Method and composition for treatment of cancer

Abstract

To provide a novel effective, uniform and successful treatment strategy for cancer.SOLUTION: Based on the surprising discovery that naloxone selectively induces cytotoxicity in tumor cells compared to non-tumor cells, methods and compositions are provided for administering OGFR locally adjacent to tumor or cancer cells in a patient. These methods and compositions can inhibit the proliferation of tumor cells while sparing surrounding normal cells.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 874,037, filed July 15, 2019, the disclosure of which is incorporated by reference in its entirety.

[0002] Technical Field The present invention relates to methods and compositions for treating cancer. [Background technology]

[0003] Cancer is a group of diseases involving abnormal cell growth that can invade or spread to other parts of the body. In 2015, approximately 90.5 million people were affected by cancer. Approximately 14.1 million new cases occur each year (excluding non-melanoma skin cancers), and cancer causes approximately 8.8 million deaths annually, or about 15.7% of all deaths. Collectively, cancer is the second leading cause of death in the United States today. The risk of cancer increases significantly with age, and many cancers occur more commonly in developed countries.

[0004] Cancer is believed to exhibit six characteristics that underlie the ability of cancer cells to form tumors and progress to a malignant state that allows cells to proliferate and invade tissues other than their tissue of origin. These six characteristics are cell growth and division in the absence of appropriate signals, continued growth and division even when signals to the contrary are given, evasion of programmed cell death, unlimited numbers of cell divisions, promotion of angiogenesis, invasion of tissues, and formation of metastases.

[0005] Although our understanding of cancer characteristics has improved significantly, there is no fundamental trait or characteristic associated with the broad range of cancers known today that would have enabled effective uniform and successful treatment strategies for cancer. Cancer can be treated by surgery, chemotherapy, radiation therapy, hormonal therapy, and targeted therapy (including immunotherapy such as monoclonal antibody therapy). Treatment options vary depending on the location and grade of the tumor, the stage of the disease, and the patient's general condition.

[0006] Despite a rapidly increasing understanding of the mechanisms underlying cancer development, many, if not most, cancers remain incurable and there remains a great medical need for new cancer treatment strategies. Summary of the Invention

[0007] One embodiment is a method of treating cancer in a subject in need thereof, comprising administering locally an opioid growth factor receptor (OGFR) antagonist to a site of the subject that contains cancer cells. In some embodiments, the OGFR antagonist comprises naloxone, naltrexone, a functional derivative or analog thereof, or a combination thereof. Another embodiment is a composition for use in a method of treating cancer in a subject in need thereof, comprising administering locally an opioid growth factor receptor (OGFR) antagonist to a site of the subject that contains cancer cells.

[0008] In some embodiments, the cancer comprises soft tissue non-osteogenic sarcoma, bone sarcoma, osteosarcoma, Ewing's sarcoma, chondrosarcoma, benign bone tumor, basal cell carcinoma, melanoma, thyroid cancer, glial blastoma, pituitary tumor, oligodendrocytoma, bladder cancer, triple negative breast cancer, breast cancer, neuroblastoma, or astrocytoma. In some embodiments, the OGFR antagonist is administered at a local dosage of about 1 mM to about 10 mM.

[0009] In one embodiment, the OGFR antagonist is administered locally by intratumoral injection.

[0010] In another embodiment, the OGFR antagonist is administered with a pharma- ceutically acceptable carrier. In some embodiments, the pharma- ceutically acceptable carrier comprises collagen sponge, powdered collagen, or collagen-based gelatin hydrogel. In some embodiments, the pharma- ceutically acceptable carrier comprises a hydrogel-based carrier. In one particular embodiment, the pharma- ceutically acceptable carrier is a collagen sponge. In some embodiments, the hydrogel-based carrier comprises poly-(butyric acid) / poly-(ethylene glycol) / poly-(butyric acid) hydrogel, poly-(ethylene glycol) / poly-(butyric acid) / poly-(ethylene glycol) hydrogel, or a combination thereof. In one particular embodiment, the hydrogel-based carrier is poly-(ethylene glycol) / poly-(butyric acid) / poly-(ethylene glycol).

[0011] In some embodiments, the OGFR antagonist is administered with a diluent, where the diluent enhances the solubility of the OGFR antagonist. In some embodiments, the diluent comprises a polar organic solvent. Preferably, the polar organic solvent is DMSO, including a dimethylsulfoxide (DMSO) based saline solution. In another embodiment, the solubility enhancing diluent is an acidified saline solution, an alcohol, a polyol, an alkane, a fatty acid, an ester, an amine, an amide, a terpene, a cyclodextrin, or a surfactant. In one embodiment, the diluent comprises a dimethylsulfoxide (DMSO) based saline. In some embodiments, the DMSO based saline comprises about 5% volume / volume (v / v) DMSO to about 80% v / v DMSO. In some embodiments, the DMSO based saline comprises about 50% v / v DMSO. In some embodiments, the acidified saline based solution exhibits a pH of about 4.5 to about 7.4. [Brief description of the drawings]

