Compositions and methods for treating solid cancer

Abstract

Compositions and methods for treating solid cancer are provided. In particular, the present disclosure provides compositions comprising a halogen peroxidase and methods comprising administering such compositions for the treatment of solid cancer.

Description

Background Art

[0001] The present disclosure generally relates to compositions and methods for treating solid cancers. Specifically, the present disclosure provides compositions comprising haloperoxidases for treating solid cancers and methods of using such compositions.

[0002] There is no doubt that cancer has and will continue to have a significant impact on society worldwide. According to current statistics from the United States National Institutes of Health, an estimated 1,806,590 new cases of cancer will be diagnosed in the United States in 2020, and 606,520 people will die from the disease (https: / / www.cancer.gov / about-cancer / understanding / statistics).

[0003] Cancer is a disease characterized by uncontrolled cell growth that can occur in almost any part of the body. Neoplasia refers to uncontrolled cell growth that occurs in solid tissues such as organs, muscles, or bones. In this regard, the majority of the most common cancers are solid neoplastic cancers, such as breast cancer, lung and bronchial cancer, prostate cancer, colon and rectal cancer, melanoma of the skin, bladder cancer, kidney and renal pelvis cancer, endometrial cancer, cervical cancer, pancreatic cancer, thyroid cancer, and liver cancer. Solid cancers include sarcomas, carcinomas, and lymphomas.

[0004] Cancer survival rates continue to improve with the development of improved treatments, but cancer mortality remains high—158.3 deaths per 100,000 men and women per year in the United States (based on deaths from 2013-2017). Therefore, improved cancer treatments are the subject of ongoing research and development in the scientific community.

[0005] For most solid cancers, diagnosis is made by biopsy of abnormal tissue. Surgery to remove as much of the tumor as possible (commonly known as debulking) may be a treatment option. Debulking can also increase the effectiveness of subsequently administered anti-cancer treatments (e.g., immunotherapy, chemotherapy, and / or radiotherapy). However, surgical interventions in cancer treatment (whether biopsy or debulking) are not without risks. In addition to uncontrolled regeneration, cancer cells lose the cohesion and organization of normal tissues and may break away from the primary tumor during a biopsy or surgery and metastasize to other parts of the body through the circulatory and lymphatic systems. Therefore, during a biopsy or surgical intervention, cancer spread (i.e., metastasis) poses a significant risk to cancer patients.

[0006] Some solid cancers, such as bladder cancer, brain cancer, or spinal cord cancer, can be difficult to biopsy and / or treat (with or without surgery) due to the lack of access to the tumor growth site. As a result, these cancers can result in high mortality rates for patients.

[0007] There is a need to minimize solid cancer metastasis and / or improve the treatment of "hard to reach" solid cancers during biopsy or surgery.

[0008] New therapeutic agents are particularly needed in controlling non-muscle invasive bladder cancer (NMIBC). The current scheme for controlling NMIBC generally involves endoscopic resection of the tumor, followed by intravesical treatment with cytotoxic chemotherapeutic agents (e.g., gemcitabine, mitomycin, docetaxel) or BCG (bacillus Calmette-Guérin) acting as an activator of local immune response. The necessary condition for the activity of these agents is the direct surface contact between the therapeutic agent and the residual cancer cells. However, these treatments have several disadvantages, including the efficacy limitations as shown by the relatively high recurrence rate, non-selective mechanism of action (which can cause cytotoxicity and related morbidity to normal epithelium) and the possibility of systemic side effects. In addition, due to the special processing requirements of these agents, application is usually carried out in a clinical setting, and repeated clinic visits are required, resulting in financial and other burdens on patients and clinical providers. These limitations highlight the need for new therapeutic agents in the treatment of NMIBC, which not only enhance therapeutic efficacy and reduce morbidity, but also have a safety feature that may potentially allow family administration.

[0009] Myeloperoxidase (MPO) is a cationic enzyme secreted by granulocytic leukocytes that has peroxidase and haloperoxidase activities and is therefore able to generate highly reactive oxidants. - ) in the presence of MPO, which catalyzes the oxidation of chloride to produce highly active hypochlorous acid (OCl - ). Hypochlorous acid can react with a second H2O2 to produce electronically excited singlet molecular oxygen ( 1 O2*). OCl - and 1 O2* has significant cytotoxic activity (not involving free radical activity). The appeal of using MPO as an antitumor agent in bladder cancer lies primarily in its selective binding ability and its local mechanism of action. Due to its cationic surface properties, MPO has a strong affinity for targets with anionic characteristics, as has been demonstrated for bladder cancer cells. Again due to their anionic surface characteristics, combined 1The millisecond half-life of O2*, the ability of MPO to avidly and selectively bind to bladder cancer cells limits the "kill zone" to a small radius of less than half a micron, enabling selective cytotoxic activity against bladder tumor cells bound to the enzyme while sparing normal adjacent urothelial cells, which have a more neutral electrostatic profile. In addition, intravesical administration of MPO and related agents will cause minimal systemic toxicity due to the large molecular weight of MPO (144 kDa) and rapid inactivation by catalase in the circulation and within the reticuloendothelial system.

[0010] Brief description of the invention

[0011] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0012] The present disclosure is based on the surprising and unexpected discovery that compositions containing haloperoxidases, including myeloperoxidase compositions, exhibit anti-cancer properties and have less toxic effects on normal cells.

[0013] According to one aspect of the present disclosure, there is provided a method of treating a solid cancer in a patient, the method comprising administering to the patient an effective amount of a pharmaceutical composition comprising a haloperoxidase.

[0014] In another aspect, the present disclosure provides a method of treating solid cancer in a patient, comprising administering to the patient an effective amount of a composition comprising a haloperoxidase, a halide, a peroxide, or a peroxide-generating oxidase and a substrate for the oxidase and a pharmaceutically acceptable carrier.

[0015] In another aspect, the present disclosure provides a method of treating solid cancer in a patient, comprising administering to the patient an effective amount of a composition comprising a haloperoxidase, a halide, a peroxide or a peroxide-generating oxidase and a substrate for the oxidase, one or more amino acids and a pharmaceutically acceptable carrier.

[0016] In another aspect, the present disclosure provides a method for treating solid cancer in a patient, the method comprising administering to the patient an effective amount of a first composition and a second composition, wherein the first composition comprises a haloperoxidase, a halide, one or more amino acids and a pharmaceutically acceptable carrier, and the second composition comprises hydrogen peroxide. The first composition and the second composition can be administered simultaneously or sequentially.

[0017] In another aspect, the present disclosure provides a method for treating solid cancer in a patient, the method comprising administering to the patient an effective amount of a first composition and a second composition, the first composition comprising a haloperoxidase, a halide, one or more amino acids, an oxidase that produces peroxides, and a pharmaceutically acceptable carrier, the second composition comprising a substrate for the oxidase that produces peroxides. The first composition and the second composition can be administered simultaneously or sequentially.

[0018] In yet another aspect, the present disclosure provides a composition for treating solid cancer in a patient, the composition comprising a haloperoxidase, a halide, a peroxide or a peroxide-generating oxidase and a substrate for the oxidase, and a pharmaceutically acceptable carrier.

[0019] In yet another aspect, the present disclosure provides a composition for treating solid cancer in a patient, the composition comprising a haloperoxidase, a halide, a peroxide or a peroxide-generating oxidase and a substrate for the oxidase, one or more amino acids and a pharmaceutically acceptable carrier.

[0020] In yet another aspect, the present disclosure provides a composition for treating a solid cancer in a patient, the composition consisting essentially of a haloperoxidase, a halide, a peroxide, one or more amino acids, and a pharmaceutically acceptable carrier.

[0021] In yet another aspect, the present disclosure provides a combination for treating a solid cancer in a patient, the combination comprising a haloperoxidase, a halide, a peroxide, one or more amino acids, and a pharmaceutically acceptable carrier.

[0022] In yet another aspect, the present disclosure provides a combination for treating a solid cancer in a patient, the combination comprising a haloperoxidase, a halide, a peroxide-generating oxidase and a substrate for the oxidase, one or more amino acids, and a pharmaceutically acceptable carrier.

[0023] In yet another aspect, the present disclosure provides a composition for treating solid cancer in a patient, the composition comprising a haloperoxidase, and optionally one or more of a halide, ethanolamine, peroxide, or a peroxide-generating oxidase and a substrate for the oxidase, and a pharmaceutically acceptable carrier.

