Methods to prevent the development of melanoma

Abstract

A method for treating melanoma or preventing the onset of melanoma, comprising administering to a subject a therapeutically effective amount of 64 Zn e A method comprising administering a composition comprising (Asp)2. Such administration may be by injection, such as intratumoral and / or intravenous injection, and may be once daily or twice or more daily. 64 Zn e A composition for treating or preventing melanoma, comprising (Asp)2. The composition may be a liquid suitable for injection.

Description

[Technical Field]

[0001] This disclosure relates to oncology, pharmacology, and veterinary medicine, and in particular to the treatment or prevention of malignant skin diseases such as melanoma. [Background technology]

[0002] Melanoma is a malignant tumor that originates primarily from melanocytes (pigment-containing cells that produce melanin) located in the basal layer of the epidermis of the skin and the middle layer of the eye (Hurst EA et al., Archives of Dermatology Research, 2003, 139: 1067-1073). This type of disease accounts for 10 percent of all malignant skin lesions. Its annual incidence is 5%. Since the 1940s, the incidence of melanoma has doubled every year. Melanoma is the sixth most common cancer among men and the seventh most common cancer among women. The average incidence of cutaneous melanoma varies from 3 to 5 cases per 100,000 people per year in Mediterranean countries to 12 to 20 cases per 100,000 people per year in Nordic countries, and continues to increase. The mortality rate is 2 to 3 cases per 100,000 people per year, with some variation depending on geographical location, and has remained relatively stable over the past decade. Increased exposure to ultraviolet radiation in genetically predisposed populations has, at least partially, led to a steady increase in melanoma incidence over the past few decades [Oncology Clinical Practice Guidelines of the European Society for Medical Oncology (ESMO), 2010, p. 294-300]. Malignant melanoma accounts for 60–80% of skin cancer deaths, and its five-year survival rate is 14%. In the United States, 2% of the population is diagnosed with this type of skin cancer, which accounts for approximately 10,000 deaths each year. At the same time, melanoma is a tumor with a very high probability of metastasizing throughout the body. [Overview of the project] [Problems that the invention aims to solve]

[0003] However, the possibility of treating or preventing melanoma is limited.

Means for Solving the Problems

[0004] In one aspect, a method for preventing the onset of melanoma, comprising administering, at a therapeutically effective dose, Zn(Asp)2 containing two molecules of aspartic acid per molecule of zinc, by intratumoral and / or intravenous administration to a subject. The Zn-enriched form is known as zinc di-aspartate and has the molecular formula Zn(C4H6NO4)2. In certain embodiments, the aspartate of Zn(Asp)2 is enriched in the L-enantiomer. In further embodiments, it is at least 90% L-enantiomer, at least 95% L-enantiomer, or at least 98% L-enantiomer. 64 Zn e (Asp)2, including intratumoral and / or intravenous administration to a subject, is provided. The non-Zn-enriched form is known as zinc di-aspartate and has the molecular formula Zn(C4H6NO4)2. In certain embodiments, the aspartate of Zn(Asp)2 is enriched in the L-enantiomer. In further embodiments, it is at least 90% L-enantiomer, at least 95% L-enantiomer, or at least 98% L-enantiomer. 64 The Zn-enriched form is known as zinc di-aspartate and has the molecular formula Zn(C4H6NO4)2. In certain embodiments, the aspartate of Zn(Asp)2 is enriched in the L-enantiomer. In further embodiments, it is at least 90% L-enantiomer, at least 95% L-enantiomer, or at least 98% L-enantiomer. 64 Zn e (Asp)2, including intratumoral and / or intravenous administration to a subject, is provided. The non-Zn-enriched form is known as zinc di-aspartate and has the molecular formula Zn(C4H6NO4)2. In certain embodiments, the aspartate of Zn(Asp)2 is enriched in the L-enantiomer. In further embodiments, it is at least 90% L-enantiomer, at least 95% L-enantiomer, or at least 98% L-enantiomer.

