Compositions and methods for treating solid tumors

Abstract

Compositions and methods for the treatment of solid cancers are provided. Specifically, the present disclosure provides compositions comprising haloperoxidase and methods of administering such compositions for the treatment of solid cancers. The present disclosure is based on the surprising and unexpected discovery that haloperoxidase-containing compositions, including myeloperoxidase compositions, exhibit anti-cancer properties and have less toxic effects on normal cells. In another aspect, the disclosure provides a method of treating solid cancer in a patient, the method 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 pharma- ceutically acceptable carrier.

Description

[Background technology]

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

[0002] Cancer will undoubtedly continue to have a significant impact on society worldwide. According to current statistics presented by the National Institutes of Health in 2020, approximately 1,806,590 new cases of cancer will be diagnosed in the United States, 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 almost anywhere in the body. Tumor formation is where uncontrolled cell growth occurs in solid tissues such as organs, muscles, or bones. To date, the majority of most common cancers are solid and tumor-forming 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, malignant tumors, and lymphomas.

[0004] Although cancer survival rates are increasing through the development of improved treatments, 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 by the scientific community.

[0005] For most solid cancers, abnormal tissue is biopsied for diagnosis. Surgery to remove as much of the tumor as possible, commonly referred to as debulking, may be a treatment option. Debulking may also improve the effectiveness of subsequently administered anti-cancer therapies, such as immunotherapy, chemotherapy and / or radiation therapy. However, surgical interventions in cancer treatment (whether biopsy or debulking) are not without risks. In addition to replicating uncontrollably, cancer cells may lose the cohesion and organization of normal tissues, detach from the primary tumor during biopsy or surgery, and migrate anywhere in the body via the circulatory and lymphatic systems. The spread of cancer (i.e. metastasis) during biopsy or surgical intervention therefore poses a significant risk to cancer patients.

[0006] Some solid cancers, such as bladder, brain, or spinal cancer, are difficult to biopsy and / or treat (surgical or non-surgical) due to the inaccessibility of the site of cancer growth, and therefore such cancers can result in a high incidence of patient mortality.

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

[0008] In particular, there is a need for new therapies in the management of non-muscle invasive bladder cancer (NMIBC). Current protocols for the management of NMIBC routinely involve endoscopic resection of the tumor followed by intravesical treatment with cytotoxic chemotherapeutic agents (e.g., gemcitabine, mitomycin, docetaxel) or BCG (bacillus Calmette-Guerin), which acts as an activator of the local immune response. The necessary activity of these agents is direct surface contact between the treatment agent and the remaining cancer cells. However, various drawbacks of these treatments exist, including limited efficacy as manifested by a relatively high recurrence rate, a nonselective mechanism of action that may result in cytotoxicity to normal epithelium and associated diseases, and the possibility of systemic side effects. Furthermore, due to the special handling requirements of these agents, administration is generally performed in a clinical setting, requiring repeated clinic visits, leading to financial and other burdens for both patients and clinical providers. These limitations highlight the need for new therapies in the treatment of NMIBC that not only enhance therapeutic efficacy and reduce morbidity, but also have a safety profile that could potentially allow administration in the home.

[0009] Myeloperoxidase (MPO) is a cationic enzyme secreted by granular leukocytes that has peroxidase and haloperoxidase activity, thus allowing the generation of the highly reactive oxidants hydrogen peroxide (HO) and the halides chloride (Cl). - In the presence of , MPO catalyzes the oxidation of chlorides to highly reactive hypochlorous acid (OCl - ), which can react with a second H2O2 to produce electronically excited singlet molecular oxygen ( 1 O2 * ) is produced. - and 1 O2 *Both have significant cytotoxic activity (free radical activity is not involved). The attractiveness of the use of 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 properties, as shown in bladder cancer cells. The ability of MPO to avidly and selectively bind bladder cancer cells, as well as its millisecond half-life, due to their anionic surface properties, 1 O2 * This confinement of the "kill zone" to a small radius of less than 0.5 micrometers allows the enzyme to bind and selectively cytotoxicity against bladder tumor cells that have a more neutral electrostatic profile while sparing the normal adjacent urothelium. Furthermore, intravesical administration of MPO and related reagents causes minimal systemic toxicity due to the large molecular weight of MPO (144 kDa) and rapid inactivation in the circulation by catalase and the reticuloendothelial system. Summary of the Invention [Means for solving the problem]

[0010] 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.

[0011] The present disclosure is based on the surprising and unexpected discovery that haloperoxidase-containing compositions, including myeloperoxidase compositions, exhibit anti-cancer properties and have less toxic effects on normal cells.

[0012] According to one aspect of the disclosure, there is provided a method of treating a solid tumor in a patient, the method comprising administering to the patient an effective amount of a pharmaceutical composition comprising a haloperoxidase.

[0013] In another aspect, this disclosure provides a method of treating a solid cancer in a patient, the method 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 pharma- ceutically acceptable carrier.

[0014] In another aspect, this disclosure provides a method of treating a solid cancer in a patient, the method 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 pharma- ceutically acceptable carrier.

[0015] In another aspect, this disclosure provides a method of treating a solid cancer in a patient, the method comprising administering to the patient an effective amount of a first composition comprising a haloperoxidase, a halide, one or more amino acids, and a pharma- ceutically acceptable carrier, and a second composition comprising hydrogen peroxide. The first composition and the second composition can be administered simultaneously or sequentially.

[0016] In another aspect, this disclosure provides a method of treating a solid cancer in a patient, the method comprising administering to the patient an effective amount of a first composition comprising a haloperoxidase, a halide, one or more amino acids, a peroxide generating oxidase, and a pharma- ceutically acceptable carrier, and a second composition comprising a substrate for the peroxide generating oxidase. The first composition and the second composition can be administered simultaneously or sequentially.

[0017] In yet another aspect, the disclosure provides a composition for treating a 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 pharma- ceutically acceptable carrier.

[0018] In yet another aspect, the disclosure provides a composition for treating a 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 pharma- ceutically acceptable carrier.

[0019] In yet another aspect, the 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 pharma- ceutically acceptable carrier.

[0020] In yet another aspect, the disclosure provides a combination for treating a solid tumor in a patient, the combination comprising a haloperoxidase, a halide, a peroxide, one or more amino acids, and a pharma- ceutically acceptable carrier.

[0021] In yet another aspect, the 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 pharma- ceutically acceptable carrier.

[0022] In yet another aspect, the disclosure provides compositions for treating a solid cancer in a patient, the compositions comprising a haloperoxidase and optionally one or more of a halide, ethanolamine, peroxide, or peroxide generating oxidase and a substrate for the oxidase, or a pharma- ceutically acceptable carrier.

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

[0024] In embodiments, the haloperoxidase is selected from the group consisting of myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), chloroperoxidase (CPO), functional derivatives thereof, and combinations thereof.

[0025] In certain embodiments, the haloperoxidase is eosinophil peroxidase.

[0026] In certain embodiments, the haloperoxidase is myeloperoxidase.

