ES2951232A1 - COMBINATION OF AN ANTI-ANGIOGENIC AND A FAK INHIBITOR IN THE TREATMENT OF UVEAL MELANOMA - Google Patents

Abstract

Combination of an antiangiogenic and a FAK inhibitor in the treatment of uveal melanoma. The present invention relates to a pharmaceutical composition comprising an antiangiogenic agent, bevacizumab, and a FAK inhibitor, for use in a specific type of intraocular cancer, uveal melanoma, preferably metastatic. Furthermore, the invention also relates to the in vitro uses of said pharmaceutical composition, such as the inhibition of tumor growth and vasculogenic mimicry. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Combination of an antianxiety agent and a FAK inhibitor in the treatment of uveal melanoma

The present invention is within the field of biomedicine. Specifically, the present invention relates to a pharmaceutical composition comprising an antiangiogenic agent, bevacizumab, and a FAK kinase inhibitor, for use in the treatment of uveal melanoma, a specific type of intraocular cancer. Furthermore, the invention also relates to the in vitro uses of said pharmaceutical composition.

BACKGROUND OF THE INVENTION

Uveal melanoma is the most common primary intraocular malignancy in adults and has an incidence of approximately 0.7 per 100,000 inhabitants, which is why it is classified as a rare disease. This type of cancer is also associated with the development of metastasis in approximately 50% of cases and 40% of patients die from metastatic disease despite successful treatment of the primary tumor.

There are different studies in the state of the art focused on therapies aimed at uveal melanoma. For example, in the review by Croce M. et al., molecules involved in the aggressiveness of this type of cancer are described as potential therapeutic targets and different clinical trials directed at uveal melanoma are compiled (Croce M. et al., Targeted Therapy of Uveal Melanoma: Recent Failures and New Perspectives. Cancers (Basel);11(6):846 (2019)).

In US2020323863A1, methods for the treatment of uveal melanoma are disclosed, which comprise administering an agent that inhibits the expression of FAK kinase ( focal adhesion kinase) and an agent that inhibits MEK kinase (MEK). English, mitogen-activated protein kinase kinase).

Despite the different known therapeutic approaches, uveal melanoma, and in particular metastatic uveal melanoma, continues to be a type of cancer in which there are no therapies that have shown a significant impact on survival until now.

Therefore, there is a need in the state of the art to provide alternative therapeutic approaches to those existing for the treatment of uveal melanoma, with the capacity to significantly reduce tumor growth and progression.

DESCRIPTION OF THE INVENTION

In the present invention, the inventors have demonstrated that the combination of PF-271 and bevacizumab reduces tumor progression and growth in a particular type of cancer, uveal melanoma. In fact, the combination of PF-271 and bevacizumab, which is not described for use in uveal melanoma, presented an unexpected improved effect in reducing tumor growth compared to their individual administration in a xenograft animal model of human uveal melanoma. metastatic (Fig. 2A). Furthermore, the inventors have also demonstrated that said combination of PF-271 and bevacizumab additively reduces tumor PAS+/CD31- regions (Fig. 2C), which are identifiers of vasculogenic mimicry, a process of blood supply of cancer cells.

The combination of PF-271 and bevacizumab presents as associated advantages an improved effect in reducing tumor growth in uveal melanoma, having potential clinical application. Furthermore, its effect against vasculogenic mimicry gives this combination a potential therapeutic use in cases of poor prognosis of uveal melanoma, since it is an alternative blood supply process to angiogenesis, which is associated with highly invasive tumor phenotypes. . In fact, there is a strong association between the histological detection of vasculogenic mimicry patterns in uveal melanoma and subsequent death due to metastasis.

Based on this, the inventors have developed a series of inventive aspects that will be described below:

Therapeutic use of the invention

In a first aspect, the invention relates to a pharmaceutical composition, comprising PF-271 and bevacizumab, hereinafter the "pharmaceutical composition of the invention", for use in the treatment of uveal melanoma.

The compound PF-271 (also known as PF-562271 or PF-562,271), is a FAK inhibitor known in the state of the art, whose chemical structure is the following:

Focal adhesion kinase, or “FAK ,” is a tyrosine kinase that regulates cell adhesion signaling and migration, and may also promote cell survival in response to stress. FAK is usually overexpressed in cancer, being a pharmacological target of great value.