[0024] In yet another aspect, the present disclosure provides a composition for treating solid cancer in a patient, the composition consisting essentially of a haloperoxidase, and optionally one or more of a halide, ethanolamine, peroxide or a peroxide-generating oxidase and a substrate for the oxidase, and a pharmaceutically acceptable carrier.

[0025] In an embodiment, the haloperoxidase is selected from the group consisting of: myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), chloroperoxidase (CPO), functional derivatives thereof, and combinations thereof.

[0026] In certain embodiments, the haloperoxidase is eosinophil peroxidase.

[0027] In certain embodiments, the haloperoxidase is myeloperoxidase.

[0028] In embodiments, the haloperoxidase catalyzes the oxidation of halides and the dismutation of peroxides to produce singlet molecular oxygen, resulting in one or more of the following effects: inhibition of cancer cell growth, inhibition of cell metastasis, and / or cancer cell death.

[0029] In an embodiment, the solid cancer is selected from the group consisting of breast cancer, lung and bronchial cancer, prostate cancer, colon and rectal cancer, skin melanoma, bladder cancer, kidney and renal pelvis cancer, endometrial cancer, cervical cancer, pancreatic cancer, thyroid cancer, liver cancer, brain cancer, and spinal cord cancer.

[0030] In certain embodiments, the solid cancer is bladder cancer.

[0031] Other embodiments of the invention will become apparent from the following detailed description of various aspects of the invention.

[0032] Detailed description

[0033] definition

[0034] The terms "a," "an," "said" or similar referents used in the context of describing the present inventive concepts (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0035] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0036] The use of any and all examples or exemplary language (eg, "such as") provided herein is intended only to provide better illustration and does not constitute a limitation on the scope of the disclosure. No language in the specification should be construed as indicating any non-protected element is essential.

[0037] Unless otherwise indicated, all numbers used in the specification and claims expressing quantities of ingredients, reaction conditions, and the like are to be understood as being modified in all instances by the term "about". Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and claims are approximate values ​​that may vary depending upon the desired properties sought to be obtained by the present disclosure. At a minimum, each numerical parameter should be interpreted in light of the number of significant figures and ordinary rounding conventions. The term "about" may be understood to refer to a range of + / -10%, for example, a range of + / -5% or + / -1% or + / -0.1%.

[0038] The listing of numerical ranges herein is intended merely to serve as a shorthand method of individually referring to each individual value within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually listed herein. For example, if a range is from about 1 to about 50, it is considered to include, for example, 1, 7, 34, 46.1, 23.7, or any other value or range within that range.

[0039] The terms "protein" and "polypeptide" are used interchangeably herein. The 3-letter code for amino acids as defined by the IUPAC-IUB Joint Commission on Biochemical Nomenclature is used in this disclosure. It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0040] Reference is made herein to "enzymes", such as haloperoxidase or glucose oxidase. In this context, an enzyme is a protein / polypeptide that acts as a catalyst to produce a specific biochemical reaction. Enzymes included within the scope of the present disclosure include enzymes isolated from natural sources (having an unmodified amino acid sequence identical to the amino acid sequence found in nature) and "functional derivatives" thereof.

[0041] The term "haloperoxidase" refers to an enzyme that catalyzes the hydrogen peroxide-dependent oxidation of a halide to produce hypohalous acid; this hypohalous acid can react with additional hydrogen peroxide to generate singlet molecular oxygen. The haloperoxidase according to the present disclosure may also be referred to as a halide: hydrogen peroxide oxidation-reductase (e.g., International Union of Biochemistry EC No. 1.11.1.7 and EC No. 1.11.1.10), for which the halide (e.g., chloride or bromide) is an electron donor or reductant and hydrogen peroxide is an electron acceptor or oxidant. Suitable haloperoxidases include myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), chloroperoxidase (CPO), functional derivatives thereof, and combinations thereof. The haloperoxidase can be obtained from any source (including human and non-human animal sources) or cell culture.

[0042] A "derivative" of an enzyme of the present disclosure generally retains the characteristic enzyme activity observed in the wild-type, native or parent form to the extent that the derivative is effective for a similar purpose as its wild-type, native or parent form.

[0043] When used in the context of the enzymes of the present disclosure, the term "functional fragment" or "functional derivative" includes naturally occurring, synthetically or recombinantly produced nucleic acids or fragments, and encodes an enzyme of the present disclosure having the functional properties of a natural, unmodified parent enzyme. "Functional derivatives" may include "substitution variants", which are variants in which at least one amino acid residue in the native sequence is removed and inserted into the same position by a different amino acid. The substitution may be a single substitution, in which only one amino acid is substituted in the molecule; or it may be a multiple substitution, in which the same molecule has more than two amino acids substituted. Multiple substitutions may be located in consecutive positions. Similarly, amino acids may be substituted by multiple residues, including substitutions and insertions. "Insertion variants" are variants in which one or more amino acids are inserted into an amino acid in the native sequence adjacent to a specific position. Adjacent to an amino acid refers to being connected to it through the α-carboxyl or α-amino functional group of the amino acid. "Deletion variants" are variants in which one or more amino acids in the native amino acid sequence are removed. Typically, deletion variants have one or two amino acids deleted in a specific region of their molecule.

[0044] The terms "isolated" or "purified" refer to the removal of a substance from its original environment (e.g., natural environment, if naturally occurring). For example, a substance is said to be "purified" when it is present in a particular composition at a higher concentration than in a naturally occurring or wild-type organism, or is combined with components not normally present when expressed from a naturally occurring or wild-type organism. For example, a naturally occurring protein / polypeptide present in a living organism is not "isolated", but the same protein / polypeptide is "isolated" when it is separated from some or all of the coexisting materials in the natural system. For example, such a protein / polypeptide can be part of a composition and still be isolated when such composition is not part of the natural environment of the protein / polypeptide.

[0045] As used herein, the term "pharmaceutically acceptable" refers to a substance that does not cause substantial adverse allergic or immune response when administered to a patient. "Pharmaceutically acceptable carriers" include, but are not limited to, solvents, coating agents, dispersants, wetting agents, isotonic and absorption delaying agents, and disintegrants.

[0046] As used herein, "treat," "treating," or "treatment" of a disease, condition, or disorder refers to achieving one or more of the following: (a) reducing severity and / or shortening duration; (b) limiting or preventing the development of characteristic symptoms; (c) inhibiting worsening of symptoms; (d) limiting or preventing recurrence; and (e) limiting or preventing recurrence of symptoms. That is, the term includes preventive or preparatory treatments (which prevent and / or slow the development of the target pathological disease, condition, or disorder) and curative, therapeutic, or disease-modifying treatments, including treatments that cure, slow, alleviate symptoms, and / or arrest progression of the disease, condition, or disorder; and treatment of patients at risk of or suspected of having the disease and patients who are ill or have been diagnosed with the disease, condition, or disorder. These terms do not necessarily mean that the patient is treated until full recovery. These terms may also refer to maintaining and / or promoting health in individuals who do not have the disease but may be susceptible to developing an unhealthy state. These terms may also include strengthening or otherwise enhancing one or more primary prevention or treatment measures. As non-limiting examples, treatment may be performed by the patient, a caregiver, a physician, a nurse, or other healthcare professional.

[0047] As used herein, "prevent", "preventing", "prevention" or "prophylaxis" of a disease or disorder refers to preventing the disorder from occurring in a patient. "Prevent" includes reducing the risk, incidence and / or severity of a disease, condition or disorder.

[0048] As used herein, the expressions "is for administration" and "is to be administered" have the same meaning as "is prepared for administration". In other words, the expression "is for administration" of an active compound should be understood as meaning that the active compound has been formulated and formed into a dosage form so that the active compound is in a state in which it can exert its therapeutic activity.

[0049] The term "effective amount" or "therapeutic amount" means the amount of a substance that elicits a desired biological or medical response in a tissue, system, animal or human. The term "prophylactically effective amount" means the amount of a drug that prevents or reduces the risk of a biological or medical event in a tissue, system, animal or human.

[0050] The terms "comprise", "comprises" or "comprising", "including" or "having" and the like in this specification and claims are used in an open sense, that is, to specify the presence of stated features but not to exclude the presence of additional or further features.

[0051] The specific embodiments disclosed herein may be further limited in the claims using language that expresses "consisting of" or "consisting essentially of." When used in a claim, whether directly filed or added by amendment, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those materials or steps that do not materially affect the basic and novel characteristics. The embodiments of the present disclosure so claimed are inherently or expressly described and enabled herein.