[0012] [Figure 1] Graph showing lactate dehydrogenase (LDH) expression with the opioid growth factor receptor (OGFR) antagonist naloxone in triple negative breast cancer cells (BT474), osteosarcoma cells (SaOS2), and breast cancer cells (BT20) compared to non-tumor cells. LDH expression is shown relative to LDH release upon naloxone treatment of non-tumor cells. [Diagram 2] 1 shows a graph depicting the change in cell number of triple-negative breast cancer cells (BT474) compared to normal non-tumor breast epithelial (HUMEC) cells and normal vascular endothelial (HUVEC) cells in response to increasing doses of naloxone. [Diagram 3]Figure 3 shows a spheroid assay demonstrating that naloxone (an OGFR antagonist) treatment reduces tumor size in vitro. Figure 3A shows tumor spheroids formed in SaOS2 (osteosarcoma) and BT474 (triple-negative breast cancer) cells in the presence of naloxone treatment at doses of 1 mM, 1.5 mM, 2 mM, 2.5 mM, and 5 mM, and in a no treatment control. Figure 3B shows a graph quantifying the size of spheroids formed in the spheroid assay described in Figure 3A as the percentage change in the percentage of spheroid area from 0 to 120 hours after treatment. Figure 3C shows a graph quantifying the size of spheroids formed in the spheroid assay described in Figure 3A as the percentage change in the percentage of cell area of ​​each spheroid at 120 hours after treatment. [Figure 4] Figure 4 shows the effect of a naloxone formulation on tumor growth in mice implanted with MDA-MB-231 triple-negative breast cancer cells. The formulation was implanted into the tibia of 8-week-old female Cr nude mice. In Figure 4A, tumor volumes were measured with calipers over a 60-day period. Tumor volumes were half as large in the formulation implant group. Regression analysis found that the slopes of the two treatment groups were significantly different (p<0.001). Figure 4B shows an assessment of survival using Kaplan-Meier statistics. Survival was two-fold higher in the formulation treatment group (p<0.0328). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The disclosure herein is based on the surprising discovery that naloxone selectively induces cytotoxicity in tumor cells compared to non-tumor cells.Therefore, the disclosure provides methods and compositions that are useful for inhibiting the proliferation of tumor cells while sparing surrounding normal cells.In some embodiments, the disclosure provides methods and compositions for administering OGFR locally adjacent to tumor or cancer cells of patients.

[0014] Illustrative examples herein show that naloxone had a selective inhibitory effect on the proliferation of breast cancer cells, including osteosarcoma and triple-negative breast cancer cells. In some embodiments, naloxone treatment selectively induced cytotoxicity in tumor cells compared to corresponding non-tumor cells, as identified by increased lactate dehydrogenase (LDH) expression. In some embodiments, naloxone induced a 6-fold increase in LDH expression in osteosarcoma and breast cancer cells compared to non-tumor cells. In some embodiments, naloxone induced a 3-fold increase in LDH expression in triple-negative breast cancer cells. In some embodiments, naloxone treatment selectively inhibited cancer cell proliferation compared to non-cancer cells, as identified by cell counting 72 hours after naloxone treatment. See Figures 1 and 2. In some embodiments, naloxone treatment reduced tumor proliferation of triple-negative breast cancer cells, as identified by spheroid assay. See Figure 3. In some embodiments, the naloxone formulation Nalovent™ inhibited tumor growth in vivo when implanted adjacent to tumors formed by triple-negative breast cancer cells in mice. See FIG. 4.

[0015] Although illustrative examples have shown the use of the methods and compositions disclosed herein to treat osteosarcoma and breast cancer, it is believed that the present disclosure is useful for treating all types of cancer. Generally, cancer refers to a condition in which abnormal cells divide uncontrollably and may invade nearby tissues. There are several main types of cancer. Carcinomas are cancers that develop in the tissues that line or cover the skin or internal organs. Sarcomas are cancers that develop in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemias are cancers that develop in blood-forming tissues, such as the bone marrow, and produce large amounts of abnormal blood cells that invade the blood. Lymphomas and multiple myelomas are cancers that develop from cells of the immune system. Central nervous system cancers are cancers that develop in tissues of the brain and spinal cord. In some embodiments, the cancer is one or more of pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neurological cancer, bone cancer, lymphoma, myeloma, gastrointestinal cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, and melanoma. In some embodiments, the cancer is soft tissue non-osteogenic sarcoma, bone sarcoma, osteosarcoma, Ewing's sarcoma, chondrosarcoma, benign bone tumor, basal cell carcinoma, melanoma, thyroid cancer, glial blastoma, pituitary tumor, oligodendrocytoma, bladder cancer, triple negative breast cancer, breast cancer, neuroblastoma, or astrocytoma.

[0016] definition The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

[0017] Technical and scientific terms used herein have the meanings commonly understood by those skilled in the art unless otherwise defined. Any suitable materials and / or methodologies known to those skilled in the art can be utilized in carrying out the methods described herein.

[0018] As used in the present specification and the appended claims, the singular forms "a," "an," and "the" are used interchangeably and are intended to include the plural forms and their respective meanings unless the context clearly indicates otherwise. Also, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combination when interpreted in the alternative ("or").

[0019] All numerical designations, including ranges, e.g., pH, temperature, time, concentration, amount, and molecular weight, are approximations that vary by 10%, 1%, or 0.1% (+) or (-), as appropriate. It should be understood, although not always explicitly stated, that all numerical designations can be preceded by the term "about." It should also be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0020] The terms "comprising" or "comprises" are intended to mean that the compositions and methods include the recited elements but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination. For example, a composition consisting essentially of the elements defined herein would not exclude other elements that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of" is intended to mean excluding minor amounts of other ingredients and substantial method steps recited in the claims. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0021] As used herein, the term "antagonist" is used interchangeably with "inhibitor" and refers to a substance that blocks or suppresses the activity, function, effect, or expression of a target. In some embodiments, the target is a compound, a protein, a gene, a cell, or a drug. As used herein, the term "expression" refers to the amount produced by a living cell from a target. In some embodiments, an inhibitor suppresses the expression of a target gene or protein. In some embodiments, an inhibitor includes a compound that prevents the binding of another molecule to an enzyme or molecular pump. In some embodiments, an inhibitor is a compound that causes downregulation of an enzyme. In some embodiments, an inhibitor may be a competitive or non-competitive inhibitor.

