ES3035164T3 - Use of dnase to improve safety and efficacy of cancer radiotherapy - Google Patents

Abstract

The invention relates to the use of a deoxyribonuclease (DNase) enzyme for preventing or ameliorating the toxicity associated with various cytostatic and/or cytotoxic chemotherapeutic compounds and radiotherapy. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Use of DNase to improve the safety and efficacy of cancer radiotherapy.

Field of the invention

The invention relates to a deoxyribonuclease (DNase) enzyme for use in preventing or ameliorating at least one side effect of radiotherapy in a subject suffering from cancer and receiving said radiotherapy, by preventing or ameliorating a toxicity associated with said radiotherapy, wherein the DNase enzyme is to be administered parenterally.

Background of the invention

Cancers are a significant cause of death in humans. Currently, the main cancer therapies are surgery, radiotherapy, and cytotoxic chemotherapy. Despite advances in chemotherapy treatments, most known chemotherapies are associated with serious side effects, such as myelopathy, liver disease, digestive disorders (e.g., nausea, vomiting, anorexia, diarrhea, constipation), pulmonary failure, dermatopathies, nervous system disorders, endocrine disorders, genital disorders, cardiovascular disorders, liver disease, pancreatic disorders, nephropathy, bladder problems, hyperuricemia, decreased immunocompetence, infections, light hypersensitivity, hair loss, etc. (2-5). These side effects are life-threatening or severely debilitating and cause significant chemotherapy-related morbidity and mortality.

One of the major complications of cancer chemotherapy is damage to bone marrow cells or suppression of their function. Specifically, chemotherapy damages or destroys hematopoietic precursor cells, which are found primarily in the bone marrow and spleen, preventing the production of new blood cells (granulocytes, lymphocytes, erythrocytes, monocytes, platelets, etc.). Many cancer patients die from infections or other consequences of hematopoietic failure following chemotherapy. Chemotherapeutic agents can also result in subnormal platelet formation, resulting in a propensity to hemorrhage. Inhibition of erythrocyte production can lead to anemia. It has also been recently recognized that the development of more potent cytotoxic therapies and more effective chemotherapy regimens for a wider range of malignancies significantly increases the frequency of a serious toxic adverse event called tumor lysis syndrome (TLS) (16). TLS is a group of metabolic complications that can occur as a result of the administration of cytotoxic therapies, most often in the context of chemotherapy for lymphomas and leukemias, and are caused by the breakdown products of dying cells.

The main reason chemotherapy is so debilitating and symptoms so severe is that chemotherapy drugs are often unable to differentiate between normal, healthy cells and the tumor cells they are designed to target. Another mechanism responsible for chemotherapy-related toxicity is the toxic effects of cellular components released by cells undergoing necrosis or apoptosis as a result of chemotherapy-induced cell death.

Side effects associated with chemotherapy drugs limit the frequency and dosage at which these drugs can be administered, leading to reduced efficacy.

As the concept of systemic cytotoxic chemotherapy has evolved, much research effort has been devoted to identifying potential approaches to attenuate chemotherapy-related toxicity and avoid cessation of patient exposure to chemotherapeutic agents. One possibility is the modulation of chemotherapeutic dosage and schedule, which would allow for the development of less toxic dosing modalities (6). Others have proposed dietary approaches, such as fasting or restriction of specific nutrients during and after chemotherapy (7); and supplementation of the patient's diet with various specific dietary amino acids (8). The use of certain specific drug metabolic antidotes, particularly acyl uridine or cytidine derivatives, for the prevention of pyrimidine nucleoside analog-induced toxicity is disclosed in U.S. Patent No. 7,776,838. The use of chromanol glucoside for the prevention of alkylating agent-induced toxicity is disclosed in U.S. Patent No. 7,462,601. The use of antioxidants has also been proposed to prevent anthracycline-induced cardiotoxicity (9). Attenuation of tissue-specific toxicity of chemotherapeutic drugs, in particular the prevention of mucositis by topical application of alpha-interferon or beta-interferon (U.S. Patent No. 5,017,371); prevention of side effects in the stomach and intestine by selective analogs of glucagon-like peptide-2 (GLP-2) (U.S. Patent No. 8,642,727); prevention of liver damage by adenosine A2B receptor antagonist (U.S. Patent No. 8,188,099); and prevention of prostate damage by inhibitor of the growth factor IGFBP-1 (U.S. Patent No. 8,211,700) have also been disclosed. The use of the enzyme alkaline phosphatase to improve the overall toxicity of chemotherapy and to maintain a healthy mass of muscle and adipose tissue in mammals receiving chemotherapy has been disclosed in U.S. Patent No. 8,460,654.

The development of new compositions and procedures to prevent or ameliorate chemotherapy-related toxicity is highly desired.

Current radiation therapies for the treatment of cancer provide significant benefits for patients with early-stage, radiosensitive cancers, but these benefits are much less significant for patients with radioresistant tumors (e.g., brain or pancreatic cancers) and for patients with late-stage tumors. In these patients, the radiation required to eradicate the tumor can cause intolerable or fatal damage. This is especially the case for pediatric patients, whose rapidly developing normal tissues are often more radiosensitive than their tumors, and therefore cannot tolerate radiation therapy that would be curative for adults with the same disease. Damage to normal tissue limits the use of radiation therapy treatments in young cancer patients, patients with central nervous system cancers, radiation-resistant cancers, and late-stage cancers with large tumors.

The development of new compositions and procedures to prevent or ameliorate radiotherapy-related toxicity is highly desired.

Summary of the invention

As specified in the Background Section, there is a great need in the art to develop new compositions and methods to prevent or ameliorate toxicity related to chemotherapy and radiotherapy.

As indicated above, the invention relates to a deoxyribonuclease (DNase) enzyme for use in preventing or ameliorating at least one side effect of radiotherapy in a subject suffering from cancer and receiving said radiotherapy, by preventing or ameliorating a toxicity associated with said radiotherapy, wherein the DNase enzyme is to be administered parenterally.

An aspect of the present disclosure is a method of preventing or ameliorating a toxicity associated with a cytostatic and/or cytotoxic chemotherapy in a subject having a cancer and receiving or contemplated to receive such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme, wherein said amount of the DNase enzyme is effective to prevent or ameliorate at least one side effect of said chemotherapy. Another aspect of the present disclosure is a method of increasing the effectiveness of a cytostatic and/or cytotoxic chemotherapy in a subject having a cancer and receiving or contemplated to receive such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme, wherein said amount of the DNase enzyme is effective to prevent or ameliorate at least one side effect of said chemotherapy and wherein the administration of DNase results in the prevention or amelioration of the toxicity associated with said chemotherapy.