[0027] In embodiments, the haloperoxidase catalyzes the oxidation of halides and the dismutation of peroxides to generate singlet molecular oxygen, resulting in one or more of the following: inhibition of cancer cell growth, inhibition of cancer cell metastasis, or cancer cell death.

[0028] In an embodiment, the solid cancer is selected from the group including: 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, liver cancer, brain cancer and spinal cancer. In certain embodiments, the solid tumor is bladder cancer.

[0029] Other embodiments of the invention will become apparent from the following detailed description of various aspects of the invention. [Brief description of the drawings]

[0030] [Figure 1] Not stated. [Diagram 2] Not stated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] As used in the context of describing inventive concepts (particularly in the context of the claims which follow), the terms "a," "an," "the," and similar supports are intended to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0032] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0033] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to provide a better description and is not intended to limit the scope of the disclosure. No language in the specification should be construed as indicating any non-claimed element as essential.

[0034] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, reaction conditions, and the like used in the specification and claims are understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present disclosure. At the very least, each numerical parameter should be construed in light of significant digits and ordinary rounding approaches. The term "about" may be understood to refer to a range of + / - 10%, for example, + / - 5% or + / - 1% or + / - 0.1%.

[0035] The description of ranges of values ​​herein is merely intended to serve as a shorthand way of referring to each separate value within the range individually.Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually described 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 the range.

[0036] The terms "protein" and "polypeptide" are used interchangeably herein. The three-letter code for amino acids, as defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature, is used throughout this disclosure. It is also understood that a polypeptide can be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0037] "Enzymes" such as haloperoxidase or glucose oxidase are referred to herein. In this context, an enzyme is a protein / polypeptide that acts as a catalyst to cause a specific biochemical reaction. The scope of the enzymes of the present disclosure includes those isolated from natural sources that have the same unmodified amino acid sequence as found in nature, and "functional derivatives" thereof.

[0038] The term "haloperoxidase" refers to an enzyme that relies on the oxidation of a halide to catalyze hydrogen peroxide to produce a hypohalous acid; this hypohalous acid can react with additional hydrogen peroxide to produce singlet molecular oxygen. According to the present disclosure, haloperoxidases may also be referred to as halide:hydrogen peroxide oxidoreductases, where a halide, such as chloride or bromide, is the electron donor or reducing agent and peroxide is the electron acceptor or oxidizing agent (e.g., under the International Union of Biochemistry, EC No. 1.11.1.7 and EC No. 1.11.1.10). Suitable haloperoxidases include myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), chloroperoxidase (CPO), functional derivatives thereof, and combinations thereof. Haloperoxidases may be derived from any source, including human and non-human animals, or cell cultures.

[0039] A "derivative" of an enzyme of this disclosure generally retains the characteristic enzymatic activity observed in the wild-type, native, or parent form to the extent that the derivative is effective for a similar purpose as the wild-type, native, or parent form.

[0040] The term "functional fragment" or "functional derivative" as used in the context of the enzyme contact of this disclosure encompasses naturally occurring, synthetically or recombinantly produced nucleic acids or fragments and encoded enzymes that have the functional characteristics of the native, unmodified parent enzyme of this disclosure. "Functional derivatives" can include "substitution variants" in which at least one amino acid residue is removed in the native sequence and a different amino acid is inserted at the same position. The substitution can be single, where only one amino acid in the molecule is replaced; or multiple, where the same molecule has two or more amino acid replacements. Multiple replacements can be located at contiguous sites. Similarly, multiple residues can replace an amino acid, including replacements and insertions. "Insertion variants" are variants in which one or more amino acids are inserted into the amino acid immediately adjacent to a particular position in the native sequence. Immediately adjacent amino acids means that they are connected via the alpha-carboxy or alpha-amino functional groups of the amino acids. "Deletion variants" are mutations in which one or more amino acids are removed in the native amino acid sequence. Generally, deletion variants have one or two amino acids removed in a particular region of the molecule.

[0041] The term "isolated" or "purified" refers to a material that has been removed from its original environment (e.g., the natural environment if it occurs in nature). For example, a material is "purified" when it is present in a particular composition at a higher concentration than it is in a naturally occurring or wild-type organism, or in a combination of components that are not naturally 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 a similar protein / polypeptide that is separated from some or all of the coexisting materials in a natural system is isolated. Such a protein / polypeptide may, for example, be part of a composition, and such a composition may still be isolated because it is not part of the protein / polypeptide's natural environment.

[0042] As used herein, the term "pharmaceutically acceptable" refers to a substance that does not cause significant adverse allergic or immunological reactions when administered to a patient. "Pharmaceutically acceptable carriers" include, but are not limited to, solvents, coatings, dispersing agents, wetting agents, isotonic and absorption delaying agents, and disintegrants.

[0043] As used herein, "treat", "treating" or "treatment" of a disease, condition or disorder means to accomplish one or more of the following: (a) reduce the severity and / or duration; (b) limit or prevent the occurrence of characteristic symptoms; (c) inhibit the worsening of symptoms; (d) limit or prevent recurrence; and (e) limit or prevent the recurrence of symptoms. That is, the term includes both preventative or prophylactic treatment of a disease, condition or disorder (which prevents and / or delays the occurrence of the targeted pathological disease, condition or disorder), and curative, therapeutic or disease modifying treatment, including therapeutic measures that cure, delay, relieve symptoms, and / or halt the progression of a disease, condition or disorder; and the treatment of patients at risk of or suspected of suffering from a disease, and patients who are ill or have been diagnosed with a disease, condition or disorder. The term does not necessarily imply that the patient is treated until complete recovery. The term may also refer to the maintenance and / or promotion of health in individuals who are not afflicted with a disease but who may be susceptible to developing an unhealthy condition. The term may also include the augmentation or other strengthening of one or more primary preventive or therapeutic measures. As a non-limiting example, treatment may be performed by the patient, a caregiver, a doctor, a nurse, or a medical professional.

[0044] As used herein, "Prevent", "Preventing", "Prevention" or "Prophylaxis" of a disease or disorder means preventing the disorder from occurring in a patient. "Prevention" includes reducing the risk, incidence and / or severity of the disease, condition or disorder.

[0045] As used herein, the expressions "for administration" and "administered" have the same meaning as "prepared for administration". In other words, the description that an active compound is "for administration" should be understood to mean that the active compound is formulated and prepared into a dosage, so that the active compound is in a state capable of exerting its therapeutic activity.

[0046] The term "effective amount" or "therapeutic amount" is intended to mean an amount of a substance that elicits a desired biological or medical response in a tissue, system, animal, or human. The term "prophylactically effective amount" is intended to mean an amount of a pharmaceutical agent that prevents the occurrence or reduces the risk of a biological or medical event in a tissue, system, animal, or human.

[0047] In this specification and in the claims, the words "comprise", "comprises", "comprised" or "comprising", "including" or "having" and the like are used in their inclusive sense, i.e. specifying the presence of mentioned features but not excluding the presence of additional or further features.