[0022] As used herein, the term "administering" includes formulations for administration as well as the actual administering, including physical administration by the subject being treated or by another.

[0023] As used herein, "subject," "patient," or "individual" refers to any subject, patient, or individual, and these terms are used interchangeably herein. In this regard, the terms "subject," "patient," and "individual" include mammals, particularly humans. When used in conjunction with "in need thereof," the term "subject," "patient," or "individual" refers to any subject, patient, or individual having or at risk for a particular condition or disorder.

[0024] As used herein, the phrase "therapeutically effective" or "effective" in the context of "dosage" or "amount" refers to a dose or amount that provides the specific pharmacological effect for which the compound is administered. Even if such a dose is considered to be a therapeutically effective amount by those skilled in the art, it is emphasized that a therapeutically effective amount is not always effective to achieve the intended effect in a given subject. For convenience, exemplary dosage amounts are provided herein. Those skilled in the art can adjust such amounts according to the methods disclosed herein to treat a particular subject suffering from a particular condition or disorder. A therapeutically effective amount can vary based on the route of administration and dosage form.

[0025] The term "treat" or "treatment" covers the treatment of cancer as described herein in a subject, such as a human, and includes: (i) inhibiting the cancer, i.e., slowing the progression of the cancer; (ii) mitigating the cancer or disorder, i.e., causing regression of the cancer; (iii) slowing the progression of the cancer; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the cancer. For example, treating cancer includes, but is not limited to, elimination of the cancer or a condition caused by the cancer, remission of the tumor, inhibition of the cancer, or reduction or elimination of at least one symptom of the tumor.

[0026] The term "analog" refers to a compound in which one or more individual atoms or functional groups are replaced with either different atoms or different functional groups, resulting in a compound that generally has similar properties. In some embodiments, an analog refers to a structure that resembles another, but differs in one or two components.

[0027] The term "derivative" refers to a compound formed from a similar starting compound by attaching another molecule or atom to the starting compound. Additionally, a derivative according to the present invention encompasses one or more compounds formed from a precursor compound by the addition of one or more atoms or molecules, or by combining two or more precursor compounds.

[0028] Opioid growth factor receptor (OGFR) antagonists By "opioid growth factor receptor (OGFR) antagonist" is meant a molecule that inhibits, suppresses, or causes the cessation of at least one OGFR-mediated biological activity, such as naloxone or a functional derivative thereof.

[0029] In some embodiments, the OGFR antagonist is an OGFR binding antagonist, ie, a molecule that interferes with, blocks, or otherwise prevents the interaction or binding of met5-ligand (OGF) to OGFR. Met-5 is derived from the prohormone proenkephalin (PENK).

[0030] The OGFR binding antagonist may compete with met5-ligand for binding to OGFR on the surface of the nuclear membrane, thereby disrupting, blocking, or otherwise preventing the binding of met5-ligand to OGFR without triggering downstream signaling otherwise induced by the binding of met5-ligand to OGFR. Alternatively, the OGFR binding antagonist may bind or sequester with proenkephalin (PENK) or met5-ligand, thereby substantially disrupting, blocking, or otherwise preventing the binding of met5-ligand to OGFR, thereby causing the inhibition, suppression, or cessation of at least one OGFR-mediated biological activity. Generally speaking, the OGFR binding antagonist may be a large molecule (e.g., an antibody) or a small molecule (e.g., a compound with a molecular weight of less than 15-kD, 12-kD, 10-kD, or even 8-kD), and may be a polypeptide, a nucleic acid, or a synthetic small molecule compound. The OGFR binding antagonist may be identified in any in vitro assay that is easily selected by one of ordinary skill in the art. For example, OGFR antagonists can be identified using the methods described in US Pat. No. 5,882,944, US Pat. No. 6,007,986, or US Pat. No. 6,270,979.

[0031] In one embodiment, the OGFR binding antagonist is naloxone or a functional derivative thereof, naltrexone or a functional derivative thereof, or a combination thereof.

[0032] As used herein, "functional derivative" refers to a derivative or analogue that is structurally and functionally similar to the original molecule (e.g., maintains the function of naltrexone or naloxone as an OGFR antagonist). Naloxone and naltrexone analogues can be synthesized using standard synthetic procedures, such as those described in March J., Advanced Organic Chemistry, 3rd Ed. (1985). Examples of functional derivatives of naltrexone and naloxone include salt forms, such as naloxone hydrochloride dihydrate or naltrexone hydrochloride. Additional examples of functional derivatives of naltrexone and naloxone suitable for use in the methods of the present invention include, for example, the naltrexone and naloxone analogues disclosed in U.S. Patent Application Publication No. 2007 / 0197573 A1, U.S. Patent No. 6,713,488.

[0033] In another embodiment, the OGFR binding antagonist is derived from oxymorphone and binds to OGFR and includes naloxone, naltrexone, nalorphine, naloxonazine, levallorphan, nalmefene, cyprodime, cyclorphan, cyclazocine, oxilorphan, LY113878, MR2266, diprenorphine, WIN 44,441-3, naltindole, or norbinaltorphimine.

[0034] In yet another embodiment, the OGFR binding antagonist is derived from trans-3,4-dimethyl-4-phenylpiperidine and binds to OGFR including LY99335, LY25506, LY117413, or LY255582.

[0035] In another embodiment, the OGFR binding antagonist is derived from a met5-enkephalin or leu-enkephalin peptide and minimally comprises the following amino acid sequence as a means of binding to and targeting the OGFR: if derived from met5-enkephalin, Tyr-Gly-Gly-Phe-Met (SEQ ID NO: 1), or if derived from leu-enkephalin, Tyr-Gly-Gly-Phe-Leu (SEQ ID NO: 2).