[0052] Compositions containing haloperoxidase

[0053] Haloperoxidases are widely present in nature and are produced by mammals, algae and fungi. US 6,294,168 discloses that haloperoxidases can be used as antimicrobial agents (especially effective against bacteria and fungi) because they selectively bind to target microorganisms and inhibit the growth of target microorganisms in the presence of peroxides and halides. Low concentrations of haloperoxidases are used to maximize the selective binding of target microorganisms without removing the desired microorganisms or causing significant damage to host cells. The selective nature of haloperoxidase binding makes it useful for therapeutic or preventive antimicrobial treatments in humans or non-human patients.

[0054] The present disclosure is based on the surprising and unexpected discovery that compositions containing haloperoxidases exhibit anticancer properties. In one aspect, the present disclosure provides a method for treating solid cancers by contacting solid cancers with compositions containing haloperoxidases. In another aspect, the present disclosure provides compositions for treating solid cancers, the compositions comprising haloperoxidases. In yet another aspect, the present disclosure provides a combination for treating solid cancers, the combination comprising haloperoxidases, and at least one of a halide, or a peroxide, or an oxidase that produces peroxides.

[0055] In certain embodiments, the haloperoxidase catalyzes the oxidation of halides and the dismutation of superoxides to singlet molecular oxygen, treating the cancer by inhibiting cancer cell growth, metastasis and / or by killing cancer cells. In embodiments, suitable haloperoxidases according to the present disclosure include eosinophil peroxidase (EPO), myeloperoxidase (MPO), lactoperoxidase (LPO), chloroperoxidase (CPO), functional derivatives thereof, and combinations thereof.

[0056] In other embodiments, the method of treatment of the present disclosure further comprises administering an effective amount of peroxide or an oxidase that produces peroxide. Optionally, a substrate for the oxidase may be administered. Preferably, the oxidase that produces peroxide is glucose oxidase and the substrate is glucose. In further embodiments, the method further comprises administering the haloperoxidase together with a halide (preferably chloride or bromide).

[0057] In further other embodiments, the haloperoxidase is used in the first composition together with at least one other composition, the other composition comprising one or more of a halogenide, a peroxide or an oxidase that produces peroxide plus the substrate of the oxidase that produces peroxide. Alternatively, the haloperoxidase can be formulated in a composition for use, the composition also comprising one or more of a halogenide, a peroxide or an oxidase that produces peroxide and the substrate of the oxidase that produces peroxide.

[0058] In further other embodiments, described haloperoxidase is used together with at least one other composition in the first composition, described other composition comprises halogenide, ethanolamine, peroxide or the oxidase that produces peroxide adds one or more in the substrate of the oxidase that produces peroxide. As an alternative, described haloperoxidase can be formulated in composition for use, described composition also comprises halogenide, ethanolamine, peroxide or the oxidase that produces peroxide and one or more in the substrate of the oxidase that produces peroxide. As an alternative, described haloperoxidase can be formulated in composition for use, described composition also comprises halogenide, one or more amino acids, peroxide or the oxidase that produces peroxide and one or more in the substrate of the oxidase that produces peroxide.

[0059] In certain embodiments, eosinophil peroxidase (EPO) and myeloperoxidase (MPO) are preferred haloperoxidases used in the disclosed compositions, combinations and methods of treatment. In further embodiments, MPO and EPO are of porcine origin. Preferably, the purified haloperoxidases (porcine MPO and EPO) are those produced by Exoxemis, Inc.

[0060] The porcine MPO preferably has a purity of 98.9% as determined by ultra-high performance liquid chromatography (RP-UPLC) and a purity of 100% as determined by size exclusion high performance liquid chromatography (SEC-HPLC). The guaiacol unit (GU) activity of the porcine MPO is preferably 404 GU / mg, and 1.0 GU of activity consumes 1.0 μmol H2O2 per minute.

[0061] Porcine EPO preferably has a purity of 99.2% as determined by reverse phase high performance liquid chromatography. The guaiacol unit (GU) activity of the porcine EPO is preferably 80 GU / mg.

[0062] Both MPO and EPO are cationic proteins. Without being bound by theory, it is believed that the cationic nature of these haloperoxidases makes them particularly adherent to the anionic surfaces of cancer cells. Therefore, at a suitable pH, the electrostatic attraction and binding of the haloperoxidase to the anionic surfaces of cancer cells (without binding to the surface of normal cells or with minimal binding to the surface of normal cells) leads to the selective binding of the haloperoxidase to the cancer cells, and in the presence of peroxide, selectively kills the cancer cells with little or no damage to the surrounding normal cells.

[0063] The physical properties of haloperoxidase and the optimal conditions for enzyme activity may be different (for example, see US9782459). For example, MPO is about 150kDa and has activity at acidic pH (4.0-6.5), while EPO is about 70kDa and has activity at acidic to neutral pH (that is, 5.0-7.4). In embodiments, the compositions of the present disclosure may include one or more haloperoxidases, wherein the characteristics of the haloperoxidases can match the conditions of the cancer treatment site. In embodiments, the selection of haloperoxidase is determined by the pH value of the treatment site. In other embodiments, the selection of haloperoxidase is determined by the accessibility of the treatment site.

[0064] The effective amount of the haloperoxidase used in the compositions, combinations or treatment methods of the present disclosure can vary widely depending on the conditions of use, the environment of use and the desired results of the compositions. In certain embodiments, the compositions of the present disclosure will comprise about 1 to about 100,000 μg / ml of haloperoxidase, more preferably about 5 to about 50,000 μg / ml, even more preferably about 10 to about 5000 μg / ml of haloperoxidase.

[0065] The oxidase that produces peroxide effectively in the present disclosure includes, for example, oxidase, such as glucose oxidase, cholesterol oxidase and galactose oxidase. As a representative example, when the oxidase is glucose oxidase and its substrate is glucose, the composition of the present disclosure may include about 0.05 to about 3000U / ml, more preferably about 0.1 to about 1000U / ml, even more preferably about 1 to about 500U / ml of glucose oxidase, and about 0.1 to about 100mM, more preferably about 0.5 to about 80mM, even more preferably from about 1 to about 50mM of glucose. Preferably, the glucose oxidase used in the present disclosure composition is derived from Aspergillus Niger. More preferably, the glucose oxidase is produced by Exoxemis, Inc, isolated from Aspergillus niger, purified to 99.8% purity as determined by RP-HPLC and 99.9% purity as determined by SECHPLC, and optionally wherein the activity per unit (U) of GO is 309 U / mg (wherein 1.0 U oxidizes 1.0 μmol of β-D-glucose to D-gluconolactone with H2O2 / minute at 35°C at pH 5.1).

[0066] As mentioned above, the haloperoxidase useful in the compositions, combinations or methods of treatment of the present disclosure, if not used in combination with the oxidase that produces peroxide, can be used in combination with peroxide. The use of peroxide, like the oxidase that produces peroxide, can be carried out simultaneously or sequentially with the use of haloperoxidase. In embodiments, peroxide can be applied to the treatment site with a concentration including but not limited to about 1 μM to about 100mM, preferably about 1mM to about 50mM, more preferably about 9mM. Use can depend on the accessibility of the treatment site. In embodiments, a large dose of peroxide of about 1ml to 1000ml, preferably 100ml to 800ml, most preferably 500ml can be used.

[0067] In certain embodiments, the haloperoxidase may be supplied to the treatment site optionally together with at least two amino acids, or at least three amino acids, selected from glycine, L-alanine, D-alanine, L-alanine anhydride, L-glutamine, L-glutamic acid, glycine anhydride, hippuric acid, L-histidine, L-leucine, D-leucine, L-isoleucine, D-isoleucine, L-lysine, L-ornithine, D-phenylalanine, L-phenylalanine, L-proline, L-hydroxyproline. , L-serine, L-taurine, L-threonine, D-threonine, L-tyrosine, L-valine, D-valine, β-amino acid (such as β-alanine, L-β-homoleucine, D-β-homoleucine, 3-aminobutyric acid, L-2,3-diaminopropionic acid monohydrochloride, D-2,3-diaminopropionic acid monohydrochloride, L-3-aminoisobutyric acid, D-3-aminoisobutyric acid, ethyl 3-aminobutyrate, sarcosine methyl ester hydrochloride and 3-piperidinecarboxylic acid), or its alkyl ester or pharmaceutically acceptable salt. In a particular embodiment, haloperoxidase can be formulated with amino acid, or as an alternative, amino acid can be supplied as a separate composition, to be premixed before application, or to be applied simultaneously or in parallel.