[0048] Certain embodiments disclosed herein may be further limited in the claims by using the language "consisting of" or "consisting essentially of." When used in the claims, and whether added at the time of filing or by amendment, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps, and those that do not materially affect basic and novel characteristics. The embodiments of the present disclosure so claimed are substantially or expressly described and enabled herein. Haloperoxidase-Containing Compositions

[0049] Haloperoxidases are widespread in nature and are produced by mammals, algae, and fungi. U.S. Patent 6,294,168 discloses that haloperoxidases can be used as antimicrobial agents (particularly effective against bacteria and fungi) because they selectively bind to target microorganisms and inhibit target microbial growth in the presence of peroxides and halides. The use of low concentrations of haloperoxidase maximizes selective binding to target microorganisms without eliminating the desired microorganisms or causing significant damage to host cells. The selective nature of haloperoxidase binding is useful in therapeutic or prophylactic antimicrobial treatment of human or non-human patients.

[0050] The present disclosure is based on the surprising and unexpected discovery that haloperoxidase-containing compositions exhibit anti-cancer properties. In one aspect, the present disclosure provides a method for treating a solid cancer by contacting the cancer with a composition comprising a haloperoxidase. In another aspect, the present disclosure provides a composition for treating a solid cancer, the composition comprising a haloperoxidase. In yet another aspect, the present disclosure provides a combination for treating a solid cancer, the combination comprising a haloperoxidase and at least one halide, or peroxide, or peroxide-generating oxidase.

[0051] In certain embodiments, haloperoxidases catalyze the oxidation of halides and the dismutation of peroxides to singlet oxygen to treat cancer by inhibiting cancer cell growth, inhibiting metastasis, or 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.

[0052] In another embodiment, the method of treatment of the present disclosure further comprises administering an effective amount of peroxide or peroxide-generating oxidase. A substrate for the oxidase can be administered as needed. Preferably, the peroxide-generating oxidase is glucose oxidase and the substrate is glucose. In a further embodiment, the method further comprises administering a haloperoxidase and a halide, preferably chloride or bromide.

[0053] In yet another embodiment, the haloperoxidase is administered in a first composition together with at least one additional composition comprising one or more of a halide, a peroxide, or a peroxide generating oxidase and a substrate for the peroxide generating oxidase. Alternatively, the haloperoxidase may be formulated in a composition for administration, the composition also comprising one or more of a halide, a peroxide, or a peroxide generating oxidase and a substrate for the peroxide generating oxidase.

[0054] In yet another embodiment, the haloperoxidase is administered in a first composition together with at least one additional composition comprising one or more of a halide, an ethanolamine, a peroxide, or a peroxide generating oxidase and a substrate for the peroxide generating oxidase. Alternatively, the haloperoxidase may be formulated in a composition for administration, the composition also comprising one or more of a halide, an ethanolamine, a peroxide, or a peroxide generating oxidase and a substrate for the peroxide generating oxidase. Alternatively, the haloperoxidase may be formulated in a composition for administration, the composition also comprising one or more of a halide, one or more amino acids, a peroxide, or a peroxide generating oxidase and a substrate for the peroxide generating oxidase.

[0055] In certain embodiments, eosinophil peroxidase (EPO) and myeloperoxidase (MPO) are the preferred haloperoxidases for use in the present compositions, combinations, and methods of treatment. In further embodiments, the MPO and EPO are porcine derived. Preferably, the purified haloperoxidases, porcine MPO and EPO, are produced by Exoxemis, Inc.

[0056] Porcine MPO is preferably 98.9% pure by ultra-high performance liquid chromatography (RP-UPLC) and 100% pure by size-exclusion high performance liquid chromatography (SEC-HPLC). The guaiacol unit (GU) activity of porcine MPO is preferably 404 GU / mg; an activity of 1.0 GU consumes 1.0 μmol of H2O2 per minute.

[0057] The porcine EPO is preferably 99.2% pure by reverse phase high performance liquid chromatography. The guaiacol unit (GU) activity of the porcine EPO is preferably 80 GU / mg.

[0058] Both MPO and EPO are cationic proteins. Without being bound by theory, it is believed that the cationic nature of such haloperoxidases makes them particularly attached to the anionic surface of cancer cells. Thus, at the appropriate pH, the electrostatic attraction and binding of haloperoxidases to the anionic surface of cancer cells results in selective binding of haloperoxidases to cancer cells with no or minimal binding to the surface of normal cells, and in the presence of peroxides, selectively kills cancer cells with little or no damage to surrounding normal cells.

[0059] Haloperoxidases may differ in their physical properties and optimum conditions for enzymatic activity (see, e.g., U.S. Patent 9,782,459). For example, MPO is about 150 kDa and is active at acidic pH (4.0-6.5), while EPO is about 70 kDa and is active at acidic to neutral pH (i.e., 5.0-7.4). In embodiments, compositions of the present disclosure may include a haloperoxidase, and the properties of the haloperoxidase may be coordinated with the conditions at the site of cancer treatment. In embodiments, the selection of the haloperoxidase is determined by the pH at the site of treatment. In another embodiment, the selection of the haloperoxidase is determined by the accessibility to the site of treatment.

[0060] The effective amount of haloperoxidase used in the compositions, combinations, or methods of treatment of the present disclosure can vary widely, depending on the conditions under which the composition is used, the environment of use, and the desired results. In certain embodiments, the compositions of the present disclosure contain from about 1 to about 100,000 μg / ml of haloperoxidase, more preferably from about 5 to about 50,000 μg / ml, and even more preferably from about 10 to about 5,000 μg / ml of haloperoxidase.

[0061] Peroxide-producing oxidases useful in the present disclosure include oxidases such as, for example, 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 contain about 0.05 to about 3,000 U / ml, more preferably about 0.1 to about 1,000 U / ml, and even more preferably about 1 to about 500 U / ml of glucose oxidase, and about 0.1 to about 100 mM, more preferably about 0.5 to about 80 mM, and even more preferably about 1 to about 50 mM of glucose. Preferably, when used in the composition of the present disclosure, the glucose oxidase is derived from Aspergillus niger. More preferably, the glucose oxidase is produced by Exoxemis, Inc., which is isolated from Aspergillus niger and purified to 99.8% by RP-HPLC and 99.9% by SECHPLC, and optionally has a unit (U) activity of GO of 309 U / mg (1.0 U oxidizes 1.0 μmol of β-D-glucose to D-gluconolactone and HO per minute at pH 5.1 and 35° C.).

[0062] As noted above, the haloperoxidases useful in the compositions, combinations, or methods of treatment of the present disclosure, when not used in combination with a peroxide-generating oxidase, may be administered in combination with a peroxide. The administration of the peroxide, as well as the peroxide-generating oxidase, may be simultaneous or sequential with the administration of the haloperoxidase. In embodiments, the peroxide may be administered to the treatment site at a concentration including, but not limited to, about 1 μM to about 100 mM, preferably about 1 mM to about 50 mM, more preferably about 9 mM. Administration may depend on the accessibility of the treatment site. In embodiments, a bolus of peroxide between about 1 ml to 1000 ml, preferably 100 ml to 800 ml, most preferably 500 ml, may be administered.