On the other hand, “bevacizumab” (also known as Avastin), is an antiangiogenic agent. It is a recombinant humanized monoclonal antibody directed against vascular endothelial growth factor (VEGF), a proangiogenic cytokine. Bevacizumab binds to VEGF and inhibits binding to its receptor, thus preventing the growth and maintenance of tumor blood vessels.

As used herein, the terms "tumor" or "cancer" refer to a condition characterized by the abnormal rapid proliferation of abnormal cells in a subject. Abnormal cells are often called "neoplastic cells," which are transformed cells that can form a solid tumor. The term "tumor" refers to an abnormal mass or population of cells (for example, two or more cells) that results from excessive or abnormal cell division, whether malignant or benign, and to precancerous and cancerous cells. Malignant tumors are distinguished from benign growths or tumors in that, in addition to uncontrolled cell proliferation, they can invade surrounding tissues and can metastasize.

In the present invention, PF-271 and bevacizumab are the active agents or principles of the pharmaceutical composition of the invention, for use in the treatment of uveal melanoma.

In the present invention, "treatment" is understood as the set of means that are used to treat, alleviate or cure a disease, in particular, uveal melanoma. The term "treat" may refer to stopping, delaying the onset (i.e., the period before the clinical manifestation of a disease) and/or reducing a subject's risk of developing or worsening a disease.

In the present invention, "subject" means any animal, preferably a mammal, more preferably a primate, in particular a human, of any race, sex or age.

The term "uveal melanoma", also known as ocular melanoma, as used in the present invention, refers to a type of cancer, in particular, a melanoma of the eye, affecting the iris, ciliary body and/or the choroid, which collectively is also called the uvea. In the case of large tumors, melanoma may occupy part or all of these regions of the eye.

In a preferred embodiment, uveal melanoma affects the iris, choroid, or ciliary body of the eye. In another preferred embodiment, uveal melanoma affects the choroid and ciliary body of the eye. In another preferred embodiment, uveal melanoma affects the choroid and iris of the eye. In another preferred embodiment, uveal melanoma affects the ciliary body and iris of the eye.

On the other hand, uveal melanoma can refer to local uveal melanoma, metastatic uveal melanoma, non-metastatic uveal melanoma, primary uveal melanoma, advanced uveal melanoma, or recurrent uveal melanoma.

As used here, “local uveal melanoma” refers to the melanoma being locally confined to the eye.

As used herein, “advanced uveal melanoma” refers to uveal melanoma that has progressed after receiving first-line therapy, second-line therapy, and/or third-line therapy. The expression "first line" or "second line" or "third "line" refers to the order of treatment a patient receives. First-line therapy regimens are the treatments that are given first, while second- or third-line therapy is given after the first-line therapy or after of second-line therapy, respectively.

The term "recurrent", as used here, in relation to uveal melanoma, refers to the pathology being present in a subject after having been treated and/or after a period of time in which it could not be detected. . Recurrent cancer may come back in the same place where it first started, or it may come back in another part of the body.

In a preferred embodiment, uveal melanoma is a primary uveal melanoma that develops in the iris, choroid and/or ciliary body.

The term "primary," as used here, refers to the cancer starting in the eye rather than spreading there from somewhere else. On the other hand, primary uveal melanoma can be stratified into four distinct, clinically relevant molecular subtypes, with a significant difference in metastasis rate and prognosis. Tumors 1A and 1B retain a phenotype of differentiated melanocytes, with a disomy of chromosome 3. Furthermore, they are distinguished by alterations in EIF1AX (eukaryotic translation initiation factor 1A, X chromosome) or SF3B1 (splicing factor subunit 1). 3b), respectively, with 1A having a lower metastasis rate compared to 1B. Class 2 uveal melanoma is associated with a high metastatic risk and is characterized by a monosomy of chromosome 3, followed by aberrations in BAP1 (BRCA1-associated protein 1) expression and global DNA methylation. A further subdivision can be made into class 2A and 2B based on chromosome 8q copy number alterations, RNA expression and cellular pathway activity profiles, with class 2B having a higher rate of metastasis compared to class 2A.

As already mentioned, the inventors have shown that the combination of PF-271 and bevacizumab reduces tumor growth in a xenograft animal model of metastatic human uveal melanoma. Thus, in another more preferred embodiment, the uveal melanoma is metastatic.