[0068] In certain embodiments, the haloperoxidase can be provided to the treatment site using two amino acids, wherein the two amino acids are glycine and L-proline.

[0069] The effective amount of the amino acid that can be optionally used in the composition / combination of the present disclosure will vary according to the amount of haloperoxidase in the composition / combination and the conditions present in the use environment. In one example, the composition can generally include 0.1 to about 500 mM, more preferably about 0.2 to about 100 mM, even more preferably about 0.3 to about 50 mM of each amino acid of the present disclosure.

[0070] Compositions / combinations of the present disclosure may optionally include halides. When the halide is chloride, the amount of chloride used in the compositions of the present disclosure will preferably fall within the range of about 10 μmol chloride to about 200 μmol / ml solution (i.e., 10 to 200 mEq chloride / L) chloride. The physiological concentration of chloride in plasma is about 105 mEq / L. When included, compositions of the present disclosure may include about 0.5 μmol bromide to about 20 μmol bromide / ml liquid composition (i.e., 0.5-20 mEq bromide / L), more preferably about 1 μmol bromide to about 10 μmol bromide / ml liquid composition (i.e., 1-10 mEq bromide / L), and most preferably about 100 nmol bromide to about 1 μmol bromide / ml liquid composition. In certain embodiments, the halide is sodium chloride.

[0071] The compositions / combinations of the present disclosure may optionally comprise ethanolamine.

[0072] The composition / combination may optionally include a pharmaceutically acceptable carrier. In certain embodiments, the composition may be conveniently provided in a liquid carrier. Any liquid carrier may generally be used for this purpose, provided that the carrier does not significantly interfere with the selective binding ability or enzymatic activity of myeloperoxidase. Alternatively, the composition may be provided in solid form, which is activated upon dissolution in a liquid.

[0073] In the embodiment of the present disclosure composition or combination including the substrate of the oxidase producing peroxide, the haloperoxidase is suitable for being constructed as a binary preparation, wherein the activating agent of the composition is formulated into two separate parts merged in use. For example, the first composition of the binary preparation may include a solution containing both the haloperoxidase and the oxidase producing peroxide, and an optional halide. In certain embodiments, the first composition may optionally include two or three amino acids. In certain embodiments, the two amino acids are amino acids and L-proline. In certain embodiments, the three amino acids are glycine, L-alanine and L-proline. The second composition of the binary preparation may include a substrate for the oxidase, such as glucose (i.e., dextrose) in the case of glucose oxidase. The substrate may be provided, for example, in the form of a solid glutinous rice capsule (wafer). In certain embodiments, the haloperoxidase composition may additionally include alcohol to promote the dissolution of the oxidase substrate and to be utilized by the oxidase.

[0074] In one embodiment, the method of the present disclosure includes a combination of applying a composition to a site in a preventive or therapeutic manner. For example, a first composition comprising a haloperoxidase and an oxidase that produces peroxide can be applied. In certain embodiments, the first composition further comprises two amino acids and a halide. A second composition comprising a substrate for an oxidase that produces peroxide can be applied separately. In some embodiments, a first composition comprising a haloperoxidase, a halide and two amino acids can be applied. A second composition comprising hydrogen peroxide can be applied separately. In certain embodiments, the first composition and the second composition are mixed before being applied to the infection site. In certain embodiments, the first composition and the second composition are applied to the site simultaneously. In certain embodiments, the first composition and the second composition are applied to the site in sequence. The first composition and the second composition can be applied in any order.

[0075] In one embodiment, the method of the present disclosure includes a combination of applying a composition to a site prophylactically or therapeutically. For example, a first composition comprising a haloperoxidase, ethanolamine and a halide can be applied. A second composition comprising hydrogen peroxide can be applied separately. In some embodiments, the first composition comprises a haloperoxidase, two amino acids and a halide. In certain embodiments, the first composition and the second composition are mixed before being applied to the infection site. In certain embodiments, the first composition and the second composition are applied to the site simultaneously. In certain embodiments, the first composition and the second composition are applied to the site in sequence. The first composition and the second composition can be applied in any order.

[0076] As an illustrative example, a composition of the present disclosure suitable for use as an anticancer treatment may include about 1 to 50000 μg / ml of a haloperoxidase, 0.01 to 500 units of glucose oxidase, and optionally the following substances: 0.1 to 500 μmol / ml (i.e., 0.1-500 mM) of glycine, 0.1 to 500 μmol / ml (i.e., 0.1-500 mM) of D-isoleucine, 0 to 100 μmol / mL (i.e., 0-100 mM) of L-alanine, and 50 to 500 mEq / L of chloride. The above composition may be combined with 1 to 500 μmol / mL (i.e., 1-500 mM) of glucose or dextrose.

[0077] As an illustrative example, a composition of the present disclosure suitable for use as an anti-cancer treatment may include about 1 to 50,000 μg / ml of a haloperoxidase, 0.1 to 500 μmol / ml (i.e., 0.1-500 mM) of glycine, 0.1 to 500 μmol / ml (i.e., 0.1-500 mM) of L-proline, 50 to 500 mEq / L of chloride, and 1 mM to about 50 mM of hydrogen peroxide.

[0078] As an illustrative example, a composition of the present disclosure suitable for use as an anti-cancer treatment may comprise eosinophil peroxidase, ethanolamine, sodium bromide, and hydrogen peroxide. Such a composition may be suitable for treating bladder cancer.

[0079] As an illustrative example, a composition of the present disclosure suitable for use as an anti-cancer treatment may include myeloperoxidase (MPO), glycine, L-proline, sodium chloride (NaCl), and hydrogen peroxide (H2O2). An illustrative example of such a composition includes 20 nM MPO, 10 mM H2O2, 100 mM NaCl, 0.21 mM glycine, and 0.21 mM L-proline. Such a composition may be suitable for treating bladder cancer.

[0080] Treatable cancer

[0081] The cancer targeted by the present invention is a solid cancer, and includes various cancers except hematological cancers (malignant lymphoma, leukemia, multiple myeloma, etc.). Typical examples of solid cancers include lung cancer, breast cancer, gastric cancer, liver cancer, colon cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer. Preferred specific examples of solid cancers include, for example, bladder cancer, colon cancer, lung cancer, pancreatic cancer, kidney cancer or breast cancer. Solid cancers may also include melanoma or glioma, but are not limited thereto.

[0082] In the case of non-surgical anticancer treatment (e.g., topical application), the anticancer composition of the present disclosure can be administered to warm-blooded animals, including human and non-human animal patients, in any effective pharmaceutically acceptable form. In this case, the composition of the present disclosure can be applied to any mucosal or epithelial surface. For example, the composition of the present disclosure can be administered in the form of a topical, lavage, oral, vaginal or rectal suppository, as a topical, oral, nasal spray, an aerosol for inhalation, or in any other effective manner.

[0083] For topical application, the pharmaceutically acceptable carrier can take the form of a liquid, cream, foam, lotion, ointment, suspension, suppository or gel, and may also include aqueous or organic solvents, buffers, emulsifiers, gelling agents, humectants, stabilizers, surfactants, wetting agents, preservatives, time release agents, and small amounts of humectants, chelating agents, dyes, perfumes and other components commonly used in topical pharmaceutical compositions. In addition, the compositions of the present disclosure can be impregnated in a dressing or covering for application to a patient.

[0084] In the case of more aggressive anticancer treatments, such as surgical debulking (reduction in size or elimination by resection), the anticancer compositions or combinations thereof of the present disclosure may be administered extratumorally. For example, extratumoral treatment may include application of the compositions / combinations of the present disclosure to the surgical site and / or the area surrounding the surgical site. In this case, the haloperoxidase may be administered as a solution or in any other dosage form, such as a subcutaneous injection or a deposit.

[0085] In non-surgical or surgical anti-cancer treatment using the compositions, combinations or methods of the present disclosure, patients in need may be treated with additional anti-cancer treatments (e.g., immunotherapy, chemotherapy and / or radiotherapy). The additional anti-cancer treatment may be administered to the patient before, simultaneously with or after treatment with the compositions / combinations of the present disclosure.