[0005] In another aspect, a method for preventing melanoma metastasis, comprising administering, at a therapeutically effective dose, Zn(Asp)2 containing two molecules of aspartic acid per molecule of zinc, by intratumoral and / or intravenous administration to a subject. 64 Zn e (Asp)2, including intratumoral and / or intravenous administration to a subject, is provided.

[0006] In yet another aspect, a composition for use in preventing the onset of melanoma and / or preventing melanoma metastasis, comprising a therapeutically effective amount of Zn(Asp)2 and at least one carrier or excipient is provided. 64 Zn e (Asp)2 and at least one carrier or excipient is provided.

[0007] In each of the above aspects, in certain embodiments, the Zn-enriched zinc of Zn(Asp)2 is at least 80% Zn, at least 85% Zn, at least 90% Zn, at least 95% Zn. 64 Zn e (Asp)2 of 64 The Zn-enriched zinc of Zn(Asp)2 is at least 80% 64 Zn, at least 85% 64 Zn, at least 90% 64 Zn, at least 95%64 Zn, or at least 99% 64 It is zinc. 64 Examples of preferred levels of Zn enrichment include 80%, 85%, 90%, 95%, 99%, and 99.8%. 64 Includes any specific value within the stated range, such as Zn. Where used herein, unless otherwise specified, X% 64 Zn has X out of 100 zinc atoms. 64 It means it is Zn. For example, 95% 64 In zinc, which is Zn, 95% of the atoms are 64 It is Zn. Unless otherwise specified, 64 Zn e The term " " is used in this specification as " 64 It is used as an abbreviation for "Zn-enriched zinc". [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows the survival data (% vs. control) of experimental animals that received an intratumoral ("i / t") injection of 64Zne(Asp)2, containing two molecules of aspartic acid / zinc atom (abbreviated as "64Zne(Asp)2" unless otherwise specified), five days after transplantation of B16 melanoma cells into mice. The survival data for the group treated with 64Zne(Asp)2 (intratumoral administration of 64Zne(Asp)2 five days after tumor transplantation) are compared with the data for the control group. [Figure 2] Figure 2 shows data on the antitumor activity of 64Zn(Asp)2 (average tumor volume in mm³ units) in mice that received intratumoral injections of 64Zn(Asp)2 five days after transplantation of B16 melanoma cells, compared to a control group. [Figure 3] Figure 3 shows data (average tumor volume in mm³ units) regarding the dynamics of B16 melanoma proliferation in mice that received intratumoral injection of 64Zn(Asp)2 five days after transplantation of B16 melanoma cells, compared to the control group. [Figure 4A-4C]Figures 4A–4C show data on the inhibition of the lung metastasis process in C57B1 mice transplanted with B16 melanoma after intravenous administration of 64Zne(Asp)2 45 minutes and 24 hours after tumor cell transplantation. Figure 4A - control, Figure 4B - intravenous injection of 64Zne(Asp)2 24 hours after tumor cell transplantation, Figure 4C - intravenous administration of 64Zne(Asp)2 45 minutes after tumor cell transplantation. [Figure 5A-5B] Figures 5A and 5B show data on the quantitative evaluation of melanoma cell metastatic activity after intravenous administration of 64Zn(Asp)2 45 minutes and 24 hours after tumor cell transplantation, expressed as the mean number of metastases (Figure 5A) and the percentage of inhibition of metastatic activity (Figure 5B). [Modes for carrying out the invention]

[0009] As used herein, the word “a” or “plural” preceding a noun refers to one or more of a particular noun.

[0010] The terms “for example” and “etc.”, and their grammatical equivalents, are understood to be followed by the phrase “and without limitation,” unless otherwise specified. Where used herein, the term “about” means to account for variations due to experimental error. All measurements reported herein, whether explicitly used or otherwise, are understood to be modified by the term “about,” unless otherwise specified. Where used herein, the singular forms “a,” “an,” and “the” include plural referents unless otherwise explicitly indicated by the context.