The term "metastatic", as used in the present invention, refers to the fact that metastasis has occurred, that is, the migration or spread of tumor cells to other parts of the body other than where the cancer originated. , for example, but not limited to, liver, lung and bones, as well as subcutaneous metastases.

On the other hand, a subject's uveal melanoma can be classified according to the size of the tumor. In a preferred embodiment, the uveal melanoma comprises at least one tumor, where the tumor comprises a height greater than 10 mm. In another preferred embodiment, the uveal melanoma comprises at least one tumor wherein the tumor comprises a height of 2.5 to 10 mm. In another preferred embodiment, the uveal melanoma comprises at least one tumor, wherein the tumor comprises a height of less than 2.5 mm.

Furthermore, a known mechanism that can occur in uveal melanoma, and that can in fact contribute to the progression of this type of cancer in a subject, is vasculogenic mimicry. The inventors have demonstrated that the combination of PF-271 and bevacizumab produces a reduction in PAS+/CD31- tumor regions, which are identifiers of the formation of vasculogenic mimicry.

Thus in a preferred embodiment, uveal melanoma further comprises vasculogenic mimicry.

The term "vasculogenic mimicry", as used in the present invention, refers to a type of blood supply or supply of cancer cells. More specifically, vasculogenic mimicry refers to the ability of cancer cells to organize into vascular-like structures for obtaining nutrients and oxygen independently of normal blood vessels or angiogenesis. In fact, vasculogenic mimicry may be related to poor survival in subjects suffering from cancer.

In another preferred embodiment, uveal melanoma further comprises angiogenesis. More preferably, uveal melanoma comprises vasculogenic mimicry and angiogenesis.

The term “angiogenesis,” as used here, refers to the process by which new blood vessels form from preexisting vessels. At the moment The invention refers to tumor angiogenesis, which is the proliferation of blood vessels that allows the supply of nutrients and oxygen to cancer cells, being a key process in tumor growth and metastasis.

As already mentioned, in the present invention, PF-271 and bevacizumab are the active agents or principles of the pharmaceutical composition of the invention.

“Pharmaceutical composition” is understood as any preparation or pharmaceutical form, whose composition formula expressed in units of the international system, is constituted by a substance or mixture of substances, with constant weight, volume and percentages, which may be prepared in pharmaceutical laboratories. legally established, packaged or labeled to be distributed and marketed as effective for diagnosis, treatment, mitigation and prophylaxis of a disease, physical abnormality or symptom, or the restoration, correction or modification of the balance of the organic functions of human beings and animals. animals. The preparation of the pharmaceutical composition can be carried out by any of the methods described in the state of the art.

In a preferred embodiment, the pharmaceutical composition of the invention comprises PF-271 and bevacizumab in a therapeutically effective amount.

As used herein, the term “therapeutically effective amount” refers to the amount of the compounds PF-271 and bevacizumab, or the pharmaceutical composition of the invention that produce the desired effect, which may be given by the nature of the compounds included in the composition, as well as the therapeutic effect to be achieved. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex or tolerance, of the mammal to which it is to be administered.

Additionally, the pharmaceutical composition of the invention may comprise an adjuvant. By "adjuvant" is meant any substance that enhances the effectiveness of the pharmaceutical composition of the invention. Examples of adjuvants include, but are not limited to, adjuvants formed by aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate or aluminum sulfate, oil-in-water or water-in-oil emulsion formulations such as Complete Adjuvant. of Freund's (ACF) as well as Freund's Incomplete Adjuvant (AIF), mineral gels; block copolymers, Avridine™, SEAM62, adjuvants formed from bacterial cell wall components such as adjuvants including liposaccharides (e.g. lipid A or monophosphoryl Lipid A (MLA), trehalose dimycolate (TDM), and skeletal components of cell wall (CWS), heat shock proteins or their derivatives, adjuvants derived from ADPribosilatinated bacterial toxins, including diphtheria toxin (DT), pertussis toxin (PT), cholera toxin (CT), labile E.coli toxins to heat (LT1 and LT2), Endotoxin A and exotoxin of Pseudomonas, exoenzyme B of B.cereus, toxin of B. sphaerieus, toxins C ₂ and C3 of C. botulinum, exoenzyme of C. limosum as well as the toxins of C. perfringens, C. spiriforma and C. diffieile, S. aureus, EDIM and toxin mutants such as CRM-197, a non-toxic mutant diphtheria toxin, saponins such as ISCOMs (immunostimulatory complexes), chemokines and cytokines such as interleukins. (IL-I IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, etc.), interferons (such as interferon gamma) macrophage colony-stimulating factor (M -CSF), Tumor Necrosis Factor (TNF), defensins 102, RANTES, MIPI -alpha, and MEP-2, muramyl peptides such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N -acetyl normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2-( 1'-2'-dipalmitoyl-s- n-glycero- 3-hydroxyphosphoryloxy)ethylamine (MTP-PE) etc; adjuvants derived from the CpG family of molecules, CpG dinucleotides and synthetic oligonucleotides comprising CpG motifs, lysosum exoenzyme from C. Limosum and synthetic adjuvants such as PCPP, cholera toxin, Salmonella toxin, alum and the like, aluminum hydroxide, N- acetyl-muramyl-Lthreonyl- D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, dimycolate of trehalose and cell wall skeleton (MPL+TDM+CWS) in a 12% squalene/Tween 80 emulsion. Other examples of adjuvants include DDA (dimethyldioctadecylammonium bromide), complete and incomplete Freund's adjuvants, QuilA, microvesicles and exosomes .