In a specific variant of the above procedures, said side effect of said chemotherapy is selected from the group consisting of body weight loss, bone marrow toxicity, catabolic changes in blood biochemistry, myocardial necrosis, gastrointestinal toxicity, suppression of immunity and neutropenia.

A further aspect of the disclosure is a method of preventing or ameliorating a catabolic state leading to body weight loss associated with cytostatic and/or cytotoxic chemotherapy in a subject having a cancer and receiving or contemplating receiving such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme, wherein said amount of the DNase enzyme is effective to prevent or ameliorate the catabolic state leading to body weight loss associated with said chemotherapy. In a specific variation, the chemotherapy is an anthracycline-containing therapy.

Yet another aspect of the present disclosure is a method of preventing or ameliorating bone marrow toxicity and/or catabolic changes in blood chemistry associated with cytostatic and/or cytotoxic chemotherapy in a subject having a cancer and receiving or contemplating receiving such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme, wherein said amount of the DNase enzyme is effective to prevent or ameliorate bone marrow toxicity and/or catabolic changes in blood chemistry associated with said chemotherapy. In a specific variation, the chemotherapy is an anthracycline-containing therapy.

Another aspect of the present disclosure is a method of preventing or ameliorating cardiotoxicity associated with cytostatic and/or cytotoxic chemotherapy in a subject having a cancer and receiving or contemplating receiving such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme, wherein said amount of the DNase enzyme is effective to prevent or ameliorate cardiotoxicity associated with said chemotherapy. In a specific variation, the cardiotoxicity is myocardial necrosis. In a specific variation, the chemotherapy is an anthracycline-containing therapy.

Another aspect of the present disclosure is a method of preventing or ameliorating gastrointestinal toxicity associated with cytostatic and/or cytotoxic chemotherapy in a subject having a cancer and receiving or contemplating receiving such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme, wherein said amount of the DNase enzyme is effective to prevent or ameliorate gastrointestinal toxicity associated with said chemotherapy. In a specific variation, the chemotherapy is a therapy containing 5-fluorouracil and/or etoposide.

Yet another aspect of the present disclosure is a method for preventing or ameliorating a suppression of immunity associated with cytostatic and/or cytotoxic chemotherapy in a subject having a cancer and receiving or contemplating receiving such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme, wherein said amount of the DNase enzyme is effective to prevent or ameliorate the suppression of immunity associated with said chemotherapy. In a specific variation, the chemotherapy is a taxane-containing therapy.

A further aspect of the present disclosure is a method of preventing or ameliorating neutropenia associated with cytostatic and/or cytotoxic chemotherapy in a subject having a cancer and receiving or contemplating receiving such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme, wherein said amount of the DNase enzyme is effective to prevent or ameliorate neutropenia associated with said chemotherapy. In a specific variation, the chemotherapy is a cyclophosphamide-containing therapy.

In a variation of any of the foregoing procedures, chemotherapy comprises the administration of one or more compounds selected from the group consisting of antimetabolites, alkylating agents, anticancer antibiotics, microtubule-targeting agents, topoisomerase inhibitors, alkaloids, and targeted therapeutics.

In a variation of any of the above procedures, chemotherapy comprises the administration of one or more compounds selected from the group consisting of anthracycline, doxorubicin, 5-fluorouracil (5-FU), etoposide, taxane, and cyclophosphamide.

In a variation of either of the above procedures, the DNase enzyme is administered during a cycle of chemotherapy. In another variation of either of the above procedures, the DNase enzyme is administered after a cycle of chemotherapy.

Another aspect of the present disclosure is a method for preventing or ameliorating a toxicity associated with radiotherapy in a subject suffering from cancer and receiving said radiotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme, wherein said amount of the DNase enzyme is effective to prevent or ameliorate at least one side effect of said radiotherapy.

Another aspect of the present disclosure is a method for increasing the effectiveness of radiotherapy in a subject suffering from cancer and receiving said radiotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme, wherein said amount of the DNase enzyme is effective to prevent or ameliorate at least one side effect of said radiotherapy and wherein the administration of DNase results in the prevention or amelioration of toxicity associated with said radiotherapy.

In a variation of the previous two procedures, the side effect of radiation therapy is selected from the group consisting of skin irritation or damage, fatigue, nausea, vomiting, fibrosis, intestinal damage, memory loss, infertility, and a second cancer.

Yet another aspect of the present disclosure is a method for preventing or ameliorating body weight loss associated with radiotherapy in a subject suffering from cancer and receiving said radiotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme, wherein said amount of the DNase enzyme is effective to prevent or ameliorate body weight loss associated with said radiotherapy.

In a variation of the three procedures above, radiotherapy is either external beam radiotherapy or systemic radioisotope therapy.

In a variation of the three procedures above, the DNase enzyme is administered during a course of radiation therapy. In another variation, the DNase enzyme is administered after a course of radiation therapy.

In one variation of any of the above methods, the DNase enzyme is DNase I (e.g., recombinant human DNase I or bovine pancreatic DNase I) or an analog thereof (e.g., DNase X, DNase gamma, or DNAS1L2). In another variation of any of the above methods, the DNase enzyme is DNase II. In one embodiment, the DNase enzyme has an extended half-life (e.g., is conjugated to polysialic acid or is protected from actin binding by modification of the actin binding site).

In a variation of any of the above methods, the therapeutically effective amount of a DNase enzyme is at least 0.5 mg/kg/day or at least 1000 Kunitz units (KU)/kg/day, preferably at least 1.5 mg/kg/day or at least 3000 KU/kg/day. In a variation of any of the above methods, the therapeutically effective amount of a DNase enzyme is 0.5 to 100 mg/kg/day or 1000 to 200000 KU/kg/day, preferably 0.5 to 50 mg/kg/day or 1000 to 100000 KU/kg/day, more preferably 1.5 to 50 mg/kg/day or 3000 to 100000 KU/kg/day, even more preferably 10 to 50 mg/kg/day or 20000 to 100000 KU/kg/day.

In a variation of either of the above procedures, the DNase enzyme is administered intravenously or intraperitoneally. In a variation of either of the above procedures, the DNase enzyme is administered enterally (e.g., orally).

In a variation of any of the above procedures, the subject is a human being.

These and other aspects of the present invention will be apparent to those of ordinary skill in the art from the following description, claims and drawings.

Brief description of the drawings

Figure 1 is a bar graph showing the area of myocardial necrosis in deceased rats challenged with sublethal IV doses of doxorubicin (7.5 mg/kg) and daily IP injections of recombinant human DNase I (15 mg/kg) (black bar) or daily IP injections of placebo (WFI) (hatched diagonal bar). Serial myocardial microscopy samples from each heart were analyzed using an automated video analyzer to quantify necrotic areas. The sum of the necrotic area (Sna; nm2) was calculated as the sum of the necrotic areas in 30 serial sections from each individual autopsied heart (n=3 for DNase I-treated rats - black bar; n=5 for placebo-treated rats - hatched diagonal bar).