[0086] Other embodiments of the present invention are described below. However, it should be noted that the present invention should not be limited to these embodiments, and the present invention may be subject to changes, modifications and / or additions different from those specifically described, and it should be understood that the present invention includes all such changes, modifications and / or additions falling within the scope of the claims. Example

[0087] Example 1 - In vivo tumor-reducing activity of porcine eosinophil peroxidase (pEPO)

[0088] Experiments were performed to determine the tumor reducing activity of porcine eosinophil peroxidase (pEPO) in subcutaneous xenografts in mice.

[0089] Tumor cell line description: Experiments were performed using HT-1080 cells (Cat#_CCL-121) purchased from the American Type Culture Collection (ATCC). Cells were grown in complete medium as described below. Cells were seeded in cell culture flasks and cultured in a fully humidified atmosphere at 37°C and 5% CO2. Once cells reached confluence, they were propagated and / or stored as described below:

[0090] Cell proliferation and storage procedures : According to standard method:

[0091] Subculture ratio: 1:4 to 1:8

[0092] · Culture medium renewal: 2 to 3 times per week

[0093] Proliferation procedure: Remove medium, wash twice with D-PBS (1X). Add 1X trypsin solution and place flask at room temperature (RT) or 37°C until cells detach. Add fresh medium, aspirate and dispense into new flasks. Record the passage number.

[0094] Storage procedure: The cells were frozen in 95% complete growth medium supplemented with 5% DMSO. The first medium used after thawing the cells was Eagle's MEM supplemented with a sufficient concentration of glucose (4500 mg / l D-glucose).

[0095] Passaging procedure: Remove medium, wash twice with D-PBS (1X). Add 1X trypsin solution and place flask at room temperature (RT) or 37°C until cells detach. Add fresh medium, aspirate and dispense into new flasks. Record the passage number.

[0096] Final harvest procedure: Harvest 6x T-150 flasks as follows: remove media and wash with 1X D-

[0097] Wash once with PBS. Add 1X trypsin solution and leave the flask at room temperature (RT) until the cells detach. Quench the trypsin with culture medium (containing bovine serum) and spin the cells at 1000 rpm for 10 min. Wash twice with 1X D-PBS and resuspend the pellet in 1X D-PBS to 52×10 6 The concentration of cells / ml D-PBS was 1.

[0098] Prior to injection into animals, the proliferation, harvesting, and viability of the HT-1080 cell line were assessed and the results were as follows: Total number of cells: 78 × 10 6 cells, cell viability before injection: 98%; live cells / mL: 51×10 6 viable cells / ml.

[0099] Tumor For the subcutaneous (SC) tumor growth model, on day 1 of the study, 5.1 × 10 6 SC injections of 10 cells / mouse were injected into the right flank of each mouse. Tumor growth was followed by measuring the three parameters of length, width and height twice a week with a caliper. Tumor volume was calculated according to the formula for ellipsoids: 4 / 3πx(La / 2)x(Wa / 2)x(Ha / 2). La, Wa and Ha are the tumor length, width and height measured in vivo minus skin thickness. Double skin thickness was subtracted from the length and width parameters, and single skin thickness was subtracted from the height measurement to determine La, Wa and Ha.

[0100] Animal Description: Charles River athymic nude mice (Nu / Nu, male) were purchased from Charles River Laboratories. Animals were allowed to acclimate for 5 days prior to the start of the study. Animals were weighed the day before injection. Starting body weights ranged from 20 to 25 grams. Animals were ear-pierced for identification and housed 5 per cage prior to randomization by tumor size. Once animals were grouped, they were housed 1 per cage.

[0101] Enzyme solution: A composition of a pEPO enzyme solution and an activator solution (peroxide) was prepared. The enzyme solution contained pEPO at a final concentration of 2.5 μg / ml, (L-alanine, L-proline, glycine 0.8-0.05 mM each, final concentration), ethanolamine (2.4-0.15 mM, final concentration), sodium bromide (2 mM) and Tween-80 (0.1%, v / v). The activator solution contained hydrogen peroxide at a concentration of 0.003%, v / v, 890 μM in phosphate buffered saline (PBS) at pH 7.4. The vehicle was PBS.

[0102] Add 15 microliters of activator solution to the vehicle or enzyme solution and allow to incubate for approximately 3-5 minutes before dosing. Activate the dosing solution for each animal. After activation, inject approximately 1 ml of dosing solution into the surgical cavity. Fill the cavity to completion until some fluid leakage from the surgical site is observed.

[0103] Experimental design

[0104] HT-1080 fibrosarcoma cells were cultured and expanded under the above conventional conditions. On the day of injection into mice, cells were collected, washed with phosphate-buffered saline, and cultured at 5 × 10 7 Thirteen athymic nude mice (13 males) were injected SC into the right flank with HT1080 cells (at a concentration of 5.1×10 6 After injection, the animals were weighed and tumor formation was monitored weekly. Once tumors were visible and reached a measurable size, they were measured twice a week using an external caliper.

[0105] When the tumor reaches 0.5-1 cm 3 The animals were randomly divided into two groups, with 5 mice in each group. Both groups underwent surgical resection of the tumor. After tumor resection, the surgical wound was closed with surgical glue, and then the cavity was filled with dosing solution (about 1 mL / animal). Group 1 received vehicle + activator treatment, and Group 2 received pEPO + activator treatment. After surgery, the animals were housed separately. One animal in Group 1 (vehicle + activator) died the day after surgery. This animal was replaced with another animal carrying a tumor. The tumor was removed from the replacement animal, the surgical wound was closed, and the cavity was treated with 1 ml of vehicle + activator.

[0106] Mortality checks and clinical observations were performed daily after treatment. Animals were weighed weekly and on the day of termination. Animals were monitored for tumor formation and, where applicable, tumors were measured twice weekly using external calipers after tumors reached a measurable size.

[0107] At the end of 4 weeks, animals were euthanized with carbon dioxide and subsequent cervical dislocation. Tumors were measured with calipers before termination. After termination, tumors were excised, weighed and fixed in 10% neutral buffered formalin.

[0108] Results and Conclusions : In vivo tumor-reducing activity of pEPO

[0109] 2. Tumor growth and clinical observations are recorded in Table 1.

[0110]

[0111]

[0112] Results do not include observations of tumor formation prior to surgical removal and treatment.

[0113] Animals injected with HT-1080 cells developed visible tumors on day 11. Tumor mass increased over time, reaching an average volume of approximately 0.3 cm on study day 21. 3 And some animals have single tumors > 0.5 cm3 When the tumor is removed surgically.

[0114] Following tumor resection and treatment, 3 of 5 animals in Group 1 (Vehicle + Activator) experienced tumor regrowth, although the tumor was not extensive in animal G1M5. Only 1 of 5 animals in Group 2 (Enzyme + Activator) had confirmed tumor regrowth. Animals G1M2 and G1M3 were terminated on study days 39 and 43, respectively, due to complications from the presence of the tumor (difficulty moving, no visible feeding, no feces, general lethargy). The remaining animals were euthanized on study day 53.

[0115] One animal died during this study. Animal G1M4 was found dead the day after tumor resection and vehicle + activator treatment. This animal was replaced with another tumor bearing animal and treated with vehicle + activator (G1M4a).

[0116] Apart from the presence of tumors, other clinical observations were of minor nature to the study animals. In addition, no significant differences in body weight were recorded between the two groups at the end of the study.

[0117] Thus, this experiment demonstrates that treatment of the tumor cavity with activated pEPO resulted in a reduction in the number of animals (1 / 5) showing tumor regrowth compared to 3 of 5 animals receiving vehicle + activator treatment.

[0118] Example 2 - Detailed evaluation of pEPO tumor-reducing activity in vivo

[0119] A similar protocol as described in Example 1 was followed with slight variations. Specifically, a larger experimental group of 38 athymic nude mice (38 males) was injected SC with HT-1080 cells (similar to the 5.0×10 cells used in Example 1) in the right flank. 6 After injection, animals were weighed and tumor formation was monitored weekly. Once tumors were visible and reached a measurable size, they were measured twice a week using an external caliper.

[0120] Although tumors up to 0.5-1 cm were allowed in Example 1 3 , but in this further embodiment, tumors are allowed to reach 0.1-0.3 cm 3 . Then, the animals were randomly divided into two groups, each with 15 mice. Both groups underwent surgical resection of the tumor. After tumor resection, the surgical wound was closed with surgical glue, and then the cavity was filled with dosing solution (about 1 mL / animal). Group 1 received phosphate buffered saline and Group 2 received pEPO activator. After surgery, the animals were housed individually. Others followed the procedure according to Example 1.