[0011] As used herein with respect to medical conditions such as melanoma, the term “to treat” means to reduce the severity and / or consequences of the condition, to slow the progression of the condition, to prevent the condition from spreading in a patient having the condition, to at least substantially prevent the condition from metastasizing, and / or to cure the condition.

[0012] As used herein with respect to medical conditions such as melanoma, the term “prevent” means to prevent the onset of the condition, to reduce, at least substantially, the severity and / or consequences of the condition, to slow the progression of the condition, to prevent the condition from spreading in a patient having the condition, and / or to prevent the condition from metastasizing.

[0013] "Effective dose," "preventive effective dose," or "therapeutic effective dose" refers to the amount of a drug or composition that provides a beneficial effect or favorable outcome to a subject, or alternatively, the amount of a drug or composition that exhibits the desired in vivo or in vitro activity. "Effective dose," "preventive effective dose," or "therapeutic effective dose" refers to the amount of a drug or composition that provides a desired biological, therapeutic, and / or preventive outcome. The outcome may be the reduction, improvement, mitigation, reduction, delay, prevention, and / or remission of one or more signs, symptoms, or causes of a patient / subject's disease, disorder, or condition, or other desirable alteration of the biological system. With respect to cellular proliferation disorders, favorable outcomes include a reduction in the effect or severity of symptoms associated with the disease or disorder, and / or an extension of life expectancy, compared to no treatment. The effective dose may be administered in one or more doses. The relationship between animal dose levels and human dose levels (based on milligrams per square meter of body surface area) is described, for example, in Freireich et al., (1966) Cancer Chemother Rep 50: 219.

[0014] For any composition, the effective dose can be initially estimated according to a cell culture assay or using an animal model, typically a mouse, rat, guinea pig, rabbit, dog, or pig. Using the animal model, an appropriate concentration range and route of administration can be determined. Such information can then be used to determine an appropriate dose and route of administration for humans. When calculating the equivalent dose for humans, it is recommended to use the conversion table provided in the Guidance for Industry and the Reviewers document (2002, US Food and Drug Administration, Rockville, MD, USA). The effective daily dose is generally 0.01 mg / kg patient weight to 2000 mg / kg patient weight of the activator, preferably 0.05 mg / kg patient weight to 500 mg / kg patient weight of the activator. The exact effective dose will vary depending on the severity of the disease, the patient's general health status, age, weight and sex, nutrition, timing and frequency of administration, drug combinations, response sensitivity, tolerance / response to administration, and other factors considered by those skilled in the art when determining the dose and route of administration for a particular patient based on their knowledge. Such dosages can be determined at the physician's discretion through conventional experimentation. The effective dose may also vary depending on the possibility of combination therapy with other medications or other treatment procedures.

[0015] As used herein, “patient” and “subject” are interchangeable terms and may refer to human patients / subjects, dogs, cats, non-human primates, etc.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to limit the scope. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, including definitions, this specification shall prevail.

[0017] melanoma Melanoma is a malignant tumor that originates primarily from melanocytes (pigment-containing cells that produce melanin) located in the basal layer of the epidermis of the skin and the middle layer of the eye (Hurst EA et al., Archives of Dermatology Research, 2003, 139: 1067-1073). This type of disease accounts for 10 percent of all malignant skin lesions. Its annual incidence is 5%. Since the 1940s, the incidence of melanoma has doubled every year. Melanoma is the sixth most common cancer among men and the seventh most common cancer among women. The average incidence of cutaneous melanoma varies from 3 to 5 cases per 100,000 people per year in Mediterranean countries to 12 to 20 cases per 100,000 people per year in Nordic countries, and continues to increase. The mortality rate is 2 to 3 cases per 100,000 people per year, with some variation depending on geographical location, and has remained relatively stable over the past decade. Increased exposure to ultraviolet radiation in genetically predisposed populations has, at least partially, led to a steady increase in melanoma incidence over the past few decades [Oncology Clinical Practice Guidelines of the European Society for Medical Oncology (ESMO), 2010, p. 294-300]. Malignant melanoma accounts for 60–80% of skin cancer deaths, and its five-year survival rate is 14%. In the United States, 2% of the population is diagnosed with this type of skin cancer, which accounts for approximately 10,000 deaths each year. At the same time, melanoma is a tumor with a very high probability of metastasizing throughout the body.