[0063] In certain embodiments, the haloperoxidase is selected from the group consisting of glycine, L-alanine, D-alanine, L-alanine anhydrous, L-glutamine, L-glutamic acid, glycine anhydrous, 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, beta amino acids, e.g., beta It may be optionally provided to the treatment site with at least two amino acids, or at least three amino acids, selected from the group including alanine, L-beta-homoleucine, D-beta-homoleucine, 3-aminobutanoic 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 nipecotic acid, or alkyl esters or pharma- ceutically acceptable salts thereof. In certain embodiments, the haloperoxidase may be formulated with the amino acids, or alternatively, the amino acids may be provided as a separate composition for premixing prior to administration, or for simultaneous or concomitant administration.

[0064] In certain embodiments, the haloperoxidase can be provided to the treatment site with two amino acids, the two amino acids being glycine and L-proline.

[0065] The effective amount of amino acids that may be optionally used in the compositions / combinations of the present disclosure may vary depending on the amount of haloperoxidase in the composition / combination and the conditions present in the environment of use. In one example, the compositions may generally contain about 0.1 to about 500 mM, more preferably about 0.2 to about 100 mM, and even more preferably about 0.3 to about 50 mM, of each of the amino acids of the present disclosure.

[0066] The compositions / combinations of the present disclosure may optionally include a halide. When the halide is chloride, the amount of chloride used in the compositions of the present disclosure may preferably range from about 10 μmol chloride to about 200 μmol chloride per ml of solution (i.e., 10-200 mEq chloride / L). The physiological concentration of chloride in plasma is about 105 mEq / L. When included, the compositions of the present disclosure may include about 0.5 μmol bromide to about 20 μmol bromide per ml of liquid composition (i.e., 0.5-20 mEq bromide / L), more preferably about 1 μmol bromide to about 10 μmol bromide per ml of liquid composition (i.e., 1-10 mEq bromide / L), and most preferably about 100 nmol bromide to about 1 μmol bromide per ml of liquid composition. In an embodiment, the halide is sodium chloride.

[0067] The compositions / combinations of the present disclosure may optionally include ethanolamine.

[0068] The composition / combination may contain a pharma- ceutically acceptable carrier if necessary. In some embodiments, the composition may be conveniently provided in a liquid carrier. Any liquid carrier may be generally 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 a solid form that is solubilized in liquid and activated.

[0069] In an embodiment of the composition or combination of the present disclosure that includes a substrate for the peroxide-generating oxidase, the haloperoxidase is suitable for configuration as a binary formulation, in which the active agents of the composition are formulated into two separate parts for integration at the time of use. For example, the first composition of the binary formulation may include a solution containing both the haloperoxidase and the peroxide-generating oxidase, and optionally a halide. In an embodiment, the first composition may optionally include two or three amino acids. In an embodiment, the two amino acids are glycine and L-proline. In an embodiment, the three amino acids are glycine, L-alanine and L-proline. The second composition of the binary formulation 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 cachet. In an embodiment, the haloperoxidase composition may additionally include alcohol to facilitate solubilization of the oxidase substrate and utilization by the oxidase.

[0070] In one embodiment, the method of the present disclosure includes administering a combination of compositions to the site, either prophylactically or therapeutically. For example, a first composition including a haloperoxidase and a peroxide generating oxidase can be administered. In an embodiment, the first composition further includes two amino acids and a halide. A second composition including a substrate for the peroxide generating oxidase can be administered separately. In an embodiment, a first composition including a haloperoxidase, a halide, and two amino acids can be administered. A second composition including hydrogen peroxide can be administered separately. In an embodiment, the first composition and the second composition are mixed before administration to the site of infection. In an embodiment, the first composition and the second composition are administered simultaneously to the site. In an embodiment, the first composition and the second composition are administered sequentially to the site. The first composition and the second composition can be administered in any order.

[0071] In one embodiment, the method of the present disclosure includes administering a combination of compositions to the site, either prophylactically or therapeutically. For example, a first composition may be administered that includes a haloperoxidase, an ethanolamine, and a halide. A second composition that includes hydrogen peroxide may be administered separately. In one embodiment, the first composition includes a haloperoxidase, two amino acids, and a halide. In one embodiment, the first composition and the second composition are mixed before administration to the infected site. In one embodiment, the first composition and the second composition are administered to the site simultaneously. In one embodiment, the first composition and the second composition are administered to the site sequentially. The first composition and the second composition may be administered in any order.

[0072] As an illustrative example, a composition of the present disclosure suitable for use as an anti-cancer treatment may contain about 1-50,000 μg / ml of haloperoxidase, 0.01-500 units of glucose oxidase, and optionally: 0.1-500 μmol / mL (i.e., 0.1-500 mM) of glycine, 0.1-500 μmol / mL (i.e., 0.1-500 mM) of D-isoleucine, 0-100 μmol / mL (i.e., 0-100 mM) of L-alanine, and 50-500 mEq / L of chloride. The above compositions may be combined with 1-500 μmol / mL (i.e., 1-500 mM) of glucose or dextrose.

[0073] As an illustrative example, a composition of the present disclosure suitable for use as an anti-cancer treatment may contain about 1-50,000 μg / ml of haloperoxidase, 0.1-500 μmol / mL (i.e., 0.1-500 mM) of glycine, 0.1-500 μmol / mL (i.e., 0.1-500 mM) of L-proline, 50-500 mEq / L of chloride, and 1 mM to about 50 mM of hydrogen peroxide.

[0074] As an illustrative example, a composition of the present disclosure suitable for use as an anti-cancer treatment may include eosinophil peroxidase, ethanolamine, sodium bromide, and hydrogen peroxide. Such a composition may be suitable for treating bladder cancer.

[0075] 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.

[0076] Treatable cancer The cancer targeted by the present invention is a solid cancer, including various cancers other than blood cancer (such as malignant lymphoma, leukemia, multiple myeloma, etc.). Representative examples of solid cancer include lung cancer, breast cancer, stomach cancer, liver cancer, colon cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, cervical cancer, and ovarian cancer. Preferred examples of solid cancer include, for example, bladder cancer, colon cancer, lung cancer, pancreatic cancer, kidney cancer, or breast cancer. Solid cancer may also include, but is not limited to, melanoma or glioma.

[0077] In the context of non-surgical anti-cancer treatment such as topical application, the anti-cancer composition of the present disclosure can be administered to warm-blooded animals, including human and non-human animal patients, in any effective pharmacologic acceptable form.In this context, the composition of the present disclosure can be administered to any mucous membrane or epithelial surface.For example, the composition of the present disclosure can be administered in the dosage form of topical, lavage, oral, vaginal or anal suppository, topical, buccal, nasal spray, aerosol for inhalation, or in any other effective way.

[0078] For topical application, the pharma- ceutically acceptable carrier may take the form of a liquid, cream, foam, lotion, ointment, suspension, suppository or gel, and may further include aqueous or organic solvents, buffers, emulsifiers, gelling agents, humectants, stabilizers, surfactants, wetting agents, preservatives, sustained-release agents, as well as small amounts of moisturizers, sequestering agents, dyes, fragrances and other compositions commonly used in pharmaceutical compositions for topical administration. Additionally, the compositions of the present disclosure may be impregnated into a dressing or covering for application to a patient.

