treatment, 3 mice were sacrificed in each group to detect cancer metastasis. The rest of the mice were kept to measure the survival rate.

[00163] Establishment of implantation of metastatic xenograft in a mouse model of human ovarian cancer SK-OV-3. Animal studies were carried out in compliance with the guidelines for the care and use of laboratory animals at the National Defense Medical Center, Taiwan. BALB / cAnN-Foxnl female nude mice, 8 weeks old, obtained from Nacional Animal Center, Taiwan, were inoculated with 5 x 106 SK-OV-3 cancer cells per mouse by intraperitoneal injection 6 days after SK implantation -OV-3, the mice were treated with fibrillar proteins (for example rVPl, 15 mg / Kg BW) or PBS (control) and the treatments repeated every other day for 60 days. The percentage of survival and body weights were recorded until the mice died or 340 days after tumor implantation.

[00164] Isolation of SK-OV-3ip.l cells. The mice were sacrificed for the present cervical dislocation 60 days after implantation of the SK-OV-3 cell. Three ml of PBS was then injected into the abdomen and the intraperitoneal cells were recovered (about 2 ml) using a 5-ml syringe and a 25 x 1 "needle. The cells were centrifuged at 200 xg for 5 minutes at 4 ° C and the collected cell granule.To remove the red blood cells, 5 times the volume of the NH4CI cell granule (0.144 M) and 1/2 times the volume of the NH4HCO3 cell granule (0.01 M) were added and incubated at 4 ° C for 5 minutes The cells were centrifuged at 200 xg for 5 minutes at 4 ° C and the cell granule was collected The cells were grown in McCoy's 5A medium with 20% FBS in a 5% humidified CO2 atmosphere at 37 ° C for 3 days prior to Western blot analysis of HER-2 receptor levels, using standard Western blot techniques following SDS-PAGE resolution on an 8-16% gradient gel (Invitrogen).

[00165] Histopathology. The organs were fixed in 10% neutral buffered formalin. The tissues were also included in paraffin, cut in 4 pm sections, stained with hematoxylin and eosin (H&E) by light microscopy.

[00166] Statistical analysis. All data were expressed as means ± standard error and estimated using Microsoft Excel. For survival data, the log classification test was used to determine the differences between groups treated with or without drugs. Student's t test and ANOVA were performed to assess the total differences between different treatments. Values of P <0.05, P <0.01, and P <0.001 were considered statistically significant. Cell invasion and / or migration suppressed by rVPl in MDA-MB-231 cells, PC-3 cells, 22Rvl cells, SK-OV-3 cells, CaSki cells, and SK-OV-3ip.l cells in vitro.

[00167] To investigate whether tumor cell invasion and / or migration suppressed by rVPl, cell invasion and / or migration were measured using Boyden chamber assay. After various concentrations (0.1 pM to 0.2 pM rVPl in human mammary adenocarcinoma cell line MDAMB-231 cells, human prostate adenocarcinoma cell line PC-3 cells, prostate carcinoma cell line 22Rvl cells human; 0.2 pM and 0.4 pM rVPl in SK-OV-3 cells of the human ovarian carcinoma cell line and SK-OV-3ip.l cells; 0.2 pM to 0.6 pM rVPl in CaSki cells from cell line of human cervical carcinoma of rVPl treatment, cell invasion and / or migration have been significantly suppressed.Note, these rVPl concentrations do not affect cell viability as determined using the MTT assay or WST-1 assay (Figs, 1- Therefore, rVPl can significantly suppress metastasis of human breast cancer, prostate cancer, cervical cancer, and ovarian cancer cell lines in vitro.

EXAMPLE 2 Metastasis tumor cell suppressed by rVPl in vivo

[00168] A. MDA-MB-231 cells treated by rVPl in vitro followed by i.v. injection in BALB / c mice. Then examined whether MDA-MB-231 human breast adenocarcinoma cells pretreated with rVPl for 24 hours to inhibit metastatic ability in vitro then injected cells intravenously into mice should reduce their metastasis in the lungs of mice as compared to cells MDA- MB-231 without rVPl pretreatment. The data show that 0.1 and 0.2 pM of the MDA-MB-231 cells treated by rVPl significantly decreased the metastatic cancer cells in the mouse lung tissue (Fig. 4A). The weight of the related lung and the number of tumor foci in the lung were also significantly decreased compared to the tumor group without the rVPl treatment (Figs. 4B-C).