Figures 2A-B show photographs of stained stomachs from rats treated with oral etoposide (200 mg/kg) and 5-fluorouracil (5-FU; 400 mg/kg) followed by (A) IV treatment with recombinant human DNase I (50 mg/kg) or (B) IV placebo (WFI). The stomach in panel (B) showed multiple erosions and ulcers, whereas the stomach in panel (A) was grossly unremarkable.

Figure 3 is a graph summarizing the white blood cell (WBC) count dynamics in the three experimental groups, in which neutropenia was induced by a single IP injection of cyclophosphamide (200 mg/kg) followed by no treatment (Group I) or IV treatment with recombinant human DNase I (25 mg/kg) (Group II) or SC treatment with recombinant human GM-CSF (200 pg/kg) (Group III). The vertical axis represents WBCs with units of 109 WBC/L. The horizontal axis represents the number of days after cyclophosphamide injection. The figure demonstrates the ameliorative effect of DNase treatment on neutropenia induced by an alkylating agent such as cyclophosphamide.

Figure 4 is a bar graph showing a summary of the survival data of animals transplanted with L1210 leukemia cells to induce cancer followed by treatment with cytosine arabinoside (AraC) and/or DNase II. The six experimental groups are represented on the horizontal axis: 1. Negative Control; 2. AraC (positive control); 3. AraC DNase II (15 mg/kg); 4. AraC DNase II (45 mg/kg); 5. DNase II (15 mg/kg); 6. DNase II (45 mg/kg). The figure demonstrates the synergistic effects of DNase and AraC treatment on the survival rates of leukemia-bearing mice.

Detailed description of the invention

The present inventors have previously demonstrated that the enzyme deoxyribonuclease (DNase) is a useful agent for treating cancer (see, e.g., U.S. Patent Application Pub. No.

2010/0150903 and US Patent No. 7,612,032), a property that has been recently confirmed by several authors (10-12). However, it has also been reported that DNase lacks a significant influence on the tolerability of antiproliferative chemotherapy (15) and may even contribute significantly to increasing chemotherapy toxicity (17).

The present invention is based on an unexpected discovery by the inventors that DNase enzymes not only increase the efficacy of cytostatic and/or cytotoxic chemotherapy, but also significantly decrease chemotherapy-related toxicity. This discovery is especially surprising in light of previous reports of the lack of influence of DNase on chemotherapy tolerability (15) and the contribution of DNase to the increased nephrotoxicity of chemotherapy (17). The ability of DNase to prevent and/or ameliorate chemotherapy-related toxicity is shared by different types of DNase enzymes and is drug- and tissue-independent, making DNases very desirable and potent candidates for adjunctive therapy to chemotherapy. In fact, as demonstrated in the Examples section below, several types of DNase enzymes (e.g., DNase I, DNase gamma, and DNase II) can be used to enhance the toxicity of various types of chemotherapeutic agents such as Doxorubicin (an anthracycline antibiotic that acts by intercalating DNA), 5-FU (antimetabolite that acts by inhibiting thymidylate synthase), Etoposide (topoisomerase inhibitor), Taxane (microtubule disruptor), and Cyclophosphamide (alkylating agent).

Definitions

The terms "cytostatic and/or cytotoxic chemotherapy" and "chemotherapy" are used interchangeably herein to refer to a therapy comprising the administration of a cytostatic and/or cytotoxic agent.

The terms "anticancer agent" and "anticancer chemotherapeutic agent" are used herein to refer to any chemical compound, which is used to treat cancer. Anticancer chemotherapeutic agents are well known in the art (see, e.g., Gilman A. G., et al., The Pharmacological Basis of Therapeutics, 8th Ed., Seq 12:1202-1263 (1990)). Specific examples of chemotherapeutic agents are provided throughout the specification.

The term "chemotherapy side effect," as used herein, refers to an unwanted and unintended, though not necessarily unexpected, result of chemotherapy.

As used herein, the terms "radiotherapy", "radiation therapy", and "RT" are used interchangeably to refer to the medical use of ionizing radiation as part of a cancer treatment to damage the DNA of malignant cells, either directly or by creating charged particles within the affected cells that damage the DNA. Commonly used types of radiotherapy encompassed by the present invention include, for example, external beam radiotherapy (EBRT or XRT), brachytherapy/sealed source radiotherapy, and systemic radioisotope radiotherapy/unsealed source radiotherapy.

The terms "side effect of radiation therapy" or "radiation therapy side effect," as used herein, refer to an unwanted and unintended, though not necessarily unexpected, result of radiation therapy. The side effects that develop will depend on the area of the body treated, the daily dose administered, the total dose administered, the patient's general medical condition, and other treatments administered at the same time. They may include, for example, skin irritation or damage, fatigue, nausea, vomiting, fibrosis, intestinal damage, memory loss, infertility, or a second cancer.

The term "catabolic state" as used herein refers to a condition characterized by rapid weight loss and loss of fat mass and skeletal muscle, which may occur in the setting of chemotherapy or chemoradiotherapy. Associated clinical events include, for example, immunosuppression, muscle weakness, predisposition to pulmonary embolism, thrombophlebitis, and altered stress response.

As used herein, the terms "deoxyribonuclease" and "DNase" are used to refer to any enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone. A wide variety of deoxyribonucleases are known and can be used in the methods of the present disclosure. Non-limiting examples of DNases useful in the methods of the present disclosure include, for example, DNase I (e.g., human recombinant DNase I or bovine pancreatic DNase I), DNase I analogs (such as, for example, DNase X, DNase gamma, and DNAS1L2), DNase II, phosphodiesterase I, lactoferrin, and acetylcholinesterase. Also encompassed by the present invention are DNase enzymes having a long half-life (e.g., DNase enzymes conjugated to polysialic acid or protected from actin binding by modification of the actin binding site; see, e.g., Gibson et al., (1992) J. Immunol. Methods, 155, 249-256). DNase I cleaves DNA preferentially at phosphodiester bonds adjacent to a pyrimidine nucleotide, resulting in 5'5'-phosphate-terminated polynucleotides with a free hydroxyl group at the 3' position, on average producing tetranucleotides. DNase I acts on single-stranded DNA, double-stranded DNA, and chromatin.

The term "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "approximately" may mean within an acceptable standard deviation, according to practice in the art. Alternatively, "approximately" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or methods, the term may mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise indicated, the term "approximately" is implied and in this context means within an acceptable error range for the particular value.