In a preferred embodiment, the pharmaceutical composition of the invention further comprises a carrier, preferably a pharmaceutically acceptable carrier, and/or a chemotherapeutic agent.

The term "vehicle" refers to a diluent or excipient with which the active ingredients are administered. Such vehicles may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solutions of saline and aqueous solutions of dextrose and glycerol are preferably used as vehicles, particularly for injectable solutions. Preferably, in the case of pharmaceutical vehicles, these are approved by the regulatory agency of a state or federal government or are listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. . The vehicles and auxiliary substances necessary to manufacture the desired pharmaceutical form of administration of the pharmaceutical composition of the invention will depend, among other factors, on the pharmaceutical form of administration chosen. Said pharmaceutical forms of administration of the pharmaceutical composition will be manufactured according to conventional methods known to those skilled in the art.

The term “pharmaceutically acceptable” refers to the fact that the vehicle or excipient must allow the activity of the compounds or agents of the pharmaceutical composition, that is, it must be compatible with said components, so that it does not cause harm to the organisms to which it is intended. that is administered.

By "chemotherapeutic agent" is meant any substance that is capable of reducing tumor progression, for example, by inhibiting cell proliferation without necessarily killing the cell, or that is capable of inducing cell death. Agents capable of inhibiting cell proliferation without causing cell death are generically called cytostatic agents, while those that are capable of inducing cell death normally by activating apoptosis are generically called cytotoxic agents. Non-limiting examples of chemotherapeutic agents suitable for use in the compositions of the invention include (i) microtubule stabilizing agents such as taxanes, paclitaxel, docetaxel, epothilones and laulimalides, (ii) kinase inhibitors such as Iressa(R), Gleevec , Tarceva™, (Erlotinib HCl), BAY-43-9006, (iii) receptor-specific antibodies with kinase activity including, without limitation, Trastuzumab (Herceptin(R)), Erlotinib, Gefitinib, Cetuximab (Erbitux(R)), Rituximab (ritusan(R)), Pertuzumab (Omnitarg™); (iv) mTOR pathway inhibitors, such as rapamycin and CCI-778; (v) Apo2L1Trail, (vi) anti-angiogenic agents such as endostatin, combrestatin, angiostatin, thrombospondin and endothelial growth inhibitor vascular (VEGI); (vii) antineoplastic vaccines including activated T cells, nonspecific immunopotentiating agents (for example interferons, interleukins) or other types of immunotherapy; (viii) antibiotic cytotoxic agents such as doxorubicin, bleomcin, dactinomycin, daunorubicin, epirubicin, mitomycin and mitozantrone; (ix) alkylating agents such as Melphalan, Carmustine, Lomustine, cyclophosphamide, ifosfamide, Chlorambucil, Fotemustine, Busulfan, Temozolomide and thiotepa; (x) antineoplastic hormonal agents such as Nilutamide, cyproterone acetate, anastrozole, Exemestane, Tamoxifen, Raloxifene, Bicalutamide, Aminoglutethimide, leuprorelin acetate, Toremifene citrate, Letrozole, Flutamide, Megestrol acetate and goserelin acetate; (xi) gonadal hormones such as cyproterone acetate and medoxyprogesterone acetate; (xii) antimetabolites such as Cytarabine, Fluorouracil, Gemcitabine, Topotecan, Hydroxyurea, Thioguanine, Methotrexate, Colaspase, Raltitrexedo and capicitabine; (xiii) anabolic agents such as nandrolone; (xiv) adrenal steroid hormones such as methylprednisolone acetate, dexamethasone, hydrocortisone, prednisolone and prednisone; (xv) antineoplastic agents such as Temozolamide, Carboplatin, Cisplatin, Oxaliplatin, Etoposide and Dacarbazine and (xvi) topoisomerase inhibitors such as topotecan and irinotecan.