[0018] Primary melanoma is resistant to chemotherapy and radiotherapy. The primary treatment for primary melanoma patients is surgical removal of the tumor and some surrounding normal tissue. The amount of tissue removed by surgical excision varies depending on the thickness of the tumor (Breslow's thickness) and the depth of tumor invasion. However, surgical treatment of melanoma has several significant drawbacks, including prolonged wound healing and the development of transplant metastases within two years of surgery. This treatment method is only used in the early stages of the disease (stages 1-2). In advanced stages, radiotherapy, chemotherapy, and immunotherapy are used in addition.

[0019] Antitumor activity is demonstrated by various compounds with different mechanisms of action and chemical structures, including alkylated compounds, antimetabolites, antibiotics, plant or animal-derived substances, hormone preparations, and enzymes. However, these chemotherapeutic drugs can have serious adverse effects on normal tissues and organs, thus limiting their usefulness.

[0020] The treatment options for melanoma are limited. Recent studies have demonstrated that isotopic compositions of tissues and organs may function as diagnostic markers. In particular, studies of Cu and Zn isotopic ratios in blood have shown promising correlations between isotopic values ​​and age, sex, and disease status. For example, measuring copper isotope ratios in serum is a novel approach for the diagnosis and prognosis of liver cirrhosis (M. Costas-Rodriguez et al., Isotopic analysis of Cu in blood serum by multi-collector ICP-mass spectrometry: a new approach for the diagnosis and prognosis of liver cirrhosis, Metallomics 2015, 7: 491-498). Zinc isotopic compositions in breast tissue may be useful in the diagnosis of breast cancer (F. Larner et al., Zinc isotopic compositions of breast cancer tissue, Metallomics 2015, 7: 112-117). Furthermore, certain patents and patent applications discuss the use of isotope-enriched compositions for therapeutic use. See, for example, U.S. Patents 9,861,659; 10,183,041; and 10,226,484.

[0021] WO2007 / 140280 suggests the use of topical anticancer compositions containing cesium and / or rubidium ion sources as pharmaceutically acceptable salts for use in the treatment of melanoma. The feasibility of using this therapy is based on an approach that involves slightly altering the acidic pH of cancer cells to alkaline, thereby impairing the survival of cancer cells and neutralizing and eliminating the formation of acidic and toxic substances normally formed in cancer cells (Sartori HE. Nutrients and cancer: an introduction to cesium therapy, Pharmacol. Biochem. Behav. 1984; 21, Suppl. 1: 7-10). Thus, mass spectrometry and isotopic studies have shown that potassium, rubidium, and cesium are most effectively absorbed by cancer cells. Glucose can still enter the cell, but oxygen cannot; therefore, the cell becomes anaerobic. In the absence of oxygen, fermentation from glucose to lactate occurs, and the cell's pH drops to 7, and eventually to 6.5. Cesium, rubidium, and potassium, which produce high pH values, can enter cells in such conditions and raise their pH. In this setting, cesium and rubidium ions can alter the ion physiology of cancer cells, including inhibiting the transmembrane movement of potassium. Cesium and rubidium also interact with potassium and bound hydrogen ions (H + It is assumed that this composition efficiently controls flux, affects all acid-dependent cancers, and provides site affinity to selectively increase the pH of the tumor microenvironment. In the authors' opinion, this provides selective tumor modulation, but the application materials do not contain any information confirming the effectiveness of treating cancer with this composition. Furthermore, the composition described in this application is for topical administration and cannot provide high efficacy in treating patients. Therefore, the use of the described approach targets cancer cells but still cannot provide effective eradication of tumor cells and can rather be used only as a dietary supplement in addition to other treatment methods.