[0121] Results and Conclusions : In vivo tumor-reducing activity of pEPO

[0122] Tumor growth and clinical observations from the further examples are recorded in Table 2.

[0123]

[0124]

[0125]

[0126]

[0127] Animals injected with HT-1080 cells developed visible tumors on day 10. Tumor masses increased over time and once tumors reached an average volume of approximately 0.15 cm 3 The tumors were surgically removed on the 24th day of the study.

[0128] Following tumor excision and treatment of the tumor cavity, 6 of 15 animals in Group 1 (phosphate buffered saline) and 5 of 15 animals in Group 2 (enzyme + activator) experienced tumor regrowth. Although the number of tumors and the time to tumor onset were similar in both groups of animals, the rates of tumor growth appeared to differ between the two groups. Tumors in Group 1 control animals increased more rapidly in volume than tumors in Group 2 enzyme + activator treated animals. On average, tumors in Group 2 animals took an additional 15 days to reach a similar size compared to Group 1 control tumors.

[0129] Similar results were observed when monitoring animal survival. Non-survival was defined as the study day when the tumor reached maximum tumor volume and the animals were euthanized. As with tumor volume, enzyme / activator treated animals survived longer than control treated animals.

[0130] After surgical resection of HT-1080 tumors and treatment of the tumor cavity with an activated enzyme solution (porcine eosinophil peroxidase, pEPO) or phosphate-buffered saline as a control, 6 of 15 animals in the phosphate-buffered saline group and 5 of 15 animals in the enzyme + activator group developed tumors. Treatment of the tumor cavity with the activated enzyme solution resulted in slower tumor growth and prolonged survival compared with phosphate-buffered saline treatment. Figure 1 As shown. Figure 1 In A, tumor volume is plotted as a function of postoperative study day until study day 42 (when any tumor reached 1 cm in maximum dimension). 3 first day of school). Figure 1The same data are shown in B, except that tumor volumes are plotted until the end of the study day. In calculating the mean tumor volume for subsequent study days, the tumor volume obtained at the time of euthanasia for each animal was used for all remaining study days.

[0131] Similar results were observed when monitoring animal survival. Non-survival was defined as the day on which the tumor reached maximum tumor volume and the animal was euthanized. The results of the survival study curves are shown in Figure 2. Figure 2 As shown. Figure 2 A shows the survival curve, where the percentage survival was calculated using all the animals studied. Figure 2 Only the survival curves of animals with tumor redevelopment are shown in B. As with tumor volume, enzyme+activator treated animals survived longer than control treated animals.

[0132] Example 3 - In vitro activity of porcine myeloperoxidase (pMPO) against bladder cancer cells

[0133] Experiments were performed as described below to determine the activity of porcine myeloperoxidase (MPO) on human bladder cancer cell lines (T24 and 5637) and human normal bladder epithelium (SV-HUC-1). The 5637 cell line is a highly invasive bladder surface tumor cell line and the T24 cell line is a moderately invasive transitional epithelial cell line.

[0134] Cell lines Two bladder cancer cell lines, 5637 (ATCC HTB-9) and T24 (ATCC HTB-4) and a normal bladder cell line SV-HUC-1 (ATCC CRL-9520) were used in the experiments.

[0135] Reagents The following stock solutions were used in the experiments: MPO (2 μM in water); H2O2 (100 mM in water); NaCl (6.2 M in water); Glycine (21.5 mM in water) and L-proline (21.5 mM in water).

[0136] Experiment 1 Experiments were performed to evaluate the in vitro cytotoxicity of individual agents on bladder cancer cell lines and normal bladder cell lines. The individual agents were myeloperoxidase (MPO), NaCl, glycine, and L-proline.

[0137] method. Bladder cancer cell lines 5637 and T24 and normal bladder cell line SV-HUC-1 were revived in the recommended culture medium. Cell lines were seeded at 2000 cells / well into 96-well plates 24 hours before the start of the assay. Six replicate wells were prepared for each reagent assay. Each cell line was seeded in four 96-well plates to monitor cell viability for four days. Cells were allowed to recover overnight before treatment.

[0138] On the day of treatment, each stock solution was diluted in McCoy's medium (vehicle) at pH 5.6 to produce the following treatment solutions:

[0139] Determination 1 .NaCl at the following concentrations: 10 mM, 20 mM, 40 mM, 80 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, and 600 mM.

[0140] Determination 2 .MPO at the following concentrations: 10 mM, 20 mM, 40 mM, 80 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, and 600 mM.

[0141] Determination 3 .The combination of glycine + L-proline, the concentration of each amino acid is as follows: 0.21mM, 0.42mM, 0.84mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM and 7mM.

[0142] For each assay, culture medium was used as a control. Culture medium was removed from each cell and well. Each well was treated with a separate treatment solution as described above. Treatment was performed for 45 minutes at 37°C, 5% CO2. Then, the treatment solution was removed and replaced with standard culture medium. After treatment, each plate was cultured for 24, 48 or 72 hours. At each post-treatment time point, Promega Cell Titer Aqueous Reagent (MTS Vitality Reagent) solution was added to each well of the plate and developed at 37°C, 5% CO2. The absorbance of each plate at 490nm was read every hour.

[0143] result The data obtained from Experiment 1 showed that each individual agent (even at higher concentrations) had little or no cytotoxic effect on cancer cells or normal cells. This result indicates that MPO alone is not cytotoxic to bladder cancer cells.

[0144] experiment 2. Experiments were conducted to evaluate the in vitro cytotoxicity of an MPO composition against bladder cancer cell lines and normal bladder cell lines. The composition contained various concentrations of MPO, H2O2, NaCl, glycine, and L-proline.

[0145] method:The cell lines used are as described in Experiment 1. 24 hours before the start of the assay, the cell lines were seeded into 96-well plates at 2000 cells / well or 4000 cells / well. Six replicate wells (a.-e., as shown below) were prepared for each treatment solution. Each cell line was seeded in four 96-well plates to monitor cell viability for four days. Cells were allowed to recover overnight before treatment.

[0146] On the day of treatment, each stock solution was diluted in McCoy's medium at pH 5.6 and then combined to produce the following treatment solutions: Composition A - medium pH 5.6 (vehicle); Composition B - medium pH 5.6 + NaCl (100 mM) + glycine (0.21 mM) + L-proline (0.21 mM) + MPO (20 nM); Composition C - medium pH 5.6 + NaCl (100 mM) + glycine (0.21 mM) + L-proline (0.21 mM) + H2O2 (1 mM); Composition D - medium pH 5.6 + NaCl (10 mM) + glycine (0.21 mM) + L-proline (0.21 mM) + MPO (20 nM) + H2O2 (1 mM); and Composition E - medium pH 5.6+NaCl (100 mM)+Glycine (0.21 mM)+L-Proline (0.21 mM)+MPO (20 nM)+H2O2 (1 mM).

[0147] Remove medium from each cell and well. If applicable, add treatment solution to each well and treat at 37°C, 5% CO2 for 45 minutes. Then, remove the treatment solution and replace it with standard medium. After treatment, each plate is cultured for 24, 48 or 72 hours. At each post-treatment time point, Promega CellTiter Aqueous Reagent (MTS Vitality Reagent) solution is added to each well of the plate and developed at 37°C, 5% CO2. Read the absorbance of each plate at 490nm every hour.

[0148] result.Data from Experiment 2 showed that culture medium alone (Composition A) and culture medium, MPO, NaCl, glycine, and L-proline (Composition B) showed no cytotoxicity to bladder cancer cells or normal bladder cells. This result is consistent with Experiment 1. Composition C (culture medium, H2O2, NaCl, glycine, and L-proline) showed some cytotoxic effects on both bladder cancer cells and normal bladder cells. Compositions D and E (culture medium, MPO, H2O2, NaCl, glycine, and L-proline) showed the strongest cytotoxic effects on both bladder cancer cells and normal bladder cells. However, this combination had a greater cytotoxic effect on cancer cell lines and a lesser cytotoxic effect on normal cell lines. In the presence of chloride, the combination of MPO and H2O2 had a cytotoxic effect on cancer cells and was less toxic to normal cells.