In the context of the present invention, as it relates to any of the disease conditions recited herein, the terms "treat," "treatment," and the like mean alleviating or mitigating at least one symptom associated with said condition, or slowing or reversing the progression of said condition. Within the meaning of the present invention, the term "treat" also denotes halting, delaying the onset (i.e., the period prior to clinical manifestation of a disease), and/or reducing the risk of developing or worsening a disease. For example, in relation to cancer, the term "treat" may mean eliminating or reducing a patient's tumor burden, or preventing, delaying, or inhibiting metastasis, etc.

As used herein, the term "therapeutically effective" as applied to dose or amount refers to that amount of a compound or pharmaceutical composition that is sufficient to give rise to a desired activity upon administration to a subject in need thereof. In the context of the present invention, when the term "therapeutically effective" is used in connection with the use of deoxyribonuclease (DNase) for ameliorating or preventing the side effects of a cytostatic and/or cytotoxic chemotherapy, it refers to an amount of DNase or a pharmaceutical composition containing DNase that is effective in ameliorating or preventing at least one side effect of said cytostatic and/or cytotoxic chemotherapy. Note that when a combination of active ingredients is administered (e.g., a combination of DNase and another compound effective in ameliorating or preventing the side effects of the chemotherapy), the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.

The phrase "pharmaceutically acceptable," as used in connection with the compositions disclosed herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce adverse reactions when administered to a subject (e.g., a mammal such as a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more specifically, humans.

The term "targeted therapeutics" is used to refer to a class of chemical and biological compounds that may be effective in patients whose cancers have a specific molecular target, but may not be effective in the absence of such a target (18).

As used herein, the term "subject" refers to any mammal. In a preferred embodiment, the subject is a human.

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

In accordance with the present invention, conventional techniques of pharmacology and molecular biology techniques within the skill of the art may be employed. These techniques are explained in detail in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985)); Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed. (1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); inter alia.

Therapeutic procedures

An aspect of the present disclosure is a method of preventing or ameliorating a toxicity associated with cytostatic and/or cytotoxic chemotherapy in a subject suffering from a cancer and receiving or contemplated to receive such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme (e.g., DNase I (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, for example, DNase X, DNase gamma or DNAS1L2), DNase II, phosphodiesterase I, lactoferrin or acetylcholinesterase), wherein said amount of the DNase enzyme is effective to prevent or ameliorate at least one side effect of said chemotherapy.

Another aspect of the present disclosure is a method for increasing the efficacy of a cytostatic and/or cytotoxic chemotherapy in a subject suffering from cancer and receiving or contemplated to receive such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme (e.g., DNase I (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, for example, DNase X, DNase gamma or DNAS1L2), DNase II, phosphodiesterase I, lactoferrin or acetylcholinesterase), wherein said amount of the DNase enzyme is effective to prevent or ameliorate at least one side effect of said chemotherapy and wherein the administration of DNase results in the prevention or amelioration of the toxicity associated with said chemotherapy.

DNase enzymes can be used to enhance the toxicity of a wide range of different chemotherapeutic agents. Non-limiting examples of such agents include antimetabolites such as pyrimidine analogs (e.g., 5-fluorouracil [5-FU], floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folate antagonists, and related inhibitors (e.g., mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents, including natural products such as vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxanes (e.g., paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine, epidipodophyllotoxins (e.g., etoposide, teniposide), DNA damaging agents (e.g., actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, nedaplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, aclarubicin, purarubicin, hexamethasone, oxaliplatin, iphosphamide, melphalan, merchlorhexamine, mitomycin, mitoxantrone, nitrosourea, nimustine, ranimustine, estramustine, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenetetraphosphate, and etoposide (VP16)]; antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin), pleomycin, peplomycin, mitomycins (e.g., mitomycin C), actinomycins (e.g., actinomycin D), zinostatin; enzymes (e.g., L-asparaginase); neocarzinostatin; antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide and analogues, imidazole carboxamide, melphalan, chlorambucil, nitrogen mustard hydrochloride-N-oxide, ifosfamide), ethyleneimines and methylmelamines (e.g., hexamethylmelamine, thiotepa, carboquone, triethylene thiophosphamide), alkyl sulfonates (e.g., busulfan, isoprosulfan tosylate), nitrosoureas (e.g., carmustine (BCNU) and analogues, streptozocin), trazenes-dacarbazinine (DTIC); epoxide compounds (e.g., mitobronitol); antiproliferative/antimitotic antimetabolites, such as folic acid analogues (e.g., methotrexate); platinum coordination complexes (e.g., cisplatin, carboplatin, cisplatin), procarbazine, hydroxyurea, mitotane, Aminoglutethimide; hormones, hormone analogues (e.g., estrogens, tamoxifen, goserelin, bicalutamide, nilutamide), and aromatase inhibitors (e.g., letrozole, anastrozole); antisecretory agents (e.g., breveldin); immunosuppressants (e.g., cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (e.g., trastuzumab); cell cycle inhibitors and differentiation inducers (e.g., tretinoin); mTOR inhibitors, topoisomerase inhibitors (e.g., doxorubicin (Adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, irinotecan); growth factor signal transduction kinase inhibitors; inducers of mitochondrial dysfunction; chromatin disruptors; sobuzoxane; tretinoin; pentostatin; flutamide; porfimer natrium; fadrozole; procarbazine; aceglatone; radioimmunotherapy (RIT) compounds (e.g., ibritumomab tiuxetan, iodine (131I) tositumomab); and targeted radionuclide therapy (TRT) compounds (e.g., samarium-153-EDTMP, strontium-89 chloride).

Non-limiting examples of side effects of cytostatic and/or cytotoxic chemotherapy that may be prevented or ameliorated by administering DNase enzymes according to the methods disclosed herein include, for example, bone marrow toxicity, neutropenia, myelopathy (e.g., leukopenia, granulocytopenia, lymphopenia, thrombocytopenia, erythropenia); hematopathy (e.g., plasma fibrinogenopenia); catabolic changes in blood biochemistry; gastrointestinal disorders (e.g., nausea, vomiting, anorexia, weight loss, fullness in the stomach, diarrhea, constipation, stomatitis, esophagitis); pulmonary insufficiency (e.g., chronic pneumonia, pulmonary fibrosis, ARDS, ALS, pulmonary emboli); dermatopathy (e.g., keratinization, pachymenia, chromatosis, epilation, rash, nail alternans, cancer-induced alopecia); Nervous system disorders (e.g., paresthesia, depression, deep areflexia, neuroparalysis, hearing disorder, allolalia, disorientation, neurological manifestation, cerebellar ataxia, somnolence, coma, vertigo, urination frequency, frequency of urge to defecate); endocrine disorders (e.g., pituitary disorder, adrenal disorder, hyperglycemia, hypoglycemia); genital disorders (e.g., hyposexuality, oligospermia, gynecomastia, menstrual disorder); cardiovascular disorders (e.g., myocardial necrosis, cardiomyopathy, arrhythmia, hypotension, tachycardia, heart failure); liver disease, pancreatic disorders, nephropathy, bladder problems, hyperuricemia, decreased immunocompetence, and infection.