[0022] method In one embodiment, a method for preventing the development of melanoma is provided. This method effectively suppresses the development of malignant tumors without surgical intervention or damage to surrounding normal tissue, and further exhibits a high anti-metastatic effect. By using the claimed method, it becomes possible to achieve effective inhibition of melanoma development without adverse effects on the body, as is characteristic of chemotherapeutic agents. This method comprises administering a light isotope of zinc in the form of aspartate to a subject in need. A pharmaceutical composition used in the disclosed method for preventing the development of melanoma is provided, which is in a therapeutically effective amount. 64 Zn e The disclosed method contains (Asp)2. The disclosed method has high efficacy in inhibiting tumor cell proliferation, along with low toxicity.

[0023] Methods for treating melanoma or preventing the development of melanoma are disclosed. These methods use a therapeutically effective dose. 64 Zn e The regimen includes intratumoral and / or intravenous administration of a composition containing (Asp)2. The administration of the composition may be single or multiple. In certain embodiments, the treatment regimen is 64 Zn e (Asp)2 comprises 5 to 10 injections. In a particular embodiment, 64 Zn e The aspartate present in (Asp)2 is at least 90%, at least 95%, at least 98% L-enantiomer, or all L-isomer. In some embodiments, the aspartate may be a D-enantiomer, and in other embodiments, the aspartate may be a mixture of two enantiomers.

[0024] A method for treating melanoma or preventing the development of melanoma, provided in a therapeutically effective amount for a patient in need. 64 Zn e A method is provided comprising administering a composition containing (Asp)2. In some embodiments, the composition is an aqueous solution. In some embodiments, the composition is administered intratumorally or intravenously. In some embodiments,64 Zn e (Asp)2 contains two molecules of aspartic acid. In some embodiments, the dosage ranges from 0.2 μg / kg patient body weight / day to 2000 mg / kg patient body weight / day. 64 Zn e (Asp)2 is administered to the patient. In some embodiments, the dose ranges from 0.01 mg / kg / day to 5 mg / kg / day. 64 Zn e (Asp)2 is administered to the patient. In some embodiments, 0.1 mg / kg / day to 1 mg / kg / day 64 Zn e (Asp)2 is administered to the patient. In some embodiments, the composition is administered once daily. In other embodiments, the composition is administered two or more times daily. In some embodiments, the composition further comprises deuterium-depleted water as a solvent. In some embodiments, the method for treating / preventing melanoma is a method for preventing, delaying, or improving melanoma metastasis.

[0025] 64 Zn e The enantiomer purity of aspartate in (Asp)2 can be determined by methods known in the art, such as chiral chromatography.

[0026] 64 Zn e The presence of zinc in (Asp)2 compounds or other zinc-containing compounds can be confirmed by methods known in the art, such as atomic emission spectroscopy using inductively coupled plasma. Before subjecting a sample to atomic emission spectroscopy using inductively coupled plasma, the sample can be treated with a mixture of mineral acids in a Teflon autoclave under microwave radiation.

[0027] 64 Zn eElemental impurities or sulfate ion impurities in (Asp)2 compounds or other zinc-containing compounds can be measured by methods known in the art, such as atomic emission spectroscopy using inductively coupled plasma. Before subjecting a sample to atomic emission spectroscopy using inductively coupled plasma, the sample can be treated with a mixture of mineral acids in a Teflon autoclave under microwave radiation.

[0028] Light isotopes can be purchased. Zn-64 oxide of the required enrichment level can be purchased, for example, from Oak Ridge National Laboratory, Oak Ridge, TN, USA.