The pharmaceutical composition of the invention, and/or its formulations, can be administered to an animal, including a mammal and, therefore, a human, in a variety of ways, including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intrathecal, intraventricular, intraarticular, intratumoral, oral, enteral, parenteral, intranasal, ocular or topical. Preferred routes of administration of the pharmaceutical composition of the invention for the treatment of uveal melanoma are the intratumoral, intraperitoneal, intraocular or oral route. In a preferred embodiment, the composition of the invention is formulated for oral, intraperitoneal, intraocular, or intratumoral administration.

The term "administration", as used herein, refers to the introduction of the therapeutic agents or active ingredients, in the present invention comprised in a pharmaceutical composition, into a subject.

Dosage forms of the pharmaceutical composition of the present invention for use in the treatment of uveal melanoma are not specifically limited, and examples include, but are not limited to, liquid dosage forms, such as a liquid medicine for injection, a liquid medicine for eye drops, a liquid medicine for nasal spray, a lotion, creams, a patch, a sublingual tablet. These dosage forms can be manufactured and administered by methods well known to one skilled in the art.

Furthermore, as understood by one skilled in the art, a healthcare professional can adjust the concentration of the active ingredients of the pharmaceutical composition for use in the treatment of uveal melanoma according to, for example, but not limited to, therapeutic and toxicity criteria.

Thus, in a preferred embodiment, the concentration of PF-271 in the pharmaceutical composition of the invention is 20 to 60 mg/Kg (including the extremes) and the concentration of bevacizumab in the pharmaceutical composition of the invention is 3.5 to 15 mg/Kg (including extremes).

In a more preferred embodiment, the concentration of PF-271 in the pharmaceutical composition of the invention is 25 mg/Kg to 50 mg/Kg (including the extremes) and the concentration of bevacizumab in the pharmaceutical composition of the invention is 5 to 10 mg/Kg (including the extremes). More preferably, the concentration of PF-271 in the pharmaceutical composition of the invention is 25, 30, 35, 40, 45, or 50 mg/Kg, and the concentration of bevacizumab in the pharmaceutical composition of the invention is 5. 6, 7, 8, 9, 10mg/Kg.

Even more preferably, the concentration of PF-271 in the pharmaceutical composition of the invention is 30 mg/Kg and the concentration of bevacizumab in the pharmaceutical composition of the invention is 5 mg/Kg.

mg/kg is understood as the concentration of the agents included in the pharmaceutical composition of the invention, PF-271 and bevacizumab, given in mg of agent per kg of the subject.

In a preferred embodiment, the pharmaceutical composition comprising bevacizumab and PF-271 is a combination preparation for simultaneous or sequential use in the treatment of uveal melanoma.

In the present invention, "combined preparation" is understood to mean that its components, PF-271 and bevacizumab, do not need to be found as a union in order to be available for sequential (separate) application. In this way, it implies that a true combination does not necessarily result, in view of the physical separation of the components.

In a preferred embodiment of the combined preparation, PF-271 and bevacizumab are administered sequentially. Preferably, PF-271 is administered orally and bevacizumab is administered intraperitoneally.

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every other day at a concentration of 30 mg/kg and bevacizumab is administered two days per week of treatment at a concentration of 5 to 7.5 mg/Kg (including the extremes).

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every other day at a concentration of 30 mg/Kg and bevacizumab is administered one day per week of treatment at a concentration of 5 to 7.5 mg/Kg (including the extremes).

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every other day at a concentration of 30 mg/Kg and bevacizumab is administered one day every 3 weeks of treatment at a concentration of 10 mg. /Kg.