[0149] Experiment 3 Experiments were performed to evaluate the in vitro cytotoxicity of an MPO composition against bladder cancer cell lines and normal bladder cell lines at various cell densities. The composition comprises MPO (1 nM), H2O2 (1 mM), NaCl (100 mM), glycine (0.21 mM) and L-proline (0.21 mM).

[0150] method : The cell lines used were as described in Experiment 1. Cell lines were seeded into 96-well plates at various cell densities 24 hours before the start of the assay. The bladder cancer cell densities tested were: 15,000 cells / well; 30,000 cells / well; 60,000 cells / well; 90,000 cells / well; 120,000 cells / well; and 400,000 cells / well. The normal bladder cell densities tested were: 4,000 cells / well; 15,000 cells / well; 30,000 cells / well; 60,000 cells / well; 90,000 cells / well; and 120,000 cells / well. Triplicate wells were prepared for each treatment solution. Each cell line was seeded in four 96-well plates to monitor cell viability for four days. Cells were allowed to recover overnight before treatment.

[0151] On the day of treatment, each stock solution was diluted in McCoy's medium, pH 5.6, and then combined to produce the following treatment solutions: Composition A - medium pH 5.6 (vehicle); Composition B - medium pH 5.6 + NaCl (100 mM) + glycine (0.21 mM) + L-proline (0.21 mM) + H2O2 (1 mM); and Composition C - medium pH 5.6 + NaCl (100 mM) + glycine (0.21 mM) + L-proline (0.21 mM) + MPO (1 nM) + H2O2 (1 mM).

[0152] Remove medium from each cell and well. If applicable, add treatment solution to each well and treat at 37°C, 5% CO2 for 45 minutes. Then, remove the treatment solution and replace it with standard medium. After treatment, each plate is incubated for 72 hours. Add Promega CellTiter Aqueous Reagent (MTS Vitality Reagent) solution to each well of the plate and develop at 37°C, 5% CO2. Read the absorbance of each plate at 490nm every hour.

[0153] result The data from Experiment 3 showed that at the tested concentrations of MPO (1 nM) and H2O2 (1 mM), the cytotoxic effect of the MPO composition on cancer cells and normal cells gradually decreased as the cell density increased.

[0154] Experiment 4 Experiments were conducted to evaluate the in vitro cytotoxicity of the MPO composition against bladder cancer cell lines and normal bladder cell lines at high cell density and higher concentrations of MPO (≥20 nM) and H2O2 (10 mM). The MPO composition also contained NaCl (100 mM), glycine (0.21 mM) and L-proline (0.21 mM).

[0155] method : The cell lines used were as described in Experiment 1. Cell lines were seeded into 96-well plates at various cell densities 24 hours before the start of the assay. The bladder cancer cell densities tested were: 120,000 cells / well and 400,000 cells / well. The normal bladder cell densities tested were: 50,000 cells / well and 100,000 cells / well. Six replicate wells were prepared for each treatment solution. Each cell line was seeded in four 96-well plates to monitor cell viability for four days. Cells were allowed to recover overnight before treatment.

[0156] On the day of treatment, each stock solution was diluted in McCoy's medium at pH 5.6 and then combined to produce the following treatment solutions: Composition A-medium pH 5.6 (vehicle); Composition B-medium pH 5.6 + NaCl (100 mM) + glycine (0.21 mM) + L-proline (0.21 mM) + MPO (20 nM) + H2O2 (10 mM); and Composition CJ, which contains the same concentrations of NaCl (100 mM), glycine (0.21 mM), L-proline (0.21 mM) and H2O2 (10 mM). The concentrations of MPO were 40 nM, 80 nM, 100 nM, 120 nM, 140 nM, 160 nM, 180 nM and 200 nM, respectively.

[0157] Remove medium from each cell and well. If applicable, add treatment solution to each well and treat at 37°C, 5% CO2 for 45 minutes. Then, remove the treatment solution and replace it with standard medium. After treatment, each plate is incubated for 72 hours. Add Promega CellTiter Aqueous Reagent (MTS Vitality Reagent) solution to each well of the plate and develop at 37°C, 5% CO2. Read the absorbance of each plate at 490nm every hour.

[0158] result The data from Experiment 4 showed that at higher cell densities, each concentration of MPO (≥20 nM) and H2O2 (10 mM) tested resulted in highly selective killing of human bladder cancer cells, while leaving normal human bladder cells unharmed.

[0159] Example 4 - In vitro activity of porcine eosinophil peroxidase (pEPO) against bladder cancer cells

[0160] Experiments are planned to determine the in vitro activity of porcine eosinophil peroxidase (pEPO) against mouse bladder cancer cells. The effects of pEPO on normal bladder cells will also be investigated. Stock and dilution solutions of EPO, hydrogen peroxide, sodium bromide, and ethanolamine will be prepared. The bladder cancer cell line T24 will be treated in the experiments, as will the benign bladder cell line SV-HUC1, which serves as a control. The cells will be treated with the following solutions: (1) cell culture medium ("culture medium"); (2) culture medium + hydrogen peroxide (H2O2); (3) culture medium + sodium bromide (NaBr); (4) culture medium + pEPO; (5) culture medium + ethanolamine; (6) culture medium + H2O2 + NaBr + ethanolamine; (7) culture medium + H2O2 + EPO + ethanolamine; (8) culture medium + NaBr + pEPO + ethanolamine); (9) culture medium + H2O2 + NaBr + pEPO; (10) culture medium + H2O2 + NaBr + pEPO; + ethanolamine. Both cell lines were exposed to each of the various treatment solutions for 15 minutes, 30 minutes and 45 minutes. Cell growth was measured at 0 hours, 24 hours, 48 ​​hours and 72 hours after treatment.

[0161] Example 5 - In vivo bladder tumor reduction activity of porcine eosinophil peroxidase (pEPO)

[0162] Experiments were planned to determine the bladder tumor reduction activity of porcine eosinophil peroxidase (pEPO) in a mouse bladder cancer model in which bladder cancer cells grow in the mouse bladder. A treatment solution containing eosinophil peroxidase, sodium bromide, and ethanolamine was mixed with an activator solution of hydrogen peroxide contained in phosphate buffered saline and activated for 3 to 5 minutes. After activation, the enzyme solution was administered intravesically into the bladder via a catheter. The reduction in tumor growth was measured over time.

[0163] Example 6 - Treatment of bladder cancer

[0164] Treatment for patients with bladder cancer is envisioned to include one or more of the following:

[0165] • Maintain the bladder environment at an optimal pH of approximately 5.0 to 6.5 through irrigation.

[0166] • Remove the bladder mucosal lining with dimethyl sulfoxide (DMSO) (optional).

[0167] - Premix a composition comprising myeloperoxidase, sodium chloride, glycine and L-proline with a hydrogen peroxide solution to produce a treatment solution.

[0168] - Allow the treatment solution to incubate for 3 to 5 minutes.

[0169] • Instill / administer the therapeutic solution directly into the bladder through a urinary catheter.

[0170] Example 7 - Treatment of bladder cancer

[0171] Phase 1 human clinical trials are planned to treat patients with bladder cancer and may include one or more of the following:

[0172] • Maintain the bladder environment at an optimal pH of approximately 5.0 to 6.5 through irrigation.

[0173] • Remove the bladder mucosal lining with dimethyl sulfoxide (DMSO) (optional).

[0174] - Washing of appropriate bladders with a neutral ionic strength solution (eg Ringer's Lactate), which can be administered without stopping the metabolism of the target cancer cells.

[0175] - Premix a composition comprising myeloperoxidase (20 nM-100 nM), sodium chloride (100 mM), glycine (0.21 mM), and L-proline (0.21 mM) with a hydrogen peroxide solution (10 mM-100 mM) to obtain a treatment solution.

[0176] - Allow the treatment solution to incubate for 3 to 5 minutes.

[0177] • Instill / administer the therapeutic solution directly into the bladder through a urinary catheter.

[0178] Example 8 - Treatment of bladder cancer

[0179] Phase 1 human clinical trials are planned to treat patients with bladder cancer and may include one or more of the following:

[0180] • Maintain the bladder environment at an optimal pH of approximately 5.0 to 6.5 through irrigation.

[0181] • Remove the bladder mucosal lining with dimethyl sulfoxide (DMSO) (optional).

[0182] - Washing of appropriate bladders with a neutral ionic strength solution (eg Ringer's Lactate) which can be administered without stopping the metabolism of the target cancer cells.