[0036] In yet another embodiment, the OGFR binding antagonist is derived from the peptide antagonist 101174864 (N,N-Diallyl-Tyr-Aib-Aib-Phe-Leu-OH, SEQ ID NO: 3; Aib = aminoisobutyric acid) or the somatostatin analog CTP (D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH.sub.2, SEQ ID NO: 4).

[0037] In other embodiments, the OGFR antagonist is, instead of being an OGFR binding antagonist, a molecule that disrupts the nuclear localization sequence found within OGFR:251 QSALDYFMFAVRCRHQRRQLVHFAWEHFRPRCKFVWGPQDKLRRFKPSSL (SEQ ID NO:5).

[0038] In yet another embodiment, the OGFR antagonist used in the methods of the present invention is a small hairpin RNA (shRNA) or small interfering RNA (siRNA) directed against the OGFR gene and effective to disrupt OGFR gene expression.

[0039] The OGFR antagonists described herein can be administered individually or in combination. Suitable combinations include, for example, naloxone and naltrexone; naloxone and / or naltrexone in combination with another OGFR binding antagonist or another OGFR antagonist.

[0040] Delivery Systems and Carriers for Topical Administration In one aspect of the disclosure, the OGFR antagonist is administered locally adjacent to the tumor site. In some embodiments, the OGFR antagonist is administered at a local dosage of about 1 μM to about 1 mM. In some embodiments, the OGFR antagonist is administered at a local dosage of about 1 mM to about 10 mM. In some embodiments, the local dosage is 1 mM, 1.5 mM, 2.0 mM, 3.0 mM, 4.0 mM, 5.0 mM, 6.0 mM, 7.0 mM, 8.0 mM, 9.0 mM, and / or 10.0 mM.

[0041] In some embodiments, the OGFR antagonist is administered with a carrier, and the volume of the carrier may be about 0.1 cubic centimeters (cc) to about 20 cc, about 0.25 cc to about 15 cc, about 0.5 cc to about 10 cc, about 1 cc to about 10 cc, about 2 cc to about 10 cc. The corresponding amount of OGFR antagonist in the carrier may be about 0.2 mg / cc, 0.5 mg / cc, 1.0 mg / cc, 2.0 mg / cc, 3.0 mg / cc, 4.0 mg / cc, 5.0 mg / cc, 6.0 mg / cc, 7.0 mg / cc, 8.0 mg / cc, 9.0 mg / cc, and / or 10.0 mg / cc.

[0042] In some embodiments, the OGFR antagonist is administered locally by intratumoral injection.

[0043] In some embodiments, the OGFR antagonist is combined with or encapsulated within a carrier for administration.

[0044] Suitable carriers may be in the form of beads, microspheres, or nanoparticles, and can be made of natural and / or synthetic biocompatible polymers. Examples of suitable biocompatible polymers include hyaluronic acid, collagen, tricalcium phosphate, chondroitin sulfate, polybutyrate, polylactide, polyglycolide, and lactide / glycolide copolymers, and mixtures or copolymers thereof. Suitable carriers also include on-polymer systems such as lipids such as carboxylic acids, fatty acids, phospholipids, amino acids, sterols, hydrogel release systems; silastic systems; peptide-based systems; implants, etc.

[0045] In one embodiment, the carrier is a bibulous collagen-based carrier, such as a collagen sponge, a collagen scaffold, powdered collagen, or a collagen-based gelatin hydrogel.

[0046] In another embodiment, the carrier is a hydrophilic hydrogel-based carrier (e.g., polylactic acid, polyglycolic acid), which allows the release of an OGFR antagonist (e.g., naloxone or naltrexone or functional derivatives thereof) injected therein over a period of time.

[0047] In another embodiment, the carrier is a carrier composed of a triblock copolymer containing a central block of PLA (poly-(lactic acid) flanked by two blocks of PEG-(poly-(ethylene glycol)).

[0048] In yet another embodiment, the carrier is albumin, an albumin derivative or fragment that maintains a naloxone / morphine binding site located at the interface between the IA and IIA domains and / or maintains a naloxone binding site around tryptophan (Trp)-214 that binds to an OGFR antagonist, such as naloxone or naltrexone or a functional derivative thereof, allowing for sustained release of the OGFR antagonist.

[0049] In yet another embodiment, it is an inert gel that binds methylcellulose and an OGFR antagonist, such as, for example, naloxone or naltrexone or functional derivatives thereof, allowing for sustained release of the antagonist.

[0050] In a further embodiment, the carrier is a bovine collagen implant.The OGFR antagonist, for example, naloxone or naltrexone or their functional derivatives, can be combined with the bovine collagen implant in a similar manner to either the INFUSE (BMP2) or OP1 (bone morphogenetic protein 1 or BMP7)-putty or OP1-implant that is supplemented with bovine collagen sponge, powdered bovine collagen, or collagen-based gelatin construct.The administration of naloxone, naltrexone or their functional derivatives can be achieved, for example, by reconstituting powdered naloxone or powdered naltrexone or their functional derivatives with sterile saline, and then adding OGFR antagonist-saline to the collagen implant; the implant can then be delivered locally to the surgical intervention site.

[0051] In further embodiments, the carrier is comprised of a cement (polymethylmethacrylate or "PMMA"), or an injectable formulation.

[0052] In another embodiment, the carrier is one comprised of PGA (poly-(glycolic acid)-PLGA (poly-(lactic acid-glycolic acid)) spheres, in which the OGFR antagonist can be encapsulated to provide immediate, delayed, or sustained release.