[0079] In the context of more invasive anti-cancer treatment, such as surgical tumor debulking (removal or reduction in size), the anti-cancer composition of the present disclosure can be administered outside of the tumor.For example, the extratumoral treatment can include applying the composition / combination of the present disclosure to the surgical site and / or the area surrounding the surgical site.In this context, the haloperoxidase can be administered in solution or in any other dosage form, such as subcutaneous injection or depot.

[0080] In either non-surgical or surgical anti-cancer treatment with the compositions, combinations or methods of the present disclosure, the patient in need may be treated with additional anti-cancer treatments such as immunotherapy, chemotherapy and / or radiation therapy. The additional anti-cancer treatments may be administered to the patient before, simultaneously with, or after treatment with the compositions / combinations of the present disclosure.

[0081] Further embodiments of the invention are described below, however it should be noted that the invention should not be limited to these embodiments and that the invention is susceptible to variations, modifications and / or additions other than those specifically described, and it should be understood that the invention includes all such variations, modifications and / or additions that fall within the scope of the claims. EXAMPLES

[0082] Example 1. In vivo tumor-reducing activity of porcine eosinophil peroxidase (pEPO) Experiments were conducted to determine the tumor-reducing activity of porcine eosinophil peroxidase (pEPO) in mouse subcutaneous xenografts.

[0083] Description of tumor cell lines: HT-1800 cells (Cat#CCL-121) purchased from American Type Culture Collection (ATCC) were used in the experiments. Cells were grown in complete medium as described below. Cells were seeded into cell culture flasks and incubated at 37°C in a well-humidified atmosphere containing 5% CO2. When cells reached confluence, they were propagated and / or maintained as described below.

[0084] Cell propagation and storage procedures: were performed according to standard methods. Passage ratio: 1:4 to 1:8 Medium replacement: 2 to 3 times per week · Propagation procedure: Remove medium and wash twice with D-PBS (1X). Add 1X trypsin solution and place flask at room temperature or 37°C until cells detach. Add fresh medium, aspirate and dispense into new bottles. Record passage number. Storage procedure: Cells were frozen in 95% complete growth medium supplemented with 5% DMSO. The first medium used after thawing the cells was Eagle's MEM medium supplemented with a sufficient concentration of glucose (4500 mg / l D-glucose). Passage procedure: Remove medium and wash twice with D-PBS (1X). Add 1X trypsin solution and place bottles at room temperature or 37°C until cells detach. Add fresh medium, aspirate and distribute into new flasks. Record passage number. Final harvesting procedure: 6x T-150 flasks were harvested by removing the medium and washing once with 1X D-PBS. 1X trypsin solution was added and flasks were left at room temperature until cells detached. Trypsin was quenched with culture medium (containing bovine serum) and cells were centrifuged at 1000 rpm for 10 minutes. After washing twice with 1X D-PBS, the pellet was collected at 52x10 6 Resuspended in 1X D-PBS to a concentration of cells / ml.

[0085] Prior to injection into animals, the HT-1080 cell line was evaluated for propagation, recovery and viability, revealing the following: Total cell number: 78x10 6 Cells; Cell viability before injection: 98%; Viable cells per mL: 51x10 6 cells / ml

[0086] Tumor: For subcutaneous (SC) tumor growth model, 5.1x10 per mouse on day 1 of the experiment. 6 A volume of 100 μL of cells was injected subcutaneously into the right flank of each mouse. Tumor growth was followed twice weekly by caliper measurements determining the three parameters length, width and height. Tumor volume was calculated according to the formula for an ellipsoid: 4 / 3πx(La / 2)x(Wa / 2)x(Ha / 2). La, Wa and Ha are the measured length, width and height of the tumor in vivo minus the skin thickness. To determine La, Wa and Ha, 2x skin thickness was subtracted from the length and width parameters and 1x skin thickness was subtracted from the height measurement.

[0087] Animal description: Charles River athymic nude (Nu / Nu) mice (male) were purchased from Charles River Laboratories. Animals were acclimated for 5 days before the start of the experiment. Animals were weighed 1 day before injection. Starting body weights ranged between 20-25 grams. Animals were ear-pierced for identification and housed 5 per cage until randomized by tumor size. Once assigned to groups, animals were housed 1 per cage.

[0088] Enzyme solutions: pEPO enzyme solution and activator solution (peroxide) compositions were prepared. The enzyme solution contained pEPO at a final concentration of 2.5 μg / ml (final concentrations of L-alanine, L-proline, and glycine were 0.8-0.05 mM, respectively), ethanolamine (final concentrations, 2.4-0.15 mM), sodium bromide (2 mM), and Tween®-80 (0.1%, v / v). The activation solution consisted of 0.003%, v / v, 890 μM hydrogen peroxide in phosphate buffered saline (PBS), pH 7.4. The vehicle was PBS.

[0089] Fifteen microliters of activation solution was added to the vehicle or enzyme solution and incubated for approximately 3-5 minutes before dosing. Dosing solutions were activated for each individual animal. After activation, approximately 1 ml of dosing solution was injected into the surgical cavity. The cavity was allowed to fill completely until some leakage of fluid was observed from the surgical area. Experimental design

[0090] HT-1080 fibrosarcoma cells were cultured and expanded under the conventional conditions described above. On the day of injection into mice, cells were harvested, washed with phosphate-buffered saline, and then cultured at 5x10 7 HT1080 cells (concentration 5.1x10 6 Cells / animal, volume 100 μl / animal) were injected subcutaneously into the right flank of 13 athymic nude mice (13 males). Animals were weighed and observed for tumor formation once a week after injection. Tumors were measured twice a week using external calipers once they were visible and reached a measurable size.

[0091] Tumor 0.5-1cm 3When the animals reached 100% CI, they were randomized into 2 groups of 5 animals each. Tumors were surgically removed from both groups. The surgical wound was closed with surgical glue after tumor excision and the cavity was then filled with dosing solution (~1 ml per animal). Group 1 received vehicle + activator, while Group 2 received pEPO + activator. After surgery, animals were housed individually. One animal from Group 1 (vehicle + activator) died the day after surgery. This animal was replaced by an extra tumor-bearing animal. The tumor was removed from the replaced animal, the surgical wound was closed, and the cavity was treated with 1 ml of vehicle + activator.

[0092] Mortality and clinical observations were performed daily after treatment. Animals were weighed weekly and on the termination day. Animals were observed for tumor formation and, where applicable, tumors were measured twice weekly with external calipers after they reached a measurable size.

[0093] At the end of the four weeks, animals were euthanized with carbon dioxide followed by cervical dislocation. Tumors were measured with calipers prior to termination. After termination, tumors were excised, weighed, and fixed in 10% neutral buffered formalin. Results and conclusions: In vivo tumor-reducing activity of pEPO Tumor growth and clinical observations are listed in Table 1. [Table 1]

[0094] Animals injected with HT-1080 cells developed visible tumors by day 11. Tumor size increased over time, and at day 21, individual tumors were >0.5 cm in some animals. 3 The average volume reached approximately 0.3 cm 3 The tumors were surgically removed when

[0095] Following tumor removal and treatment, animal G1M5 did not develop widespread tumors, but 3 of 5 animals in group 1 (vehicle + activator) had tumor regrowth. Only 1 of 5 animals in group 2 (enzyme + activator) had tumor regrowth. Animals G1M2 and G1M3 were terminated on study days 39 and 43, respectively, due to complications arising from the presence of tumors (difficulty moving, lack of visible food, lack of feces, general lethargy). The remaining animals were euthanized on study day 53.