Another aspect of the present disclosure is a method of preventing or ameliorating a toxicity associated with radiotherapy in a subject suffering from cancer and receiving said radiotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme (e.g., DNase I (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, e.g., DNase X, DNase gamma or DNAS1L2), DNase II, phosphodiesterase I, lactoferrin or acetylcholinesterase), wherein said amount of the DNase enzyme is effective to prevent or ameliorate at least one side effect of said radiotherapy.

Another aspect of the present disclosure is a method of increasing the effectiveness of a radiation therapy in a subject suffering from cancer and receiving said radiation therapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme (e.g., DNase I (e.g., recombinant human DNase 1 or bovine pancreatic DNase I), DNase I analogs (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II, phosphodiesterase I, lactoferrin, or acetylcholinesterase), wherein said amount of the DNase enzyme is effective to prevent or ameliorate at least one side effect of said radiation therapy and wherein the administration of DNase results in the prevention or amelioration of toxicity associated with said radiation therapy.

In accordance with the present invention, DNase enzymes can be used to improve the toxicity and increase the efficacy of various types of radiotherapy, including, for example, external beam radiotherapy (EBRT or XRT), brachytherapy/sealed source radiotherapy, and systemic radioisotope radiotherapy/unsealed source radiotherapy.

Non-limiting examples of side effects of radiation therapy that may be prevented or ameliorated by administering DNase enzymes according to the methods disclosed herein include, for example, skin irritation or damage, fatigue, nausea, vomiting, fibrosis, intestinal damage, memory loss, infertility, and a second cancer.

The methods disclosed herein may be used in subjects suffering from a wide range of cancers, which subjects are undergoing anti-cancer chemotherapy treatments that result in deleterious side effects. Non-limiting examples of relevant cancers include, for example, breast cancer, prostate cancer, multiple myeloma, transitional cell carcinoma, lung cancer (e.g., non-small cell lung cancer [NSCLC]), kidney cancer, thyroid cancer, leukemia (e.g., chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia), lymphoma (e.g., B-cell lymphoma, T-cell lymphoma, non-Hodgkins lymphoma, Hodgkins lymphoma), head and neck cancer, esophageal cancer, stomach cancer, colon cancer, intestinal cancer, colorectal cancer, rectal cancer, pancreatic cancer, liver cancer, bile duct cancer, gallbladder cancer, ovarian cancer, uterine endometrial cancer, vaginal cancer, cervical cancer, bladder cancer, neuroblastoma, sarcoma, osteosarcoma, malignant melanoma, cancer squamous cell, bone cancer, including primary bone cancers (e.g., osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant fibrous histiocytoma, adamantinoma, giant cell tumor, and chordoma) and secondary (metastatic) bone cancers, soft tissue sarcoma, basal cell carcinoma, angiosarcoma, hemangiosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, testicular cancer, uterine cancer, gastrointestinal cancer, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Waldenstroom's macroglobulinemia, papillary adenocarcinomas, cystadenocarcinoma, bronchogenic carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, epithelial carcinoma, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, medullary carcinoma, thymoma, sarcoma, etc.

In one embodiment, a method is provided for preventing or ameliorating a catabolic state leading to body weight loss associated with cytostatic and/or cytotoxic chemotherapy (e.g., doxorubicin therapy) in a subject having a cancer and receiving or contemplated for receiving such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme (e.g., DNase I (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II, phosphodiesterase I, lactoferrin, or acetylcholinesterase), wherein said amount of the DNase enzyme is effective to prevent or ameliorate the catabolic state leading to body weight loss associated with said chemotherapy.

In another variation, a method of preventing or ameliorating bone marrow toxicity and/or catabolic changes in blood chemistry associated with cytostatic and/or cytotoxic chemotherapy (e.g., doxorubicin therapy) in a subject having a cancer and receiving or contemplated for receiving such chemotherapy is disclosed, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme (e.g., DNase I (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, for example, DNase X, DNase gamma, or DNAS1L2), DNase II, phosphodiesterase I, lactoferrin, or acetylcholinesterase), wherein said amount of the DNase enzyme is effective to prevent or ameliorate bone marrow toxicity and/or catabolic changes in blood chemistry associated with said chemotherapy.

In yet another embodiment, a method is disclosed for preventing or ameliorating cardiotoxicity (e.g., myocardial necrosis) associated with cytostatic and/or cytotoxic chemotherapy (e.g., doxorubicin therapy) in a subject having a cancer and receiving or contemplated to receive such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme (e.g., DNase I (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II, phosphodiesterase I, lactoferrin, or acetylcholinesterase), wherein said amount of the DNase enzyme is effective to prevent or ameliorate cardiotoxicity associated with said chemotherapy.

In a further variation, a method is disclosed for preventing or ameliorating gastrointestinal toxicity associated with a cytostatic and/or cytotoxic chemotherapy (e.g., 5-fluorouracil/etoposide combination chemotherapy) in a subject having a cancer and receiving or contemplated for receiving such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme (e.g., DNase I (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, for example, DNase X, DNase gamma, or DNAS1L2), DNase II, phosphodiesterase I, lactoferrin, or acetylcholinesterase), wherein said amount of the DNase enzyme is effective to prevent or ameliorate gastrointestinal toxicity associated with said chemotherapy.

In another variation, a method is disclosed for preventing or ameliorating immune suppression associated with cytostatic and/or cytotoxic chemotherapy (e.g., taxane therapy) in a subject having a cancer and receiving or contemplated to receive such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme (e.g., DNase (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, for example, DNase X, DNase gamma, or DNAS1L2), DNase II, phosphodiesterase I, lactoferrin, or acetylcholinesterase), wherein said amount of the DNase enzyme is effective to prevent or ameliorate immune suppression associated with said chemotherapy.

In a further variation, a method is disclosed for preventing or ameliorating neutropenia associated with cytostatic and/or cytotoxic chemotherapy (e.g., cyclophosphamide therapy) in a subject having a cancer and receiving or contemplated for receiving such chemotherapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme (e.g., DNase I (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, for example, DNase X, DNase gamma, or DNAS1L2), DNase II, phosphodiesterase I, lactoferrin, or acetylcholinesterase), wherein said amount of the DNase enzyme is effective to prevent or ameliorate neutropenia associated with said chemotherapy.