[0029] In a particular embodiment, it is administered to a subject that requires it. 64 Zn e The effective dose may be 0.2 μg / kg patient body weight / day to 2000 mg / kg patient body weight / day. This range of 0.2 μg / kg / day to 2000 mg / kg / day corresponds to the amount of zinc present in the composition as part of the aspartate. In further embodiments, the administered dose is 64 Zn e The range is 0.01 mg / kg / day to 5 mg / kg / day, more preferably 0.1 mg / kg / day to 1 mg / kg / day. The composition for use in the disclosed method contains the corresponding amount. For example, the composition for use in the disclosed method contains the composition in a single dose. 64 Zn e (Asp)2 form, 1-100 mg 64 Zn e For example, 1, 5, 10, 20, 30, 40, 50, or 100 mg 64 Zn e an amount containing 64 Zn e It contains 1 mg per 1 ml of solution. An example composition contains 1 mg 64 Zn e Contains 64 Zn eThis is a solution of (Asp)2. In some embodiments, the solvent is deuterated water. This solution is formulated for oral or parenteral administration, for example, for administration by injection, such as intratumoral or intravenous administration. The injectable composition may be an aqueous solution, such as a solution whose salinity and pH are optimized for the injection route. This solution may contain excipients such as DMSO. DMSO may be present at a concentration of 1%. Another exemplary composition for use in the disclosed method is 30 mg 64 Zn e The composition is an orally administered tablet or other solid composition containing [the specified substance]. The composition may also be an aqueous composition, such as an orally administered liquid such as a syrup.

[0030] The treatment regimen of the disclosed method may include either intratumoral administration or intravenous administration, or both, of the composition for use in the disclosed method. When both routes are used for a patient, the same composition may be administered via both routes, or different compositions may be administered. In some embodiments, a combination of intratumoral and intravenous administration routes is used.

[0031] In this example, a melanoma model system is used, 64 Zn e The efficacy of compositions containing (Asp)2 will be studied to simulate in vivo multiple processes of tumor dissemination in warm-blooded animals, including surgical intervention, when tumor cells may spread to distant sites and / or local niches. Postoperative administration of the composition and / or administration of the composition to prevent the potential melanoma metastatic process (and thus tumor progression) will suppress the metastatic process, which has significant advantages.

[0032] I have no intention of linking this to scientific theory, 64 Zn e The effectiveness of (Asp)2 is explained below. Cancer tissue mainly contains heavy isotopes (Zn 70 (etc.) are concentrated, and light isotopes of the basic elements ( 64 Zn e (etc.) are depleted.64 Zn e of 70 Zn e ( 70 Substitution with zinc (Zn-enriched zinc) can lead to isotopic-induced changes in the chirality of one or more amino acids within a protein structure, potentially affecting the conformation of receptors, ligands, and signaling molecules. Loss of structural precision, protein depletion, and degradation can disrupt intracellular and intercellular homeostasis, resulting in various pathological conditions. Depending on the characteristics of the pathological chiral amplification of amino acids by autocatalytic reactions in living cells, the rate of disease onset and symptom manifestation can be slow or rapid. Generally, asymmetric autocatalytic reactions have a nonlinear nature in the relationship between yield and time. The most dramatic consequences occur when the above changes occur in the p53 protein, known as the "guardian of the genome," which is composed of two-thirds zinc fingers. Zinc has five stable isotopes. Indeed, "improper" conformation of p53 can lead to cell cycle arrest failure, differentiation dysfunction, apoptosis, metabolic problems, genomic stability issues, angiogenesis, DNA repair, aging, and other processes. What is unknown is that the very onset of the pathological changes is due to chirality induced by isotopic substitution, which leads to a change in the protein's three-dimensional structure. Since these changes are apparently reversible, recovery to the normal state can be achieved by using light isotopes. In the case of p53, this is 64 It could be a Zn isotope.