[0096] One animal died during the course of this experiment. Animal G1M4 died the day after the tumor was surgically removed and treated with vehicle plus activator. This animal was replaced with one of the remaining tumor-bearing animals and treated with vehicle plus activator (G1M4a).

[0097] Apart from the presence of tumors, other clinical observations were of a marginal nature for the experimental animals. Furthermore, no obvious differences in body weight were noted between the two groups at the end of the experiment.

[0098] Consequently, this experiment demonstrated that treatment of the tumor cavity with activated pEPO reduced the number of animals showing tumor regrowth (1 of 5) compared to 3 of 5 animals receiving vehicle + activator. Example 2 - Refined evaluation of the in vivo tumor-reducing activity of pEPO

[0099] A similar protocol as described in Example 1 was followed with a slight variation: a larger cohort of 38 athymic nude mice (38 males) was inoculated with HT-1080 cells (5.0×10 cells, the same concentration used in Example 1). 6 Cells / animal, volume 100 μl / animal) were injected subcutaneously into the right flank. Animals were weighed and observed for tumor formation every week after injection. Tumors were measured twice weekly using external calipers once they were visible and reached a measurable size.

[0100] In Example 1, the tumor was 0.5 to 1 cm 3In this further embodiment, the tumors were grown to 0.1 to 0.3 cm 3 The animals were then randomized into two groups of 15 animals. The tumors were surgically removed from both groups. The surgical wound was closed with surgical glue after tumor resection, and the cavity was then filled with the dosing solution (approximately 1 ml / animal). Group 1 received phosphate buffered saline, while Group 2 received the pEPO activator. The animals were individually housed after surgery. The rest of the procedure in Example 1 was followed. Results and conclusions: In vivo reducing activity of pEPO Tumor growth and clinical observations from this further example are set out in Table 2. [Table 2-1] [Table 2-2]

[0101] Animals injected with HT-1080 cells developed visible tumors by day 10. Tumor size increased over time, with the average tumor volume reaching approximately 0.15 cm. 3 On day 24 of the experiment, when the tumor size reached 100 mg / kg, the tumor was surgically excised.

[0102] Following tumor removal and treatment of the tumor cavity, tumors re-emerged in 6 of 15 animals from Group 1 (phosphate buffered saline) and 5 of 15 animals from Group 2 (enzyme + activator). The number of animals developing tumors and the time to tumor appearance were similar in both groups, but the tumor growth rates in the two groups were clearly different. Tumors in Group 1 control animals increased in volume more quickly than tumors in Group 2 enzyme + activator treated animals. On average, tumors in Group 2 animals required an additional 15 days to reach a similar size compared to Group 1 control tumors.

[0103] Similar results were observed when observing animal survival. Non-survival was defined as the experimental day when the tumor reached maximum tumor volume and the animal was euthanized. Similar to tumor volume, enzyme / activator-treated animals survived longer than control-treated animals.

[0104] Following surgical removal of HT-1080 tumors and treatment of the tumor cavity with either an activated enzyme solution (porcine eosinophil peroxidase, pEPO) or phosphate-buffered saline as a control, 6 of 15 animals from the phosphate-buffered saline group and 5 of 15 animals from the enzyme plus activator group developed tumors. Animals whose tumor cavities were treated with the activated enzyme solution resulted in slower tumor growth and prolonged survival compared to phosphate-buffered saline treatment. The results are shown graphically in Figure 1. In Figure 1A, tumor volume is plotted as a function from the experimental day after surgery to experimental day 42 (on the first day all tumors were up to 1 cm 3 (Tumor volumes reached a size of 100 μg / kg / day.) The same data are shown in Figure 1B, except that tumor volumes were plotted up until the final experimental day. When calculating the average tumor volume on subsequent experimental days, the tumor volumes measured at the time of euthanasia for each animal were used for all remaining experimental days.

[0105] Similar results were observed when observing the survival of the animals. Non-survival was defined as the experimental day when the tumor reached the maximum tumor volume and the animal was euthanized. The results of the survival experimental curves are shown in Figure 2. In Figure 2A, the survival curve shows the survival percentage calculated using all experimental animals. In Figure 2B, the survival curve is only for animals that re-developed the tumor. Similar to the tumor volume, the animals treated with enzyme + activator survived longer than the control treated animals. Example 3 - In vitro activity of porcine myeloperoxidase (pMPO) on bladder cancer cells

[0106] To determine the activity of porcine myeloperoxidase (MPO) on human bladder cancer cell lines (T24 and 5637) and human normal bladder epithelial tissue (SV-HUC-1), experiments were performed as described below: The 5637 cell line is a highly invasive bladder surface tumor cell line, and the T24 cell line is an intermediately invasive, transitional epithelial cell line.

[0107] Cell lines. Two bladder cancer cell lines, 5637 (ATCC HTB-9) and T24 (ATCC HTB-4), and one normal bladder cell line, SV-HUC-1 (ATCC CRL-9520), were used in the experiments.

[0108] 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).

[0109] Experiment 1. Experiments were performed to evaluate the in vitro cytotoxicity of individual reagents against bladder cancer and normal bladder cell lines. The individual reagents were myeloperoxidase (MPO), NaCl, glycine, and L-proline.

[0110] Methods: Bladder cancer cell lines 5637 and T24, and normal bladder cell line SV-HUC-1 were resurrected in the recommended culture medium. 24 hours before the start of the assay, cell lines were plated in 96-well plates at 2000 cells / well. Six replicate wells were prepared for each reagent assay. Each cell line was plated in four 96-well plates and cell viability was observed for four days. Cells were allowed to recover overnight before treatment.

[0111] On the day of treatment, each stock solution was diluted in McCoy's culture medium (vehicle) at pH 5.6 to generate the following treatment solutions:

[0112] Assay 1. NaCl concentrations: 10 mM, 20 mM, 40 mM, 80 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, and 600 mM.

[0113] Assay 2. MPO concentrations: 10 mM, 20 mM, 40 mM, 80 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, and 600 mM.

[0114] Assay 3. Glycine + L-proline combination at respective amino acid concentrations: 0.21 mM, 0.42 mM, 0.84 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, and 6 mM and 7 mM.

[0115] For each assay, medium was used as a control. Medium was removed from the cells and wells. Wells were treated with the individual treatment solutions indicated above. Treatments were performed for 45 minutes at 37°C, 5% CO2. Treatment solutions were then removed and replaced with standard culture medium. Plates were incubated for 24, 48, or 72 hours after treatment. At each post-treatment time point, Promega CellTiter Aqueous Reagent (MTS Viability Reagent) solution was added to each well of the plate and development was performed at 37°C, 5% CO2. Plates were read every hour at absorbance at 490 nm.