[0053] Pharmaceutical Compositions and Administration In some embodiments, the pharmaceutical compositions disclosed herein include one or more "pharmaceutical acceptable carriers," such as aqueous carriers, buffers, antioxidants, and / or diluents. In some embodiments, the pharmaceutical compositions include an OGFR antagonist in dimethyl sulfoxide (DMSO)-based saline. In some embodiments, the pharmaceutical compositions include greater than about 100 mM of an OGFR antagonist in dimethyl sulfoxide (DMSO)-based saline. In some embodiments, the pharmaceutical compositions include about 10 mM to about 150 mM of an OGFR antagonist in dimethyl sulfoxide (DMSO)-based saline. In some embodiments, the pharmaceutical compositions include about 20 mM to about 140 mM of an OGFR antagonist in dimethyl sulfoxide (DMSO)-based saline. In some embodiments, the pharmaceutical compositions include about 30 mM to about 130 mM of an OGFR antagonist in dimethyl sulfoxide (DMSO)-based saline. In some embodiments, the pharmaceutical composition comprises about 40 mM to about 120 mM of the OGFR antagonist in dimethyl sulfoxide (DMSO) based saline. In some embodiments, the pharmaceutical composition comprises about 50 mM to about 110 mM of the OGFR antagonist in dimethyl sulfoxide (DMSO) based saline. In some embodiments, the pharmaceutical composition comprises about 50 mM to about 110 mM of the OGFR antagonist in dimethyl sulfoxide (DMSO) based saline. In some embodiments, the pharmaceutical composition comprises more than 100 mM of the OGFR antagonist in dimethyl sulfoxide (DMSO) based saline. In some embodiments, the OGFR antagonist comprises about 100 mM of naloxone, naltrexone, functional derivatives or analogs thereof in dimethyl sulfoxide (DMSO) based saline.

[0054] In some embodiments, the acidified saline solution has a pH of about 4.5 to about 7.4. In some embodiments, the acidified saline solution has a pH of about 5.5 to about 7.4. In some embodiments, the acidified saline solution has a pH of about 6.5 to about 7.4.

[0055] In some embodiments, the diluent is dimethyl sulfoxide (DMSO)-based saline. In some embodiments, the DMSO-based saline comprises about 1% volume / volume (v / v) DMSO to about 80% v / v DMSO. In some embodiments, the DMSO-based saline comprises about 5% volume / volume (v / v) DMSO to about 80% v / v DMSO. In some embodiments, the DMSO-based saline comprises about 10% volume / volume (v / v) DMSO to about 80% v / v DMSO. In some embodiments, the DMSO-based saline comprises about 15% volume / volume (v / v) DMSO to about 80% v / v DMSO. In some embodiments, the DMSO-based saline comprises about 25% volume / volume (v / v) DMSO to about 80% v / v DMSO. In some embodiments, the DMSO-based saline comprises about 30% volume / volume (v / v) DMSO to about 80% v / v DMSO. In some embodiments, the DMSO-based saline contains about 35% volume / volume (v / v) DMSO to about 80% v / v DMSO. In some embodiments, the DMSO-based saline contains about 40% volume / volume (v / v) DMSO to about 80% v / v DMSO. In some embodiments, the DMSO-based saline contains about 40% volume / volume (v / v) DMSO to about 70% v / v DMSO.

[0056] In some embodiments, the DMSO-based saline comprises about 1% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 2% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 3% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 4% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 5% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 6% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 7% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 8% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 9% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 10% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 20% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 30% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 40% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 50% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 60% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 70% volume / volume (v / v) DMSO. In some embodiments, the DMSO-based saline comprises about 80% volume / volume (v / v) DMSO.

[0057] In some embodiments, the dimethyl sulfoxide (DMSO)-based saline comprises phosphate buffered saline, borate buffered saline, Tris buffered saline, or carbonate buffered saline.

[0058] In some embodiments, the saline solution is comprised of salt and water. In some embodiments, the salt in the saline solution comprises sodium chloride or potassium chloride. In some embodiments, the saline solution comprises about 0.7% w / w salt to about 1.5% w / w salt. In some embodiments, the saline solution comprises about 0.7% salt. In some embodiments, the saline solution comprises about 0.8% salt. In some embodiments, the saline solution comprises about 0.9% salt. In some embodiments, the saline solution comprises 1.0% salt. In some embodiments, the saline solution comprises about 1.1% salt. In some embodiments, the saline solution comprises about 1.2% salt. In some embodiments, the saline solution comprises about 1.3% salt. In some embodiments, the saline solution comprises about 1.4% salt. In some embodiments, the saline solution comprises about 1.5% salt.

[0059] The OGFR antagonist can be administered in combination or in coordination with a suitable carrier or vehicle depending on the route of administration. The term "pharmaceutical acceptable carrier" refers to a carrier that is conventionally used in the art to facilitate the storage, administration, and / or therapeutic effect of an active agent in a pharmaceutical composition.

[0060] Aqueous liquid carriers may contain pharma- ceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffers, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending agents and / or thickening agents, tonicity agents, wetting agents or other biocompatible materials. Tables of ingredients listed in the above categories can be found in the United States Pharmacopeia National Formulary, 1857-1859, and (1990). Some examples of materials that function as pharma- ceutically acceptable carriers include sugars (such as lactose, glucose, and sucrose); cyclodextrins, including alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin; starches (such as corn starch and potato starch); cellulose and its derivatives (such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate); powdered tragacanth; malt; gelatin; talc; excipients (such as cocoa butter and suppository wax); oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil). oils, etc.); glycols, such as propylene glycol; polyols (such as glycerin, sorbitol, mannitol, and polyethylene glycol); esters (such as ethyl oleate and ethyl laurate; agar; buffers (such as magnesium hydroxide and aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffers, and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers, lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents, and fragrances.