[0033] The disclosed method results in isotopic-selective protein regulation, which not only makes it possible to restore damaged negative feedback in cellular communication systems by restoring transmit and receive receptors and signaling molecules, but more importantly, opens up the possibility of eliminating biomolecular mutations by reactivating the normal function of the p53 protein and associated pathways. Confirmation is made by mass spectrometry studies as shown in the examples.

[0034] The present invention will be described more fully below by reference to the following examples, which are presented for illustrative purposes only and should not be construed as limiting the scope of the invention. [Examples]

[0035] [Example 1]

[0036] In vivo studies conducted in mouse models supporting the effectiveness of the claimed method ( 64 Zn e (Intratumoral administration of Asp2) B16 melanoma cells with the following characteristics were used in the experiment. Origin: House mouse (Mus musculus) skin (C57BL / 6 mouse) Characteristics of tumor growth: short incubation period, rapid growth, and absence of spontaneous metastasis. The tumor inoculation rate is 100%. The minimum dose of cells that induce tumor growth in this melanoma is only 100-1000 per subcutaneous injection in mice. The average life expectancy of animals is 21 to 31 days. The tumor cell population is heterogeneous, containing both highly pigmented areas and fragments with low melanin content. Karyotype: Chromosome number varies from 30 to 80, with 2n=40, modal number being 72 chromosomes (14%), and ploidy occurring in 3% of cells. All cells contain 2 to 8 interchromosomal associations due to Robertsonian translocations.

[0037] The distinguishing features of B16 melanoma cells are the extremely low mRNA expression levels of the c-myc, c-jun, and c-fos oncogenes, and the absence of expression of the c-ras, c-abl, c-erb-B2, B-lym, c-sis, and c-myb oncogenes. Another characteristic unique to these melanoma cells is that they produce large amounts of factors exhibiting procoagulant activity in vitro.

[0038] Immunocytochemical analysis of the phenotypic features of melanoma cells revealed that the cells express VE-cadherin, N- and E-cadherin, Twist and Slug transcription factors, P-glycoprotein, ERCC, DAB2, and TAP1, and weakly express CD44.

[0039] All studies involving the use of mice were conducted in accordance with the rules of the European Convention on the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes [Commission of the European Communities: Council Directive of 18 December 1986 (87 / 18 / EEC) on the Law Regulating the Application of Principles of Good Laboratory Practice and the Verification of Their Applications for Tests on Chemical Substances. The Rules Governing Medicinal Products in the European Community. - 1991. - V. 1. - P. 145-146].

[0040] Animals were selected according to the purpose of the experiment, in accordance with generally accepted requirements for preclinical studies of test products, including biological drugs exhibiting antitumor activity.

[0041] The animals were maintained according to the standards described in The Guide for Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press, 1996). During the experiment, the animals were housed in plastic cages, had a natural day-night cycle, were given a standard diet, and had free access to food and water.

[0042] Clinical observation All animals in the cage were examined daily to determine mortality or any signs of abnormalities in their health status. Thorough examinations were conducted whenever any abnormalities were detected. All abnormalities were recorded.

[0043] Statistical processing of the results was performed using the STATISTISA 6.0 software package designed for the analysis of medical and biological statistics using Student's t-test; differences with a probability of over 95% were considered significant (p < 0.05).

[0044] In the experiment, 15 female С 57 Bl / J6 mice aged 10 - 12 weeks and weighing 18 - 22 g (5 mice per group) were used.

[0045] B16 melanoma cells were cultured in vitro under standard conditions. For transplantation, tumor cells in the exponential phase were removed from the substrate with 0.02% versen solution, and the cell density and viability of the suspension were evaluated using a hemocytometer in the presence of trypan blue. The suspension was diluted with saline to adjust the concentration to 10 7 cells / ml. The melanoma cells were injected intradermally (i.c.) into the dorsal region of the animals with 0.05 ml of the suspension (0.5×10 6 cells / mouse). Twenty-four hours before the injection of the tumor cells, the wool cover on the back of each mouse was removed with a depilatory cream.