[0116] Results. Data from experiment 1 showed that each individual agent had little or no cytotoxic effect on cancer or normal cells, even at higher concentrations. The results indicate that MPO alone is not cytotoxic to bladder cancer cells.

[0117] Experiment 2. An experiment was conducted to evaluate the in vitro cytotoxicity of MPO compositions against bladder cancer and normal bladder cell lines. The compositions contained MPO, H2O2, NaCl, glycine, and L-proline at various concentrations.

[0118] Methods: Cell lines were used as described in experiment 1. 24 hours before the start of the assay, cell lines were plated in 96-well plates at 2000 or 4000 cells / well. Six replicate wells were prepared for each treatment solution (a.-e., as indicated below). Each cell line was plated in four 96-well plates to observe cell viability over a 4-day period. Cells were allowed to recover overnight before treatment.

[0119] On the day of treatment, each stock solution was diluted in McCoy's culture 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) + MPO (20 nM). 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).

[0120] Media was removed from the cells and wells. Treatment solutions were added to the wells and, where applicable, treatments were performed for 45 minutes at 37°C, 5% CO2. Treatment solutions were then removed and replaced with standard culture media. Plates were incubated for 24, 48, or 72 hours after treatment. At each post-treatment time point, Promega CellTiter Aqueous Reagent (MTS Viability Reagent) solution was added to each well of the plate and development was performed at 37°C, 5% CO2. Plates were read every hour at absorbance at 490 nm.

[0121] Results. Data from experiment 2 showed that medium alone (composition A) and 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 (medium, H2O2, NaCl, glycine, and L-proline) showed some cytotoxicity to both bladder cancer cells and normal bladder cells. Compositions D and E (medium, MPO, H2O2, NaCl, glycine, and L-proline) showed the greatest cytotoxic effect on both bladder cancer cells and normal bladder cells. However, the cytotoxic effect of the combination was greater on the cancer cell line and less on the normal cell line. These results showed that 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.

[0122] Experiment 3. An experiment was conducted to evaluate the in vitro cytotoxicity of MPO compositions against bladder cancer and normal bladder cell lines at various cell densities. The compositions included MPO (1 nM), H2O2 (1 mM), NaCl (100 mM), glycine (0.21 mM), and L-proline (0.21 mM).

[0123] Methods: Cell lines were used as described in experiment 1. 24 hours before the start of the assay, cell lines were seeded in 96-well plates at various cell densities. 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. 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. Three wells were prepared for each treatment solution. Each cell line was seeded in four 96-well plates and cell viability was observed for four days. Cells were allowed to recover overnight before treatment.

[0124] On the day of treatment, each stock solution was diluted in McCoy's culture 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) + 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).

[0125] Media was removed from the cells and wells. Treatment solutions were added to the wells and, where applicable, treatments were performed for 45 minutes at 37°C, 5% CO2. Treatment solutions were then removed and replaced with standard culture media. Plates were incubated for 72 hours after treatment. Promega CellTiter Aqueous Reagent (MTS Viability Reagent) solution was added to each well of the plate and development was performed at 37°C, 5% CO2. Plates were read every hour at absorbance of 490 nm.

[0126] Results. Data from experiment 3 showed that at the concentrations of MPO (1 nM) and H2O2 (1 mM) tested, the cytotoxic effects of the MPO composition on both cancer and normal cells gradually decreased as cell density increased.

[0127] Experiment 4. Experiments were conducted to evaluate the in vitro cytotoxicity of MPO compositions against bladder cancer and normal bladder cell lines at high cell density and higher concentrations of MPO (≧20 nM) and H2O2 (10 mM). The MPO compositions also contained NaCl (100 mM), glycine (0.21 mM), and L-proline (0.21 mM).

[0128] Methods: Cell lines were used as described in experiment 1. Cell lines were plated in 96-well plates at various cell densities 24 hours prior to the start of the assay. Bladder cancer cell densities tested were: 120,000 cells / well and 400,000 cells / well. 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 plated in four 96-well plates and cell viability was observed over a four-day period. Cells were allowed to recover overnight before treatment.

[0129] On the day of treatment, each stock solution was diluted in McCoy's culture 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.

[0130] Media was removed from the cells and wells. Treatment solutions were added to the wells and, where applicable, treatments were performed for 45 minutes at 37°C, 5% CO2. Treatment solutions were then removed and replaced with standard culture media. Plates were incubated for 72 hours after treatment. Promega CellTiter Aqueous Reagent (MTS Viability Reagent) solution was added to each well of the plate and development was performed at 37°C, 5% CO2. Plates were read every hour at absorbance of 490 nm.

[0131] Results. Data from experiment 4 showed that at higher cell densities, the concentrations of MPO (≥20 nM) and H2O2 (10 mM) tested highly selectively killed human bladder cancer cells with minimal effects on normal human bladder cells. Example 4 - In vitro activity of porcine eosinophil peroxidase (pEPO) on bladder cancer cells

[0132] An experiment is designed to determine the in vitro activity of porcine eosinophil peroxidase (pEPO) on mouse bladder cancer cells. Additionally, the effect of pEPO on normal bladder cells is examined. Stock and dilution solutions of EPO, hydrogen peroxide, sodium bromide, and ethanolamine are prepared. The bladder cancer cell line T24 will be treated in the experiment, as well as the benign bladder cell line SV-HUC1 as a control. The cells will be treated with the following solutions: (1) cell culture medium ("medium"); (2) medium + hydrogen peroxide (H2O2); (3) medium + sodium bromide (NaBr); (4) medium + pEPO; (5) medium + ethanolamine; (6) medium + H2O2 + NaBr + ethanolamine; (7) medium + H2O2 + EPO + ethanolamine; (8) medium + NaBr + pEPO + ethanolamine; (9) medium + H2O2 + NaBr + pEPO; (10) medium + H2O2 + NaBr + pEPO; + ethanolamine. Both cell lines are exposed to the various treatment solutions for 15, 30, and 45 minutes, respectively. Cell growth is measured at 0, 24, 48, and 72 hours after treatment. Example 5 - In vivo bladder tumor reducing activity of porcine eosinophil peroxidase (pEPO)

[0133] An experiment is designed to determine the bladder tumor shrinkage activity of porcine eosinophil peroxidase (pEPO) in a mouse bladder cancer model in which bladder cancer cells are grown in the bladder of mice. A treatment solution containing eosinophil peroxidase, sodium bromide, and ethanolamine is mixed with an activation solution containing hydrogen peroxide in phosphate buffered saline and allowed to activate for 3-5 minutes. After activation, the enzyme solution is applied intravesically through a catheter to the bladder. The decline in tumor growth over time is measured. Example 6 - Treatment of Bladder Cancer

[0134] Treatment of patients with bladder cancer is envisioned, which treatment may include one or more of the following: · Maintenance of optimal pH of approximately 5.0 to 6.5 in the bladder environment through irrigation. Remove the mucosal lining of the bladder with dimethyl sulfoxide (DMSO) (optional). A composition including myeloperoxidase, sodium chloride, glycine, and L-proline is premixed with a solution of hydrogen peroxide to produce a treatment solution. Allow the treatment solution to incubate for 3-5 minutes. · Direct infusion / administration of treatment solutions into the bladder via a urinary catheter. Example 7 - Treatment of Bladder Cancer