[0061] In some embodiments, the pharmaceutical composition comprises a preservative and an antioxidant. Examples of pharma-ceutically acceptable antioxidants include water-soluble antioxidants (such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, and sodium sulfite); oil-soluble antioxidants (such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol); and metal chelators (such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid).

[0062] The pharmaceutical composition according to the present invention may also include one or more binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art. Examples of fillers include lactose monohydrate, anhydrous lactose, and various starches; examples of binders include various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose (such as Avicel™, PH101 microcrystalline cellulose, and / or silicified microcrystalline cellulose such as Avicel™, PH102 microcrystalline cellulose, and ProSolv SMCC™). Suitable lubricants, including agents that affect the flowability of the powder to be tableted, may include colloidal silicon dioxide such as Aerosil® 200 (colloidal silicon dioxide), talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweetening agents may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame. Examples of flavoring agents are monoammonium glycyrrhizinate, such as Magnasweet™ (a flavoring composition containing monoammonium glycyrrhizinate and the MAFCO trademark), bubble gum flavor, and fruit flavor. Examples of preservatives include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid, such as butylparaben, alcohols, such as ethyl or benzyl alcohol, phenolic compounds, such as phenol, or quaternary compounds, such as benzalkonium chloride.

[0063] Any pharmaceutical preparation used for therapeutic administration may be sterile.Sterilization can be easily achieved, for example, by filtration through a sterile filtration membrane (for example, 0.2 micron membrane).Any pharmaceutically acceptable sterilization method can be used in the composition of the present invention.

[0064] Pharmaceutical compositions containing an OGFR antagonist derivative or a salt thereof will be formulated and administered in a manner consistent with good medical practice, taking into account the clinical condition of the individual patient, the method of administration, the schedule of administration, and other factors known to the practitioner.

[0065] Various routes of administration can be utilized. The pharmaceutical composition of the present invention can be administered using any mode of administration that is medically acceptable, i.e., any mode of administration that produces an effective level of the active ingredient without causing clinically unacceptable side effects. Thus, the pharmaceutical composition can be administered to a subject parenterally, orally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, buccally, intranasally, intraliposomally, via minicells, via antibody binding, via cell targeting peptides, via inhalation, intravaginally, intraocularly, via local delivery by catheter or stent, subcutaneously, intraadipose, intraarticularly, or intrathecally.

[0066] Modes of administration include oral, rectal, topical, nasal, intradermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term treatment and prophylaxis. Oral administration is used for prophylactic treatment due to patient convenience and dosing schedule.

[0067] Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed 25 oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases. Lower doses would result from other forms of administration, such as intravenous administration. If the subject responds inadequately to the initial dose applied, higher doses (or effectively higher doses via a different, more localized delivery route) can be used, as tolerated by the patient. Multiple doses per day are contemplated to achieve appropriate systemic levels of the compound.

[0068] Compositions suitable for oral administration can be presented as discrete units, such as capsules, tablets, or lozenges, each containing a predetermined amount of the active agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as syrup, elixir, or emulsion.

[0069] Other delivery systems may include time-release, delayed release, or sustained release delivery systems. Such systems can avoid repeated administration of the pharmaceutical composition of the present invention, enhancing the convenience of the subject and the physician. Many types of release delivery systems are available and known to those skilled in the art. They include polymer-based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the aforementioned polymers containing drugs are described, for example, in U.S. Pat. No. 5,075,109. Delivery systems also include non-polymeric systems such as: lipids including sterols such as cholesterol, cholesterol esters, fatty acids, or neutral fats such as monoglycerides, diglycerides, and triglycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.

[0070] The methods and compositions herein may be provided in the form of a kit. A "kit" is defined herein as a package, which includes several individual parts that exhibit complementary effects when applied together. In this embodiment, the effects achieved by the kit and the pharmaceutical composition are similar. The kit may optionally include instructions for using the pharmaceutical composition.

[0071] The present invention is further illustrated by the following non-limiting examples. EXAMPLES

[0072] Example 1 - Naloxone exhibits selective cytotoxic effects on tumor cells compared to non-tumor cells. The purpose of this experiment was to measure the antitumor proliferation activity of naloxone solubilized in acidified saline containing 50% (v / v) DMSO, relative to other non-tumor cell phenotypes likely to be located adjacent to tumors. The naloxone formulation of this example is referred to herein as "naloxone."

[0073] The effect of naloxone treatment at doses of 1 mM, 1.5 mM, 2 mM, 2.5 mM, and 5 mM on lactate dehydrogenase (LDH) in triple-negative breast cancer cells (BT474), osteosarcoma cells (SaOS2), and breast cancer cells (BT20) was compared with non-tumor breast epithelial (HUMEC) cells and normal vascular endothelial (HUVEC) cells, as shown in Figure 1. Naloxone was added for 3 h, and LDH release was evaluated 24 h after treatment. At 24 h, a significant increase in LDH, corrected for changes in cell number caused by the treatment, was seen in triple-negative breast cancer cells (BT474), osteosarcoma cells (SaOS2), and breast cancer cells (BT20) compared with normal cells. As shown in Figure 1, a six-fold increase in LDH values ​​was observed in SaOS2 and BT20 cells, and a three-fold increase in cellular LDH values ​​was observed in BT474 (*p<0.001). At the highest dose, cells were reduced in number by 60% in SaOS2 cells, while BT20 and 90 cell numbers were reduced by 90% after 72 hours. See Figure 2.