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every three days at a concentration of 50 mg/Kg and bevacizumab is administered two days per week of treatment at a concentration of 5 to 7.5 mg/Kg (including the extremes).

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every three days at a concentration of 50 mg/Kg and bevacizumab is administered one day per week of treatment at a concentration of 5 to 7.5 mg/Kg (including the extremes).

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every three days at a concentration of 50 mg/Kg and bevacizumab is administered one day every 3 weeks of treatment at a concentration of 10 mg. /Kg.

Even more preferably, PF-271 is administered every other day at a concentration of 30 mg/Kg and bevacizumab is administered two days per week of treatment at a concentration of 5 mg/Kg.

Use of the pharmaceutical composition of the invention in the manufacture of a medicine

The inventors have demonstrated that the combination of PF-271 and bevacizumab reduces tumor progression and growth in uveal melanoma. This allows the use of the pharmaceutical composition of the invention, including the combined preparation, in the preparation of a medicament for the treatment of uveal melanoma.

Thus, in another aspect, the invention relates to the use of the pharmaceutical composition of the invention in the preparation of a medication for the treatment of uveal melanoma. Techniques and procedures for preparing medicines are widely known in the field.

The terms "pharmaceutical composition", "treatment" and "uveal melanoma" have already been previously defined or explained, and both they and their preferred embodiments are applicable to the present aspect of the invention.

Thus, in a preferred embodiment, uveal melanoma is a primary uveal melanoma that develops in the iris, choroid and/or ciliary body.

In another preferred embodiment, the uveal melanoma is metastatic.

In another preferred embodiment, uveal melanoma further comprises vasculogenic mimicry.

The terms used to define the present aspect of the invention have already been previously explained, and both they and their preferred embodiments are applicable to the present aspect of the invention.

In vitro uses of the invention

In addition to the therapeutic applications mentioned above, the application of the pharmaceutical composition of the invention in in vitro tests is also possible, hereinafter, the " in vitro uses of the invention."

The combination of PF-271 and bevacizumab showed the ability to reduce tumor size in uveal melanoma in an improved manner compared to their individual administration.

Thus, another aspect of the invention refers to the use of the pharmaceutical composition of the invention to inhibit the growth of uveal melanoma tumor cells in vitro .

The terms “pharmaceutical composition” and “uveal melanoma” have already been explained, and both they and their preferred embodiments are applicable to the in vitro uses of the invention.

The expression "inhibit", referring to "the growth of tumor cells", refers to stopping, blocking, reducing, decreasing or attenuating the proliferation of tumor cells, and therefore, stopping and even reducing the volume of the tumor of which they are part. , in the present invention, of uveal melanoma.

Preferably, the uveal melanoma tumor cells are metastatic. More preferably, the metastatic uveal melanoma tumor cells are MUM2B cells.

Furthermore, the combination of PF-271 and bevacizumab reduces PAS+/CD31-tumor regions. Thus, another aspect of the present invention relates to the use of the pharmaceutical composition of the invention to reduce PAS+/CD31- regions of a tumor in vitro .

As previously explained, the PAS+/CD31- regions of a tumor are vasculogenic mimicry identifiers. Thus, another aspect of the invention relates to the use of the pharmaceutical composition of the invention to inhibit in vitro the vasculogenic mimicry of a tumor, preferably where the tumor is uveal melanoma.

The term “vasculogenic mimicry” has already been previously explained herein, and both it and preferred embodiments thereof are applicable to the present aspect of the invention.

In a preferred embodiment of the " in vitro uses of the invention", the pharmaceutical composition comprises a vehicle and/or a chemotherapeutic agent.

In another preferred embodiment of the " in vitro uses of the invention", the concentration of PF-271 in the pharmaceutical composition is 0.50 mg/mL to 2.54 mg/mL (including the extremes) and the concentration of bevacizumab in the pharmaceutical composition it is 0.25 mg/mL to 1 mg/mL (including the extremes).