[0183] - Premix a composition containing myeloperoxidase (20 nM-100 nM), sodium chloride (100 mM), glycine (0.21 mM), and L-proline (0.21 mM).

[0184] - Instill / administer the composition directly into the bladder via a urinary catheter.

[0185] - Allow the composition to remain in the bladder for approximately 10 minutes.

[0186] • Instill / administer hydrogen peroxide (10 mM-100 mM) directly into the bladder via a urinary catheter to activate the myeloperoxidase composition.

[0187] Example 9 - Treatment of bladder cancer

[0188] Treatment for patients with bladder cancer is envisioned to include one or more of the following:

[0189] • Maintain the bladder environment at an optimal pH of approximately 5.0 to 6.5 through irrigation.

[0190] • Remove the bladder mucosal lining with dimethyl sulfoxide (DMSO) (optional).

[0191] - Washing of appropriate bladders with a neutral ionic strength solution (eg Ringer's Lactate) which can be administered without stopping the metabolism of the target cancer cells.

[0192] • Direct instillation / administration of a composition comprising a haloperoxidase, a halide (eg bromide or chloride), a peroxide or a peroxide-generating oxidase and an oxidase substrate via a urinary catheter.

[0193] Example 10 - Treatment of bladder cancer

[0194] Treatment for patients with bladder cancer is envisioned to include one or more of the following:

[0195] • Maintain the bladder environment at an optimal pH of approximately 5.0 to 6.5 through irrigation.

[0196] Remove the bladder mucosal lining with dimethyl sulfoxide (DMSO) (optional)

[0197] - Washing of appropriate bladders with a neutral ionic strength solution (eg Ringer's Lactate) which can be administered without stopping the metabolism of the target cancer cells.

[0198] - Premix a composition comprising eosinophil peroxidase, sodium chloride, and ethanolamine with a hydrogen peroxide solution to produce a treatment solution.

[0199] - Allow the treatment solution to incubate for 3 to 5 minutes.

[0200] • Instill / administer the therapeutic solution directly into the bladder through a urinary catheter.

[0201] Example 11 - Treatment of bladder cancer

[0202] Treatment for patients with bladder cancer is envisioned to include one or more of the following:

[0203] • Maintain the bladder environment at an optimal pH of approximately 5.0 to 6.5 through irrigation.

[0204] • Remove the bladder mucosal lining with dimethyl sulfoxide (DMSO) (optional).

[0205] - Washing of appropriate bladders with a neutral ionic strength solution (eg Ringer's Lactate) which can be administered without stopping the metabolism of the target cancer cells.

[0206] • Premix a composition containing eosinophil peroxidase, sodium bromide and ethanolamine.

[0207] - Instill / administer the composition directly into the bladder via a urinary catheter.

[0208] - Allow the composition to remain in the bladder for approximately 10 minutes.

[0209] • Instillation / administration of hydrogen peroxide directly into the bladder via a urinary catheter to activate the myeloperoxidase composition.

[0210] Although exemplary embodiments have been shown and described, including the best mode known to the inventors for carrying out the invention, those skilled in the art will recognize that the present disclosure may be practiced by making variations in the disclosed structures, materials, compositions, and methods, and such variations are considered to be within the scope of the present disclosure.

[0211] Any discussion or reference to the prior art in this specification should not be taken as an admission that the prior art is part of the common general knowledge of the skilled person receiving the specification.

[0212] The contents of all references, published patents, and patent applications cited in this application are hereby incorporated by reference.

Claims

1. A method of treating a solid cancer in a patient, the method comprising: An effective amount of a pharmaceutical composition comprising myeloperoxidase, hydrogen peroxide or a source of hydrogen peroxide, and a halide is administered to the patient.

2. The method of claim 1, wherein the source of hydrogen peroxide is a peroxide-generating oxidase that produces hydrogen peroxide in the presence of a substrate for the oxidase.

3. The method of claim 2, wherein the peroxide-generating oxidase is glucose oxidase and the substrate is glucose.

4. The method of claim 1, wherein the halide is sodium chloride.

5. The method of claim 1, wherein the composition further comprises one or more amino acids.

6. The method of claim 5, wherein the one or more amino acids are selected from the group consisting of glycine, L-alanine, D-alanine, L-alanine anhydride, L-glutamine, L-glutamic acid, glycine anhydride, hippuric acid, L-histidine, L-leucine, D-leucine, L-isoleucine, D-isoleucine, L-lysine, L-ornithine, D-phenylalanine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-taurine , L-threonine, D-threonine, L-tyrosine, L-valine, D-valine, β-amino acids such as β-alanine, L-β-homoleucine, D-β-homoleucine, 3-aminobutyric acid, L-2,3-diaminopropionic acid monohydrochloride, D-2,3-diaminopropionic acid monohydrochloride, L-3-aminoisobutyric acid, D-3-aminoisobutyric acid, ethyl 3-aminobutyrate, sarcosine methyl ester hydrochloride and 3-piperidinic acid, or alkyl esters or pharmaceutically acceptable salts thereof.

7. The method of claim 6, wherein the amino acids are glycine and L-proline.

8. The method of claim 1, wherein the solid cancer is selected from the group consisting of breast cancer, lung and bronchial cancer, prostate cancer, colon and rectal cancer, skin melanoma, bladder cancer, cervical cancer, kidney and renal pelvis cancer, endometrial cancer, pancreatic cancer, thyroid cancer, liver cancer, brain cancer, and spinal cord cancer.

9. The method of claim 8, wherein the solid cancer is bladder cancer.

10. A method of treating a solid cancer in a patient, the method comprising: An effective amount of a pharmaceutical composition comprising myeloperoxidase, sodium chloride, glycine, L-proline, and hydrogen peroxide is administered.

11. The method of claim 1, wherein the pharmaceutical composition comprises (a) a first composition comprising myeloperoxidase, a halide, and an amino acid; and (b) a second composition comprising a peroxide.

12. The method of claim 11, wherein the first composition and the second composition are premixed prior to administration.

13. The method of claim 11, wherein said first composition and said second composition are administered simultaneously or sequentially.

14. A method of treating bladder cancer in a patient, the method comprising the steps of: Maintain the bladder environment at a pH of approximately 5.0 to 6.5 by lavage; Use an appropriate bladder wash solution with neutral ionic strength; premixing a composition comprising myeloperoxidase, sodium chloride, glycine, and L-proline with a hydrogen peroxide solution to obtain a treatment solution; The treatment solution was incubated for 3 to 5 minutes; it was instilled into the bladder via the urinary catheter.

15. The method of claim 12, wherein the treatment solution comprises myeloperoxidase at a concentration of 20 nM-100 nM, sodium chloride at a concentration of 100 mM, glycine at a concentration of 0.21 mM, L-proline at a concentration of 0.21 mM, and hydrogen peroxide at a concentration of 10 mM-100 mM.

16. A method of treating bladder cancer in a patient, the method comprising the steps of: Maintain the bladder environment at a pH of approximately 5.0 to 6.5 by lavage; Use an appropriate bladder wash solution with neutral ionic strength; premixing a composition comprising myeloperoxidase, sodium chloride, glycine, and L-proline; instilling the composition into the bladder through a urinary catheter; allowing the composition to remain in the bladder for approximately 10 minutes; and instilling hydrogen peroxide into the bladder through the urinary catheter to activate the myeloperoxidase composition.

17. The method of claim 16, wherein the composition comprises myeloperoxidase at a concentration of 20 nM-100 nM, sodium chloride at a concentration of 100 mM, glycine at a concentration of 0.21 mM, and L-proline at a concentration of 0.21 mM.

18. The method of claim 16, wherein the concentration of hydrogen peroxide is 10 mM to 100 mM.

19. A pharmaceutical composition for treating a solid cancer in a patient, the composition comprising myeloperoxidase, a halide, and hydrogen peroxide.

20. The pharmaceutical composition of claim 19, further comprising one or more amino acids.

21. A pharmaceutical composition for treating bladder cancer in a patient, the composition comprising myeloperoxidase, sodium chloride, glycine, L-proline, hydrogen peroxide, and a pharmaceutically acceptable carrier.

22. A pharmaceutical composition for treating bladder cancer in a patient, the composition comprising myeloperoxidase, sodium chloride, glycine, L-proline, glucose oxidase, glucose, and a pharmaceutically acceptable carrier.

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