In yet another embodiment, a method is disclosed for preventing or ameliorating body weight loss associated with radiation therapy in a subject having a cancer and receiving radiation therapy, said method comprising administering to the subject a therapeutically effective amount of a DNase enzyme (e.g., DNase I (e.g., recombinant human DNase I or bovine pancreatic DNase I), DNase I analogs (such as, e.g., DNase X, DNase gamma, or DNAS1L2), DNase II, phosphodiesterase I, lactoferrin, or acetylcholinesterase), wherein said amount of the DNase enzyme is effective to prevent or ameliorate body weight loss associated with said radiation therapy.

In a variation of any of the above methods, the therapeutically effective amount of a DNase enzyme is at least 0.5 mg/kg/day or at least 1000 Kunitz units (KU)/kg/day, preferably at least 1.5 mg/kg/day or at least 3000 KU/kg/day. In one embodiment of any of the above methods, the therapeutically effective amount of a DNase enzyme is 0.5 to 100 mg/kg/day or 1000 to 200000 KU/kg/day, preferably 0.5 to 50 mg/kg/day or 1000 to 100000 KU/kg/day, more preferably 1.5 to 50 mg/kg/day or 3000 to 100000 KU/kg/day, even more preferably 10 to 50 mg/kg/day or 20000 to 100000 KU/kg/day.

In a variation of any of the disclosed procedures, the subject is a human being.

DNase administration procedures and DNase compositions

In the methods disclosed herein, a DNase enzyme may be administered before, during, and/or after administration of a chemotherapeutic agent or during or after administration of a radiotherapy. Preferably, (i) a DNase enzyme and a chemotherapeutic agent (or a radiotherapy) are administered at the same time, and/or (ii) a DNase enzyme is administered shortly after administration of a chemotherapeutic agent (or a radiotherapy). The DNase may be administered to the patient all at once or in a series of treatments; once or more times per day.

The DNase doses useful in the methods disclosed herein depend on the type of chemotherapy or radiation therapy side effects being treated, the severity and course of those side effects, prior therapy, the patient's medical history and response to chemotherapy (or radiation therapy) and DNase, as well as the discretion of the treating physician. Non-limiting examples of useful dosage ranges include 0.5 to 100 mg/kg/day or 1,000 to 200,000 KU/kg/day, preferably 0.5 to 50 mg/kg/day or 1,000 to 100,000 Kunitz units (KU)/kg/day, more preferably 1.5 to 50 mg/kg/day or 3,000 to 100,000 KU/kg/day, even more preferably 10 to 50 mg/kg/day or 20,000 to 100,000 KU/kg/day.

Administration of a DNase enzyme according to the methods disclosed herein can be carried out by any suitable route, including systemic administration, as well as administration directly to the disease site (e.g., to a primary tumor). Specific, non-limiting examples of useful routes of administration include intravenous (IV), subcutaneous (SC), intraperitoneal (IP), oral, or inhalation.

In certain embodiments, a DNase enzyme is formulated in a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient.

The formulations used in the methods disclosed herein may conveniently be presented in unit dosage form and may be prepared by methods known in the art. The amount of active ingredients that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredients that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect.

In general, formulations can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaped into the product.

Pharmaceutical compositions suitable for parenteral administration may comprise one or more active ingredients (a DNase and optionally another compound effective to ameliorate or prevent the side effects of cytostatic and/or cytotoxic chemotherapy or radiotherapy) in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders capable of being reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes rendering the formulation isotonic with the blood of the intended recipient, or thickening or suspending agents. Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Adequate fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

These compositions may also contain preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, sorbic phenol, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

Injectable depot forms can be manufactured by forming microencapsule matrices of one or more active ingredients in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of active ingredient to polymer and the nature of the particular polymer used, the rate of release of the active ingredient can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the active ingredients in liposomes or microemulsions that are compatible with body tissue.

Formulations for oral administration may be presented in the form of capsules, sachets, pills, tablets, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid (for example, as a mouthwash, as a swallowing composition, or as an enema), or as an oil-in-water or water-in-oil liquid emulsion, and the like, each containing a predetermined amount of one or more active ingredients.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), one or more active ingredients may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) dissolution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Suspensions, in addition to one or more active ingredients, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Powders and aerosols may contain, in addition to one or more active ingredients, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Aerosols may also contain common propellants, such as chlorofluorohydrocarbons and unsubstituted volatile hydrocarbons, such as butane and propane.

Examples

The present invention is also described and demonstrated by the following examples. However, the use of these and other examples elsewhere in the specification is for illustrative purposes only and in no way limits the scope and meaning of the invention or any exemplified terms.

EXAMPLE 1: Improvement of acute toxicity of doxorubicin in rats (reference example).

Materials and Procedures

Forty-two male Wistar rats (180–200 g) (obtained from the Stolbovaya nursery of the Russian Academy of Medical Sciences) were used in this experiment. The animals were maintained under standard conditions with free access to food and drinking water. The animals were randomly divided into seven groups (six animals in each group) as follows:

1. Group I: control group (no Doxorubicin, no DNase);

2. Group II: animals treated with daily intravenous (IV) injections of doxorubicin (LENS) at a dose of 3.75 mg/kg/day for 5 days plus daily intraperitoneal (IP) injections of recombinant human DNase I (Pharmsynthez OJSC) at a dose of 15 mg/kg/day (30,000 KU/kg/day); doxorubicin and DNase I injections were administered concurrently;

3. Group III: animals treated with daily IV injections of Doxorubicin (LENS) at a dose of 3.75 mg/kg/day for 5 days plus daily IP injections of placebo (water for injection [WFI]);

4. Group IV: animals treated with daily IV injections of doxorubicin (LENS) at a dose of 7.5 mg/kg/day for 5 days plus daily IP injections of recombinant human DNase I (Pharmsynthez OJSC) at a dose of 15 mg/kg/day (30,000 KU/kg/day); doxorubicin and DNase I injections were administered concurrently;

5. Group V: animals treated with daily IV injections of Doxorubicin (LENS) at a dose of 7.5 mg/kg/day for 5 days plus daily IP injections of placebo (WFI);

6. Group VI: animals treated with daily IV injections of doxorubicin (LENS) at a dose of 10.0 mg/kg/day for 5 days plus daily IP injections of recombinant human DNase I (Pharmsynthez OJSC) at a dose of 15 mg/kg/day (30,000 KU/kg/day); doxorubicin and DNase I injections were administered concurrently;

7. Group VII: animals treated with daily IV injections of Doxorubicin (LENS) at a dose of 10.0 mg/kg/day for 5 days plus daily IP injections of placebo (WFI).