More preferably, the concentration of PF-271 in the pharmaceutical composition is 0.50 mg/mL, 0.55 mg/mL, 0.60 mg/mL, 0.65 mg/mL, 0.70 mg/mL, 0.75 mg/mL, 0.80 mg/mL, 0.85 mg/mL, 0.90 mg/mL, 0.95 mg/mL, 1.00 mg/mL, 1.05 mg/mL, 1 ,10 mg/mL, 1.15 mg/mL, 1.20 mg/mL, 1.25 mg/mL, 1.30 mg/mL, 1.35 mg/mL, 1.40 mg/mL, 1, 45 mg/mL, 1.50 mg/mL, 1.55 mg/mL, 1.60 mg/mL, 1.65 mg/mL, 1.70 mg/mL, 1.75 mg/mL, 1.80 mg/mL, 1.85 mg/mL, 1.90 mg/mL, 2.00 mg/mL, 2.05 mg/mL, 2.10 mg/mL, 2.15 mg/mL, 2.20 mg /mL, 2.25 mg/mL, 2.30 mg/mL, 2.35 mg/mL, 2, 40 mg/mL, 2.45 mg/mL, 2.50 mg/mL, or 2.54 mg /mL, and the concentration of bevacizumab is 0.25 mg/mL, 0.30 mg/mL, 0.35 mg/mL, 0.40 mg/mL, 0.45 mg/mL, 0.50 mg/mL mL, 0.55 mg/mL, 0.60 mg/mL,0.65 mg/mL, 0.70 mg/mL, 0.75 mg/mL, 0.80 mg/mL, 0.85 mg/mL , 0.90 mg/mL, 0.95 mg/mL or 1 mg/mL.

Treatment method of the invention

In another aspect, the invention relates to a method for the treatment of uveal melanoma in a subject, comprising the administration of PF-271 and bevacizumab. Preferably, the uveal melanoma is metastatic.

In a preferred embodiment, PF-271 and bevacizumab are administered simultaneously or sequentially. Preferably, PF-271 is administered orally and bevacizumab is administered intraperitoneally.

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every other day at a concentration of 30 mg/kg and bevacizumab is administered two days per week of treatment at a concentration of 5 to 7.5 mg/Kg (including the extremes).

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every other day at a concentration of 30 mg/Kg and bevacizumab is administered one day per week of treatment at a concentration of 5 to 7.5 mg/Kg (including the extremes).

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every other day at a concentration of 30 mg/Kg and bevacizumab is administered one day every 3 weeks of treatment at a concentration of 10 mg. /Kg.

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every three days at a concentration of 50 mg/Kg and bevacizumab is administered two days per week of treatment at a concentration of 5 to 7.5 mg/Kg (including the extremes).

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every three days at a concentration of 50 mg/Kg and bevacizumab is administered one day per week of treatment at a concentration of 5 to 7.5 mg/Kg (including the extremes).

In another more preferred embodiment, when PF-271 and bevacizumab are administered sequentially, PF-271 is administered every three days at a concentration of 50 mg/Kg and bevacizumab is administered one day every 3 weeks of treatment at a concentration of 10 mg. /Kg.

Even more preferably, PF-271 is administered every other day at a concentration of 30 mg/Kg and bevacizumab two days per week of treatment at a concentration of 5 mg/Kg.

References to in vivo treatment methods by therapy in this description should be construed as references to the compounds, pharmaceutical compositions and medicaments of the present invention for use in said methods.

The terms defined and explained for the rest of the aspects of the invention, as well as the preferred embodiments thereof, are also applicable to the treatment method of the invention.

DESCRIPTION OF THE FIGURES

Fig. 1 . Representation of vasculogenic mimicry (VM) in Matrigel after treatment of 1 μM for 24 h of PF-271 in MUM2B cells. Scale: 50 μm.

Fig. 2. A: Representation of the progression of tumor growth on different days. The annotations regarding tumor volume were made every two days, with day 12 post-injection of MUM2B cells being the start of the different treatments. B: Graph representing the final tumor growth with the treatment groups (N=6). C: Representative images and statistical graph of PAS+/CD31- regions of control group vs Bevacizumab+PF-271. Asterisks indicate statistical significance in a Students t test (p<0.05, p<0.01, p<0.001), and error bars indicate standard deviation.

Fig. 3. Representative images of different tumors from the treatment groups.

Fig. 4. Global tumor representation of the different tumors of the post-fixation treatment groups. Scale: 1mm.

EXAMPLES

Next, the invention will be illustrated through tests carried out by the inventors, which demonstrate the effectiveness of the invention.

1. Materials and methods

Cellphone line

MUM 2B metastatic uveal melanoma cells were maintained with RPMI medium ( Roswell Park Memorial Institute) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1% penicillin/streptomycin (PAA laboratories). . The cells were maintained at 37°C and 5% CO ₂ in a cell incubator.