[0074] As seen in Figure 1, exposure of triple-negative breast cancer (TNBC) cells BT474 for 72 hours significantly reduced the number of metabolically active cells in a dose-dependent manner compared to control non-tumor cells, breast epithelial (HUMEC) cells or vascular endothelial (HUVEC) cells. These data indicated that naloxone exhibited significant anti-tumor activity and may provide a non-tumor cell sparing effect up to doses of 2.5 mM.

[0075] It was also demonstrated that naloxone treatment reduced tumor size in vitro. Malignant tumors are characterized by invasion into surrounding normal tissues, and the spheroid assay is a simple microplate method (based on uniform self-organizing 3D tumor spheroids) that is routinely used to evaluate tumor growth. The antitumor effect of naloxone was evaluated using the spheroid assay, as shown in Figure 3. The antitumor effect was determined by quantifying the size of the spheroids formed in vitro and calculating the percentage change in spheroid area from time 0 to 120 h after treatment, as shown in Figure 3B. The antitumor effect was also determined by quantifying the percentage change in the cellular area fraction of each spheroid at 120 h after treatment, as determined by staining the cells with crystal violet, as shown in Figure 3C. The results shown in Figure 3 indicate that naloxone dose-dependently reduced the tumor mass size and tumor mass cellularity of SaOS2 osteosarcoma cells and BT474 TNBC cells.

[0076] Thus, the results herein demonstrated that naloxone has a selective cytotoxic effect on tumor cells while sparing non-tumor cells. Furthermore, the results demonstrated that naloxone can inhibit tumor cell growth in vitro.

[0077] Example 2 - This example demonstrates that local administration of a formulation of 50% DMSO (50%) in approximately 0.9% saline in a collagen sponge adjacent to a growing tumor effectively inhibits tumor growth in vivo and enhances survival in mice inoculated with breast cancer.

[0078] To study the effect of this formulation on in vivo tumor growth, MDA-MB-231 triple-negative breast cancer cells were implanted into female NCr nude mice. The formulation was implanted into the mice 3 weeks after inoculation of MDA-MB-231 triple-negative breast cancer cells in mice with tumors confirmed to be actively growing. The formulation was formulated by dissolving naloxone in acidified saline containing 50% (v / v) DMSO in a collagen gel-based carrier. The formulation was implanted into the tibia of 8-week-old female NCr nude mice bearing actively growing tumors formed by implanted MDA-MB-231 triple-negative breast cancer. Figure 4 shows the effect of the formulation on tumor growth in mice implanted with MDA-MB-231 triple-negative breast cancer cells. In Figure 4A, tumor volumes are shown measured with calipers over a 60-day period. Tumor volumes were half as large in the formulation implant group. Regression analysis found that the slopes of the two treatment groups were significantly different (p<0.001). Figure 4B shows the assessment of survival using Kaplan-Meier statistics. Survival was 2-fold higher in the formulation-treated group (p<0.0328).

[0079] All references cited herein are specifically incorporated by reference in their entirety to the same extent as if individually incorporated. While preferred embodiments have been illustrated and described, it will be understood that changes and modifications can be made according to those skilled in the art without departing from the invention in its broader aspects defined herein.

[0080] Example 3 - Local injection of OGFR antagonists directly into or adjacent to the tumor inhibits tumor growth in human patients. This example demonstrates that delivery of an OGFR antagonist directly to or adjacent to a tumor can inhibit tumor growth in human patients suffering from cancer.

[0081] The OGFR antagonist is administered to a human patient suffering from a cancer, such as breast cancer, liver cancer, lung cancer, skin cancer, brain cancer, or prostate cancer. The OGFR antagonist is administered as a pharmaceutical formulation of naloxone, dimethyl sulfide, saline, a pharma- ceutically acceptable excipient, and a hydrogel-based carrier, as described herein. The volume of the pharmaceutical formulation is between 0.25 cubic centimeters (cc) and 10 cc; or between 10% and 150% of the tumor volume. The dose of the OGFR antagonist naloxone is between 2 milligrams (mg) per cc of carrier and about 8 mg per cc of carrier. In particular, the dose of the OGFR antagonist naloxone is 2 mg per cc of carrier, 4 mg per cc of carrier, or 8 mg per cc of carrier. The amount of carrier and the amount of OGFR antagonist vary depending on the type of cancer and the delivery method, as described below.

[0082] In some patients, the pharmaceutical preparation comprising OGFR antagonist is adapted for injection.The injectable OGFR antagonist composition is directly injected into solid primary or metastatic tumor.For some patients suffering from lung cancer, the pharmaceutical preparation comprising OGFR antagonist is adapted for inhalation, and the pharmaceutical preparation comprising OGFR antagonist is delivered via inhalation.

[0083] In liver patients, the pharmaceutical preparation containing the OGFR antagonist is injected into the viscera and deep tissue of the liver of patients suffering from liver cancer using fluoroscopically guided injection. Alternatively, to treat patients with liver cancer, the pharmaceutical preparation containing the OGFR antagonist is delivered to the tumor via local perfusion via a catheter. Furthermore, the liver tumor is removed, and the pharmaceutical preparation containing the OGFR antagonist is delivered to the tissue layer adjacent to the tumor site to control the recurrence of the tumor.

[0084] The pharmaceutical preparation containing the OGFR antagonist is also used for the treatment of lung cancer. The pharmaceutical preparation containing the OGFR antagonist is injected into the visceral and deep tissues of the lung of a patient suffering from lung cancer. Thus, in a patient suffering from lung cancer, the pharmaceutical preparation containing the OGFR antagonist is injected directly into or adjacent to the tumor during tumor resection or lobectomy. The pharmaceutical preparation containing the OGFR antagonist is optionally adapted for inhalation and is delivered to a patient suffering from lung cancer via inhalation.