[0135] A Phase 1 human clinical trial is designed to treat patients with bladder cancer, which may include one or more of the following: · Maintenance of optimal pH of approximately 5.0 to 6.5 in the bladder environment through irrigation. Remove the mucosal lining of the bladder with dimethyl sulfoxide (DMSO) (optional). · Adequate bladder irrigation with a solution of neutral ionic strength, such as lactated Ringer's solution, that can be applied without halting the metabolism of the targeted cancer cells. A composition comprising myeloperoxidase (20 nM-100 nM), sodium chloride (100 mM), glycine (0.21 mM), and L-proline (0.21 mM) is premixed with a solution of hydrogen peroxide (10 mM-100 mM) to produce a treatment solution. Allow the treatment solution to incubate for 3-5 minutes. · Direct infusion / administration of treatment solutions into the bladder via a urinary catheter. Example 8 - Treatment of Bladder Cancer

[0136] A Phase 1 human clinical trial is designed to treat patients with bladder cancer, which may include one or more of the following: · Maintenance of optimal pH of approximately 5.0 to 6.5 in the bladder environment through irrigation. Remove the mucosal lining of the bladder with dimethyl sulfoxide (DMSO) (optional). · Adequate bladder irrigation with a solution of neutral ionic strength, such as lactated Ringer's solution, that can be applied without halting the metabolism of the targeted cancer cells. Premix a composition containing myeloperoxidase (20 nM-100 nM), sodium chloride (100 mM), glycine (0.21 mM), and L-proline (0.21 mM). Direct injection / administration of the composition into the bladder via a urinary catheter. Allow the composition to remain in the bladder for approximately 10 minutes. Direct infusion / administration of hydrogen peroxide (10 mM to 100 mM) into the bladder via a urethral catheter to activate the myeloperoxidase composition. Example 9 - Treatment of Bladder Cancer

[0137] Treatment of patients with bladder cancer is envisioned, which treatment may include one or more of the following: · Maintenance of optimal pH of approximately 5.0 to 6.5 in the bladder environment through irrigation. Remove the mucosal lining of the bladder with dimethyl sulfoxide (DMSO) (optional). · Adequate bladder irrigation with a solution of neutral ionic strength, such as lactated Ringer's solution, that can be applied without halting the metabolism of the targeted cancer cells. Direct injection / administration via a urinary catheter of a composition comprising a haloperoxidase, a halide, e.g. bromide or chloride, a peroxide, or a peroxide generating oxidase and a substrate for the oxidase. Example 10 - Treatment of Bladder Cancer

[0138] Treatment of patients with bladder cancer is envisioned, which treatment may include one or more of the following: · Maintenance of optimal pH of approximately 5.0 to 6.5 in the bladder environment through irrigation. Remove the mucosal lining of the bladder with dimethyl sulfoxide (DMSO) (optional). · Adequate bladder irrigation with a solution of neutral ionic strength, such as lactated Ringer's solution, that can be applied without halting the metabolism of the targeted cancer cells. A composition containing eosinophil peroxidase, sodium bromide, and ethanolamine is premixed with a solution of hydrogen peroxide to produce a treatment solution. Incubate the treatment solution for 3-5 minutes. · Direct infusion / administration of treatment solutions into the bladder via a urinary catheter. Example 11 - Treatment of Bladder Cancer

[0139] Treatment of patients with bladder cancer is envisioned, which treatment may include one or more of the following: · Maintenance of optimal pH of approximately 5.0 to 6.5 in the bladder environment through irrigation. Remove the mucosal lining of the bladder with dimethyl sulfoxide (DMSO) (optional). · Adequate bladder irrigation with a solution of neutral ionic strength, such as lactated Ringer's solution, that can be applied without halting the metabolism of the targeted cancer cells. · Premix a composition containing eosinophil peroxidase, sodium bromide and ethanolamine. Direct injection / administration of the composition into the bladder via a urinary catheter. Allow the composition to remain in the bladder for approximately 10 minutes. Direct infusion / administration of hydrogen peroxide into the bladder via a urinary catheter activates the myeloperoxidase composition.

[0140] Although illustrative embodiments have been illustrated and described, including the best mode known to the inventors for carrying out the invention, those of ordinary skill in the art will recognize that the disclosure can be practiced with variations in the disclosed structures, materials, compositions, and methods and that such variations are considered within the scope of the disclosure.

[0141] The discussion or reference of any piece of prior art in the specification shall not be taken as an admission that that prior art is part of the common general knowledge of the parties to the specification.

[0142] The contents of all references and published patents and patent applications cited throughout the application are hereby incorporated by reference.

Claims

1. A pharmaceutical composition for treating solid tumors in patients, comprising myeloperoxidase, hydrogen peroxide or a source of hydrogen peroxide, sodium chloride, and one or more amino acids.

2. The pharmaceutical composition according to claim 1, wherein the hydrogen peroxide source is the oxidase that produces hydrogen peroxide in the presence of a substrate for the peroxide-producing oxidase.

3. The pharmaceutical composition according to claim 2, wherein the peroxide-producing oxidase is glucose oxidase and the substrate is glucose.

4. The one or more amino acids mentioned above are glycine, L-alanine, D-alanine, L-alanine anhydrous, L-glutamine, L-glutamic acid, glycine anhydrous, 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- The pharmaceutical composition according to claim 1, selected from the group comprising valine, beta-amino acids such as beta-alanine, L-beta-homoleucine, D-beta-homoleucine, 3-aminobutanoic acid, L-2,3-diaminopropionic acid monohydrochloride, D-2,3-diaminopropionic acid monohydrochloride, L-3-aminoisobutyric acid, D-3-aminoisobutyric acid, ethyl 3-aminobutyrate, methyl sarcosine hydrochloride, and nipecotinic acid, or their alkyl esters or pharmaceutically acceptable salts.

5. The pharmaceutical composition according to claim 4, wherein the amino acids are glycine and L-proline.

6. The pharmaceutical composition according to claim 1, wherein the solid tumor is a sarcoma, malignant tumor, or lymphoma.

7. The pharmaceutical composition according to claim 1, wherein the solid tumor is selected from the group including breast cancer, lung and bronchial cancer, prostate cancer, colon and rectal cancer, malignant melanoma of the skin, bladder cancer, cervical cancer, kidney and renal pelvis cancer, endometrial cancer, pancreatic cancer, thyroid cancer, liver cancer, brain cancer, and spinal cord cancer.

8. The pharmaceutical composition according to claim 7, wherein the solid tumor is bladder cancer.

9. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises (a) a first composition comprising myeloperoxidase, sodium chloride, glycine, and L-proline; and (b) a second composition comprising a peroxide.

10. The pharmaceutical composition according to claim 9, wherein the first composition and the second composition are pre-mixed before administration.

11. The pharmaceutical composition according to claim 1, comprising myeloperoxidase at a concentration of 20 nM to 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 to 100 mM.

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

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

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