Reagents

The following reagents were used: Avastin/Bevacizumab (Roche) 5 mg/Kg intraperitoneally two days a week, PF-271 1 μM for 24 h in in vitro assays and 30 mg/Kg by oral gavage every two days in tumor xenograft assay.

In vitro vasculogenic mimicry assay

The effect of PF-271 after 24 h on the formation of tube-like structures in Matrigel (BD Biosciences) was determined according to the manufacturer's instructions. Briefly, 96-well plates were coated with 50 μL of BD Matrigel™ Basement Membrane Matrix and allowed to solidify at 37 °C in 5% CO ² for 30 min. After 24 h of incubation, images were acquired using an Olympus CKX41 microscope (4X and 10X lenses). The images of the formation of tubular structures were processed by the Wimasis program.

Tumor xenograft trial

Male Swiss Nude (SwN) mice were purchased from Charles River Laboratories and maintained at the IPBLN-CSIC animal facility in accordance with institutional guidelines (Approved Ethical Committee). For xenograft generation, 2*106 MUM 2B cells in 100 μL of PBS were injected subcutaneously into the flank of 7-week-old mice. Animals (n=6 per group) were monitored every other day after cell injection until the final time point, when they were sacrificed and tumors were dissected for further analysis. Tumor volume was calculated as: ongoing tumor volume = (^*length*width2)/6 and tumor volume end = (^ x length * width * height) /6).

2. Results

In vitro vasculogenic mimicry assay

The inhibitor PF-271 demonstrated a reduction of tube-like structures in Matrigel compared to the control (RPMI medium 10% fetal bovine serum + 2 mM L-glutamine, and 1% penicillin/streptomycin, without any treatment) (Fig. 1) .

Tumor xenogeneic assay

Tumor growth was evaluated for 26 days after MUM 2B inoculation in xenograft models (N=6 per group). The combination of Bevacizumab plus PF-271 globally reduced tumor progression as well as final tumor growth (Fig. 2A, 2B, 3 and 4). The reduction of the PAS+/CD31- regions of the tumors, identifying vasculogenic mimicry, after treatment with the combination of bevacizumab plus PF-271 is represented in Fig 2C.

Claims (14)

1. Pharmaceutical composition comprising PF-271 and bevacizumab, for use in the treatment of uveal melanoma. 2. Pharmaceutical composition for use according to claim 1, wherein the uveal melanoma is a primary uveal melanoma that develops in the iris, choroid and/or ciliary body. 3. Pharmaceutical composition for use according to claim 1, wherein the uveal melanoma is metastatic. 4. Pharmaceutical composition for use according to any one of claims 1 to 3, wherein uveal melanoma further comprises vasculogenic mimicry. 5. Pharmaceutical composition for use according to any one of claims 1 to 4, which further comprises a vehicle and/or a chemotherapeutic agent. 6. Pharmaceutical composition for use according to any one of claims 1 to 5, wherein the pharmaceutical composition is formulated for oral, intraperitoneal, intraocular, or intratumoral administration. 7. Pharmaceutical composition for use according to any one of claims 1 to 6, wherein the concentration of PF-271 is 20 to 60 mg/Kg and the concentration of bevacizumab is 3.5 to 15 mg/Kg. 8. Use of a pharmaceutical composition comprising PF-271 and bevacizumab, to inhibit the growth of uveal melanoma tumor cells in vitro . 9. Use according to claim 8, wherein the uveal melanoma tumor cells are metastatic. 10. Use of a pharmaceutical composition comprising PF-271 and bevacizumab, to reduce PAS+/CD31- regions of a tumor in vitro . 11. Use of a pharmaceutical composition comprising PF-271 and bevacizumab, to inhibit vasculogenic mimicry of a tumor in vitro . 12. Use according to claim 10 or 11, wherein the uveal melanoma tumors. 13. Use according to any one of claims 8 to 12, wherein the pharmaceutical composition further comprises a vehicle and/or a chemotherapeutic agent. 14. Use according to any one of claims 8 to 13, wherein the concentration of PF-271 in the pharmaceutical composition is 0.50 mg/mL to 2.54 mg/mL and wherein the concentration of bevacizumab in the composition pharmaceutical is 0.25 mg/mL to 1 mg/mL.