[0085] In patients with brain tumors, a pharmaceutical formulation containing an OGFR antagonist is injected during tumor resection or administered via a port or catheter before or after tumor resection.

[0086] The pharmaceutical formulation containing the OGFR antagonist is also used to treat skin cancer. The pharmaceutical formulation containing the OGFR antagonist is injected into or adjacent to a skin tumor to treat a patient suffering from skin cancer. In patents where the tumor is a skin tumor located on the skin, the skin tumor is treated by tumor scraping or tumor excision, after which the pharmaceutical formulation containing the OGFR antagonist is delivered via conventional means that do not involve injection (i.e., hand placement and / or hand packing into the excision). During an incisional biopsy or partial excision of a skin tumor, such as melanoma or squamous cell carcinoma or basal cell carcinoma, the pharmaceutical formulation containing the OGFR antagonist is injected into the excision space and / or adjacent to the tumor. To treat skin cancer, the volume of the pharmaceutical composition containing the OGFR antagonist is between 0.25cc and 2.5cc, or between 10% and 150% of the tumor volume. The dose of the OGFR antagonist naloxone is 2 milligrams (mg) per cc of carrier to about 8 mg per cc of carrier. In one patient with skin cancer, the corresponding dose of the OGFR antagonist naloxone is 2 mg per cc of carrier. In another patient with skin cancer, the corresponding dose of the OGFR antagonist naloxone is 4 mg per cc of carrier. In another patient with skin cancer, the corresponding dose of the OGFR antagonist naloxone is 8 mg per cc of carrier.

[0087] To treat breast tumors, the pharmaceutical formulation containing the OGFR antagonist is delivered directly to the breast tumor during a wire or needle localization procedure (i.e., stereotactic wire localization), in which a thin, hollow needle is guided to the tumor using a mammogram or ultrasound. In this procedure, a portion of breast tissue is removed for biopsy, and then a second needle containing the preferred formulation is injected into and adjacent to the tumor mass. To treat breast cancer, the volume of the pharmaceutical composition containing the OGFR antagonist is between 1 cc and 10 cc, or between 10% and 150% of the tumor volume. The dose of the OGFR antagonist naloxone is between 2 milligrams (mg) per cc of carrier to approximately 8 mg per cc of carrier. For a single patient suffering from breast cancer, the corresponding dose of the OGFR antagonist naloxone is 2 mg per cc of carrier. In another breast cancer patient, the corresponding dose of the OGFR antagonist naloxone is 4 mg per cc of carrier. In another breast cancer patient, the corresponding dose of the OGFR antagonist naloxone is 8 mg per cc of carrier.

Claims

1. A method of treating cancer in a subject in need thereof comprising locally administering an opioid growth factor receptor (OGFR) antagonist to a site in the subject containing cancer cells.

2. 2. The method of claim 1, wherein the OGFR antagonist comprises naloxone, naltrexone, a functional derivative or analog thereof, or a combination thereof.

3. 3. The method of claim 1 or 2, wherein the cancer comprises soft tissue non-osteogenic sarcoma, bone sarcoma, osteosarcoma, Ewing's sarcoma, chondrosarcoma, benign bone tumor, basal cell carcinoma, melanoma, thyroid cancer, glial blastoma, pituitary tumor, oligodendroglioma, bladder cancer, triple negative breast cancer, breast cancer, neuroblastoma, or astrocytoma.

4. The method of any one of claims 1 to 4, wherein the OGFR antagonist is administered at a local dosage of about 1 mM to about 10 mM.

5. The method of any one of claims 1 to 4, wherein the OGFR antagonist is administered locally by intratumoral injection.

6. The method of any one of claims 1 to 5, wherein the OGFR antagonist is administered with a pharma- ceutically acceptable carrier.

7. 7. The method of claim 6, wherein the pharma- ceutically acceptable carrier comprises a collagen-based carrier in the form of a collagen sponge, powdered collagen, or a collagen-based gelatin hydrogel.

8. The method of claim 7 , wherein the pharma- ceutically acceptable carrier is the collagen sponge.

9. The method of claim 6 , wherein the pharma- ceutically acceptable carrier comprises a hydrogel-based carrier.

10. 10. The method of claim 9, wherein the hydrogel-based carrier comprises a poly-(butyric acid) / poly-(ethylene glycol) / poly-(butyric acid) hydrogel, a poly-(ethylene glycol) / poly-(butyric acid) / poly-(ethylene glycol) hydrogel, or a combination thereof.

11. The method of claim 10, wherein the hydrogel-based carrier is poly-(ethylene glycol) / poly-(butyric acid) / poly-(ethylene glycol).

12. The method of any one of claims 1 to 11, wherein the OGFR antagonist is administered with a diluent, wherein the diluent enhances the solubility of the OGFR antagonist.

13. 13. The method of claim 12, wherein the diluent comprises a dimethylsulfoxide (DMSO) based saline solution or an acidified saline solution.

14. 14. The method of claim 13, wherein the diluent is a dimethylsulfoxide (DMSO)-based saline solution.

15. 15. The method of claim 14, wherein the DMSO-based saline comprises from about 1% volume / volume (v / v) DMSO to about 80% v / v DMSO.

16. 15. The method of claim 14, wherein the DMSO-based saline comprises about 50% v / v DMSO.

17. The method of claim 13, wherein the acidified saline-based solution exhibits a pH of about 4.5 to about 7.

4.

18. 20. A composition for use in a method of treating cancer in a subject in need thereof comprising locally administering an opioid growth factor receptor (OGFR) antagonist to a site in the subject containing cancer cells.

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