The animals were monitored for 2 weeks from the start of the experiment for survival and weight.

Results

Table 1: Animal survival data among the different experimental groups

The results summarized in Table 1 demonstrate that DNase suppresses the lethality of doxorubicin and increases the lifespan of rats challenged with sublethal doses of doxorubicin. The suppression of lethality, as measured by the number of surviving animals, occurred in a dose-dependent manner.

Table 2: Body weight of surviving rats (in grams) after administration of doxorubicin at a dose of 3.75 mg/kg, measured before doxorubicin administration and on days 4, 7 and 14 of treatment.

The results summarized in Table 2 demonstrate that DNase treatment improved body weight loss in rats that survived an acute challenge with doxorubicin at the LD50 dose of 3.75 mg/kg.

Table 3: Blood cell counts and blood biochemical data of surviving rats after administration of doxorubicin at a dose of 3.75 mg/kg on day 14 of treatment.

The results summarized in Table 3 indicate that DNase treatment improved bone marrow toxicity and catabolic changes in blood chemistry in rats that survived an acute challenge with doxorubicin at the LD50 dose of 3.75 mg/kg.

The hearts of deceased rats challenged with sublethal doses of doxorubicin (7.5 mg/kg) from Groups IV and V were autopsied. Serial myocardial microscopy samples from each heart were analyzed using an automated video analyzer to quantify necrotic areas. The sum of the necrotic areas (Snai nm2) was calculated as the sum of the necrotic areas in 30 serial sections from each individual autopsied heart (n=3 for Group IV (Figure 1, black bars); n=5 for Group V (Figure 1, hatched diagonal bars)). Group IV received daily IP injections of recombinant human DNase I at a dose of 15 mg/kg, while Group V received daily IP injections of placebo (WFI).

The results presented in Figure 1 demonstrate that DNase treatment decreases the area of myocardial necrosis in rats challenged with sublethal doses of doxorubicin.

EXAMPLE 2: Improvement of gastrointestinal toxicity of combination chemotherapy with 5-fluorouracil/etoposide (reference example).

Materials and Procedures

Sixty-four male Wistar rats (170–200 g) (obtained from the Stolbovaya nursery of the Russian Academy of Medical Sciences) were used in this experiment. The animals were maintained under standard conditions with free access to food and drinking water. On day 1, etoposide (LANCE) 200 mg/kg and 5-fluorouracil (5-FU; EBEWE) 400 mg/kg were administered orally via a feeding needle in 500 μl of a 9% glucose solution. The animals were divided into 4 groups of 16 rats each. Two hours after the etoposide/5-FU challenge, the rats were treated as follows:

1. Group I: IV placebo (WFI);

2. Group II: Human recombinant DNase I (Pharmsynthez OJSC) at 1.5 mg/kg (3000 KU/kg) IV dose; 3. Group III: Human recombinant DNase I (Pharmsynthez OJSC) at 50 mg/kg (100000 KU/kg) IV dose;

4. Group IV: cimetidine solution (Gedeon Richter) at a dose of 50 mg/kg IV.

Thirty-six hours later, all animals were euthanized, their stomachs removed, and analyzed for erosions and hemorrhages using an automated video analysis system.

Results

Table 4: Effects of DNase I or cimetidine administration on gastrointestinal lesions in animals treated with 200 mg/kg etoposide and 400 mg/kg 5-FU.

The results summarized in Table 4 demonstrate that DNase I suppresses chemotherapy-induced ulceration and increases the number of animals free of gastrointestinal erosion in a dose-dependent manner in animals treated with etoposide and 5-FU. The 50 mg/kg DNase I dose was significantly superior in terms of protection achieved, compared to both the 1.5 mg/kg DNase I dose and the 50 mg/kg cimetidine dose.

Figure 2 shows the stained stomach of a Group III rat (A) and a Group I rat (B). The stomach of the Group I animal showed multiple erosions and ulcers, whereas the stomach of the Group III animal showed no gross abnormalities.

EXAMPLE 3: Improvement of suppression of immunity induced by portaxanes (reference example).

Materials and Procedures

A single dose of 20 mg/kg paclitaxel (Paclitaxel-Teva) was administered IV to 10 6-week-old F1 male mice (CBAxC57Bl6) (obtained from the Rappolovo nursery). A group of 5 paclitaxel-treated mice were injected IP 1 hour later with recombinant mouse DNase gamma (USCN Life Science Inc.) at a dose of 2 mg/kg IP. A group of 5 untreated mice were injected IP at the same time with 1 ml of placebo (WFI) to serve as a control. All mice were sacrificed 24 hours later by cervical dislocation. Thymi were collected from each group and homogenized in a separator in RPMI 1640 medium supplemented with 10% fetal calf serum. The thymocyte suspension was filtered and pelleted in a centrifuge at 400xg for 2 minutes and then resuspended with the same medium. Thymocytes were seeded in 96-well plates (900,000 cells per well in 200 pl of RPMI 1640 supplemented with 10% fetal calf serum (FCS)) and incubated for 32 hours at 37°C, 100% humidity, and a 5% CO2 atmosphere. Each well was then supplemented with 3H thymidine (UC/10pl/well) and Concanavalin A (ConA; 50pl; 1.25 pg/ml). After an additional 24 hours of incubation, thymocytes were harvested and dried. 3H-labeled thymidine incorporation was measured using a Beckman scintillation counter. The data on Con Ase-induced blast transformation of lymphocytes are summarized in Table 5 below.

Results

Table 5: Effect of DNase gamma on Con A-induced blast transformation of lymphocytes.

The results summarized in Table 5 demonstrate that DNase enhances portaxane-induced suppression of thymocyte proliferative activity.

EXAMPLE 4: Improvement of cyclophosphamide-induced neutropenia (reference example).

Materials and Procedures

Thirty 10- to 12-week-old female BALB/C mice (obtained from the Rappolovo nursery) were used in this experiment. The animals were maintained under standard conditions with free access to food and drinking water. On day 1, neutropenia was induced by a single IP injection of 200 mg/kg of cyclophosphamide (Cyclophosphanum-LANS) in 200 μl of WFI. From day 2, the mice were treated as follows:

1. Group I (n=10): control (no treatment);

2. Group II (n=10): animals were treated with recombinant human DNase I (Pharmsynthez OJSC) at a dose of 25 mg/kg/day (50000 KU/kg/day) injected IV on days 2-5;

3. Group III (n=10): Animals were treated with recombinant human GM-CSF (Neostim, Pharmsynthez OJSC) at a dose of 200 μg/kg/day injected subcutaneously (SC) on days 2–5. The white blood cell (WBC) count in each animal was monitored daily during the 10-day experiment using a blood cell counter.