[00169] B. Orthotropic PC-3 xenograft model. We also examined whether rVPl should suppress prostate cancer metastasis in vivo. The PC-3 human prostate adenocarcinoma cells were first orthotopically implanted into the prostate of nude mice and 4 weeks or 10 weeks after implantation, rVPl (25 mg / kg) was then intravenously injected three times every week for 6 weeks. At the end of the rVPl treatment, we sacrifice the mice for autopsy. Their data show that rVPl significantly suppresses PC-3 cells that have metastasized (Table 1, Fig. 5A) and pelvic bones, and inhibited osteolysis in pelvic bones (Fig. 5B). Table 1. The percentage and location of metastatic lymph nodes with rVPl treatment in nude mice implanted by PC-3.

[00170] C. SK-OV-3 metastatic xenograft model. In vitro data show that cell invasion of SK-OV-3 ovarian carcinoma suppressed by rVPl. To examine whether rVPl should also suppress SK-OV-3 ovarian cancer metastasis in vivo, implantation of metastatic xenograft in a murine model of human SK-OV-3 ovarian cancer has been established. We observed that after 60 days, SK-OV-3 livers treated by PBS lead the mice to be surrounded by tumor cells. The mice conduct SK-OV-3 injected with rVPl (15 mg / kg) intraperitoneally every other day for 60 days, otherwise, they do not observe any apparent tumor invasion of their livers (Fig. 6).

EXAMPLE 3

[00171] F-HSA-suppressed tumor cell invasion and / or migration in MDA-MB-231 cells, PC-3 cells, 22Rvl cells and CaSki cells in vitro [00171]. To examine whether other fibrillar proteins such as F-HSA also suppressed cell invasion and / or cancer migration, the effect of F-HSA on cell invasion and / or cancer migration was measured using the Boyden chamber assay. After being treated with various concentrations of F-HSA (0.1 pM to 0.2 pM F-HSA in MDA-MB-231 cells; 0.025 to 0.1 pM F-HSA in PC-3 cells; 0.025 to 0 , 05 pM F-HSA in 22Rvl cells; 0.2 to 0.4 pM F-HSA in CaSki cells), the invasion and / or migration capabilities of a variety of cancer cells have been significantly suppressed. It is noted that at these concentrations, F-HSA does not affect cell viability using the MTT assay (Fig. 7).

EXAMPLE 4 Metastasis tumor cell suppressed by F-HSA in vivo

[00172] Still to examine whether F-HSA should suppress tumor cell metastasis in vivo, murine mammary adenocarcinoma TS / A cells were intravenously injected into the lateral tail vein of BALB / c mice or MDA-MB-231 cells of human breast adenocarcinoma were injected into the nude mice respectively. F-HSA (1 mg / kg) was then injected intravenously on the next day and then once every other day for ten times. At the end of the F-HSA treatment, the mice were sacrificed to detect lung metastasis. Their results show that F-HSA significantly suppressed the metastasis of TS / A cells and MDA-MB-231 cells to the lung (Figs. 8A and 9A) as compared to TS / A or MDA-MB-231 lead the mice treated with medium control only. The relative lung weight and the number of tumor foci in the lung of F-HSA treated by TS / A or MDAMB-231 lead the mice were significantly less than that of TS / A or MDA-MB-231 lead the mice without any drug treatment (Figs. 8B-C and 9B-C).

EXAMPLE 5 CaSki cell invasion suppressed by F-BSA in vitro

[00173] Also examined if other fibrillar proteins such as cell invasion of human cervical carcinoma CaSki suppressed by F-BSA. Cell invasion was measured using the Boyden chamber assay. We observed that at 0.1 pM - 0.2 pM, although F-BSA did not affect cell viability as indicated by the MTT assay, CaSki cell migration / invasion significantly suppressed (Fig. 10).

Example 6

[00174] F-HSA Displaying the enhanced fluorescence levels of specific amyloid pigment ThT in a dosage-dependent manner.

[00175] Fig. 12 shows an implementation of the experimental data showing that F-HSA, Aβ-like amyloid fibrils (1-42), exhibit the enhanced fluorescence level of the specific pigment by amyloid ThT in a dosage-dependent manner as compared to BS A not processed by the Superdex-200 column. This result shows that F-HSA has a fibrillar structure similar to Aβ (1-42), whereas HSA has a globular structure, (binding to ThT is one of the characteristics of amyloid-like proteins.)

Example 7 F-HSA has a cytotoxic effect on breast cancer cells

[00176] Fig. 13 shows the F-HSA cytotoxic effect on TS / A cells and Fig. 14 shows the F-HSA cytotoxic effect on MDA-MB-231 cells. Each respective cell type was treated for 16 hours in serum-free culture medium with various concentrations of F-HSA. Cell viability was determined by the MTT assay. Globular HSA has no cytotoxic effect on cancer or normal cells.

Example 8

[00177] F-HSA has a cytotoxic effect on cervical and ovarian cancer cells.

[00178] Fig. 15 shows the cytotoxic effect of F-HSA on SKOV-3 cells and Fig. 16 shows the cytotoxic effect of FHSA on CaSki cells. Each respective cell type was treated for 16 hours in serum-free culture medium with various concentrations of F-HSA. Cell viability was determined by the MTT assay.

Example 9

[00179] F-HSA has a cytotoxic effect on prostate cancer cells.

[00180] Fig. 17 shows the cytotoxic effect of F-HSA on PC-3 cells and Fig. 7 shows the cytotoxic effect of F-HSA on 22 Rvl cells. The respective cell type was treated for 16 hours in serum-free culture medium with various concentrations of F-HSA. Cell viability was determined by the MTT assay.

Example 10

F-HSA has a cytotoxic effect on lung cancer cell lines

[00181] According to implementations shown in Fig. 19, F-HSA has been shown to induce cytotoxicity in adenocarcinoma cell lines A549, CLl-0, Cll-5, H1299, PC13, and PC14; lung cancer cell line from H520 squamous cell carcinoma; and H661 wide cell lung cancer carcinoma cell line. Fig. 19 shows the IC50 of each of the respective cell lines.

Example 11 F-HSA Suppresses tumor cell invasion and in Vitro migration

[00182] F-HSA has also been shown to be effective in suppressing tumor cell invasion and migration in vitro, as shown according to the implementations of the experimental data in Fig. 20. As shown in Figs. 9A, 9C, 9E, and 9G, F-HSA significantly reduce tumor cell invasion / migration capabilities, at concentrations that do not affect the viability of cancer or normal cells.

Example 12 F-HSA Suppress inactive tumor cell metastasis

[00183] Breast cancer suppresses by F-HSA the TS / A and MDA-MB-231 tumor cell lines in vivo. FIGS. 21A and 22A show F-HSA significantly suppress breast cancer metastasis. TS / A cells and MDA-MB-231 cells to the lung compared to TS / A or MDA-MB-231 lead mice without F-HSA treatment. Figs. 21B, 21C, 22B, and 22C measure the weight and focus number of tumor cells in lung tissues, which further confirm the effectiveness of F-HSA in vivo.

[00184] According to implementations, breast cancer cells were injected through the individuals' tail vein. The focus of the tumor cell detected in the lung tissue indicates that the breast cancer cells have metastasized in the lung.

Materials and methods

[00185] Preparation of F-HSA. Twenty milligrams of HSA was dissolved in 10 ml of PBS with 1% SDS (w / v). The HSA solution was sonified for 5 minutes and subsequently applied to a Superdex-200, which was previously equilibrated with the elution of the solution (25 mM TrisHCI (pH 8.0), 1 mM EDTA, 0.1 M NaCl, and 0.05% SDS). The column was eluted at a rate of 1 ml / minute and fractions C3 to C7 containing HSA were joined. The combined fractions were concentrated to 2-3 mg / ml then dialyzed against PBS with Cellu-Sep T4 / Nominal dialysis membrane (MWCO: 12,000-14,000 Da). The new PBS buffer was changed every two hours at room temperature three times. The yield of HSA-5200 was about 75%.

[00186] Thioflavin T (ThT) fluorescence assay. Binding to ThT is one of the characteristics of proteins similar to amyloid. For fluorescence measurements, increased protein concentrations were incubated with 20 pM ThT for 1 hour at room temperature, then fluorescence was measured in triplicate on a Wallac Victor2 1420 Multilabel counter (Perkin Elmer Life Science, Waltham, MA, USA ). Excitation and emission lengths were 430 nm and 486 nm, respectively. The ThT ground signal from the buffer solution was subtracted from the corresponding measurements.