Results

The graph in Figure 3 summarizes the dynamics of the white blood cell (WBC) count in the three experimental groups. The vertical axis represents the WBC count, with units of 109 WBC/L. The horizontal axis represents the number of days after cyclophosphamide injection.

The results in Figure 3 demonstrate that DNase ameliorates neutropenia induced by an alkylating agent such as cyclophosphamide. Of particular importance is that DNase treatment prevents the typical early-phase decline in white blood cell count, whereas GM-CSF treatment fails to do so.

EXAMPLE 5: Synergy between DNase treatment and cancer chemotherapy (reference example).

Materials and Procedures

To evaluate a possible synergy of the combined use of DNase and the nucleoside analogue chemotherapeutic agent cytosine arabinoside (AraC), DBA2 mice (8–10 weeks old; 24–26 g; obtained from Rappolovo nursery) were transplanted IP with L1210 leukemic cells (1*105 cells per mouse; cells obtained from the collection of Petrov Institute of Oncology) on Day 0 to induce cancer. DNase and AraC treatment was started on Day 1 of the experiment. Cytosine arabinoside (AraC; Cytosar; Pfizer) was injected IP at a dose of 1000 mg/kg on Days 2, 5, and 8. The injected doses were considered appropriate for the model and injection route based on previous experiments (13–14). DNase II (Wornington) was injected IP at doses of 15 mg/kg/day and 45 mg/kg/day on days 1, 3, 5, 8, 10, 12, 15, 17, and 19.

The animals were randomly distributed into 6 experimental groups of 6 mice per group as follows:

1. Group I: negative control (no treatment);

2. Group II: positive control (animals treated with AraC);

3. Group III: animals treated with AraC and 15 mg/kg of DNase;

4. Group IV: animals treated with AraC and 45 mg/kg of DNase;

5. Group V: animals treated with 15 mg/kg of DNase, without AraC;

6. Group VI: animals treated with 45 mg/kg of DNase, without AraC.

Results

Survival data are summarized in Figure 4. The vertical axis of Figure 4 represents the survival measured in days after implantation with L1210 leukemic cells. The six experimental groups are represented on the horizontal axis: 1 - Negative control; 2 - AraC (positive control); 3 - AraC DNase II 15 mg/kg; 4 - AraC DNase II 45 mg/kg; 5 - DNase II 15 mg/kg; 6 - DNase II 45 mg/kg.

The results summarized in Figure 4 show that treatment with either AraC alone (Group II) or DNase II alone (Groups V and VI) resulted in improved survival rates compared to untreated mice (Group I). However, the combination of AraC and DNase resulted in a clear survival advantage over either treatment alone. The synergistic effect of the combined treatment on survival rates depended on increasing DNase doses.

EXAMPLE 6: DNase treatment increases the efficacy and decreases the toxicity of cancer radiotherapy.

Materials and Procedures

To evaluate the possible synergy of the combined use of DNase and radiotherapy, 60 SHR mice (12–14 weeks old; 28–32 g; obtained from the Rappolovo nursery) were transplanted with Erlich carcinoma cells (Ix io 6 cells per mouse; cells obtained from the collection of the Petrov Institute of Oncology) on day 0 to induce ascites tumor growth. Radiotherapy was performed using a cobalt-60 teletherapy device. The abdominal fields of the mice were treated by external irradiation. Fractionation was 200 cGy/day for 5 days. The mice were divided into 6 groups treated as follows: Group 1: radiotherapy only (external irradiation of 200 cGy/day for 5 days); Group 2: recombinant human DNAse 1 (Pharmsynthez) 20 mg/kg/day administered intramuscularly in 2 daily doses; Group 3: recombinant human DNAse I (Pharmsynthez) 40 mg/kg/day administered intramuscularly in 2 daily doses; Group 4: radiotherapy (external irradiation 200 cGy daily for 5 days) plus recombinant human DNAse I (Pharmsynthez) 20 mg/kg/day administered intramuscularly in 2 daily doses; Group 5: radiotherapy (external irradiation 200 cGy daily for 5 days) plus recombinant human DNAse I (Pharmsynthez) 40 mg/kg/day administered intramuscularly in 2 daily doses. Group 6 was a control group receiving placebo (water for injection).

Results

Animal survival and body weight dynamics on day 16 after Erlich carcinoma transplantation are summarized in Table 6:

The results summarized in Table 6 show that treatment with either radiotherapy alone (Group 1) or DNase I alone (Groups 2 and 3) resulted in improved survival rates compared to untreated mice (Group 6). However, the combination of radiotherapy and DNase I resulted in a clear survival advantage over either treatment alone. The synergistic effect of the combined treatment on survival rates depended on increasing DNase doses. DNase treatment also clearly prevented radiotherapy-induced body weight loss.

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Claims (12)

1. A DNase enzyme for use in preventing or ameliorating at least one side effect of radiotherapy in a subject suffering from cancer and receiving such radiotherapy, by preventing or ameliorating a toxicity associated with such radiotherapy, wherein the DNase enzyme is administered parenterally. 2. The DNase enzyme for use according to claim 1, wherein said side effect of said radiotherapy is selected from the group consisting of body weight loss, skin irritation or damage, fatigue, nausea, vomiting, fibrosis, intestinal damage, memory loss, infertility and a second cancer. 3. The DNase enzyme for use according to claim 1 or 2, wherein said radiotherapy is external beam radiotherapy or systemic radioisotope therapy. 4. The DNase enzyme for use according to any one of claims 1 to 3, wherein the DNase enzyme is to be administered during a course of radiotherapy. 5. The DNase enzyme for use according to any one of claims 1 to 3, wherein the DNase enzyme is administered after a course of radiotherapy. 6. The DNase enzyme for use according to any one of claims 1 to 5, wherein the DNase enzyme is DNase I, preferably human recombinant DNase I. 7. The DNase enzyme for use according to any one of claims 1 to 5, wherein the DNase enzyme is an analogue of DNase I, preferably DNase gamma. 8. The DNase enzyme for use according to any one of claims 1 to 5, wherein the DNase enzyme is a DNase II. 9. The DNase enzyme for use according to any one of claims 1 to 8, wherein the DNase enzyme has a long half-life. 10. The DNase enzyme for use according to any one of claims 1 to 9, wherein the DNase enzyme is to be administered within the range of 0.5-50 mg/kg/day, preferably 1.5-50 mg/kg/day, even preferably 10-50 mg/kg/day. 11. The DNase enzyme for use according to any one of claims 1 to 10, wherein the DNase enzyme is to be administered intravenously or subcutaneously. 12. The DNase enzyme for use according to any of claims 1 to 11, wherein the subject is a human.