[00187] Cell survival was determined by the MTT colorimetric assay. The exponentially developed cells (2 x 104 cells / well per TS / A) were seeded in a 96 well plate in the medium with 10% FBS and incubated for 24 hours. The treatment of cells with a series of protein concentrations was carried out in a serum-free medium for 16 hours at 37 ° C. After treatment, the MTT solution was added to each reservoir (0.5 mg / ml), followed by an incubation period of 4 hours. The viable cell number is targeted proportional to the production of formazan, which, following solubilization with isopropanol, can be spectrophotometrically measured at 570 nm by an ELISA plate reader.

[00188] Cell viability tests. Cell viability was measured by the WST-1 assay according to the manufacturer's instructions (Roche, Mannheim, Germany). Soon, 2 x 104 cells were added to 100 pl medium per well in a 96 well plate and incubated at 37 ° C in 5% CO2 overnight in a humidified incubator. The cells bound to the cells were incubated in serum-free medium and treated with various concentrations of F-HSA. After incubation at 37 ° C in 5% CO2 for 16 hours allowing the drug to take effect, 10 µl of the WST-1 reagent was added to each reservoir. The plate was then placed on a shaking table and stirred at 150 rpm for 1 min. After incubation at 37 ° C in 5% CO2 for an additional 2 hours allowing the reagent WST-1 to be metabolized, the proportion of cell survival was determined by the optical density (450 nm test wavelength, 690 nm reference wave). The percentage of cell survival was calculated as (ODtreatment / OD.COntroie) x 100% while the percentage of development inhibition was calculated as [1- (θ-D.treatment / θ-D.control)] X 100% . According to these experiments, IC50 is the concentration at which the reagent produces 50% inhibition of cell viability.

[00189] Tests of cell migration and invasion. Cell migration and invasion were determined using the Boyden chamber migration and invasion (Corning) assay. Soon, the 8-pm pore membranes of the upper chambers were coated with 20 pg / ml of fibronectin (for the cell migration assay) or 40 pg / ml of Matrigel (for the cell invasion assay) and placed in a reservoir with 1 ml of PB S and incubated for 2 hours at 37 ° C. The cancer cells (1 x 105) in 100 pl of serum-free culture medium were seeded in the upper chamber for 1 hour. The serially diluted concentration of F-HSA was added in the upper chamber and then the culture medium containing 10% FBS was added in the lower chamber. The cells were incubated for 24 hours at 37 ° C. After incubation, the cells on the top side of the membrane were removed by cleaning it with a cotton swab and cells that migrated on the bottom membrane surface were dissociated using the cell dissociation solution (Sigma) and counted by flow cytometry (BD company ). At these concentrations, however, F-HSA does not affect cell viability when measured with the MTT assay, and as shown in Figs. 9B, 9D, 9F, and 9H. Viability and cytotoxicity were measured using the MTT or WST-1 assay. These kits are designed by spectrophotometric measurement of cell development as a function of mitochondrial activity in living cells (Roche).

[00190] Breast cancer cell metastasis in vivo. The murine TS / A mammary adenocarcinoma cells were intravenously injected into the lateral tail vein of the BALB / c mice or MDA-MB-231 human mammary adenocarcinoma cells were injected into nude mice. The F-HSA (1 mg / kg) was then injected intravenously on the next day and then once every other day for ten times. At the end of the F-HSA treatment, the mice were sacrificed to detect lung metastasis. 1/1

Claims (7)

1. Pharmaceutical composition, characterized by the fact that it is to suppress cancer metastasis in an individual, comprising a fibrillar protein and a pharmaceutically acceptable excipient, in which the fibrillar protein is fibrillar albumin or human fibrillar serum albumin. 2. Composition according to claim 1, characterized by the fact that said cancer is characterized by the overexpression of α5βl and / or avβ3 integrin. Composition according to claim 1, characterized by the fact that said fibrillar protein is a chimeric protein. 4. Composition according to claim 1, characterized by the fact that cancer is at least one chosen from breast cancer, ovarian cancer, cervical cancer, prostate cancer and lung cancer. 5. Composition according to claim 1, characterized by the fact that it is adapted for intravenous injection, subcutaneous injection, intraperitoneal injection, intraarterial injection, intramuscular injection, intralesional injection into the tumor, intralesional injection adjacent to the tumor, intravenous infusion, and infusion intra-arterial. 6. Composition according to claim 1, characterized by the fact that it also comprises a second therapeutic agent. Composition according to claim 6, characterized by the fact that said second therapeutic agent is a chemotherapeutic agent.

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