[0046] Grouping. The animals were grouped as follows: Group number 1: Control group, mice injected i.c. with B16 melanoma cells; Group number 2: Mice injected i.c. with B16 melanoma cells and injected intratumorally with 64 Zn e (Asp)2 on the 5th day after tumor cell inoculation; Group number 3: Mice injected i.c. with B16 melanoma cells and injected intratumorally with 64 Zn eMice injected with (Asp)2 into the tumor

[0047] The composition to be administered was prepared immediately before its administration. 64 Zn e (Asp)2 was dissolved in deuterium-depleted water with 1% DMSO added. For the animals in group number 2, after the tumor reached a diameter of 0.5 cm, 20 μl / mouse of a composition containing 200 μg / mouse of 64 Zn e (Asp)2 was injected (on the 5th day after i.c. administration of tumor cells). When the experimental animals had new tumor growth, the following 64 Zn e (Asp)2 injection was given according to the above scheme.

[0048] For the animals in group number 3, on the 11th day after i.c. administration of tumor cells, 30 μl / mouse of a composition containing 300 μg / mouse of 64 Zn e (Asp)2 was injected. When the experimental animals had new tumor growth, a follow-up injection of 20 μl / mouse of a composition containing 200 μg / mouse of 64 Zn e (Asp)2 was given. A series of injections was given 24 hours after the first injection and then every other day. 64 Zn e (Asp)2 was administered intratumorally 5 times at a dose of 100 μg / mouse every other day for 10 days.

[0049] For the third group: The mice in group 3 received 4 Zn e in the form of aspartate at 300 mcg / mouse on days 13, 15, 17, and 19 at 200 mcg / mouse of 64 Zn e aspartate. 4 Zn e For the second group: The mice in group 2 received 200 mcg / mouse of

[0050] Zn 4 Zne If an experimental animal receives aspartate and then develops new tumor growth, administer 200 mcg / ml. 4 Zn e I was given aspartate.

[0051] 64 Zn e The antitumor effect of (Asp)2 was evaluated based on the growth dynamics of melanoma tumors estimated based on changes in tumor volume according to generally accepted rules.

[0052] When the experimental tumors reached a diameter of 0.3–0.5 cm, the size of each tumor nodule in the experimental animals was measured every 2–3 days thereafter in three orthogonal planes (width × length × height (W × L × H)), and the tumor volume was calculated using the following formula for the volume of an ellipse: V = 4 / 3 × πabc; where V is the tumor volume (mm³). 3 ) and; a, b, and c are tumor radii (mm): a is the radius along the x-axis, b is the radius along the y-axis, and c is the radius along the z-axis.

[0053] Statistical data processing: Student's t-test was used to determine if there was a statistically significant difference between the group means. The calculation was performed using the STATISTICA 6.0 software package.

[0054] 64 Zn e During studies of the antitumor activity of methods including the administration of (Asp)2, 64 Zn e Results showed that intratumoral administration of a composition containing (Asp)2 suppressed the development of melanoma in mice (see Table 1). Figures 1-4 show the results of the claimed method's effectiveness in mice. 64 Zn e The survival rates of experimental animals that received intratumoral injections of compositions containing (Asp)2 were also determined during the experiment (see Table 2).

[0055] [Table 1]

[0056] In group number 2 (administration of the therapeutic composition 5 days after inoculation of tumor cells), 64 Zn e It should also be noted that in 2 out of 5 experimental animals, melanoma growth was 100% suppressed after a single intratumoral injection of (Asp)2.

[0057] [Table 2]

[0058] [Table 3]

[0059] [Table 4]

[0060] As can be seen from the presented data, 5 days after inoculation of tumor cells 64 Zn e Intratumoral administration of (Asp)2 resulted in a significant reduction in tumor volume. 64 Zn e The tumor volume of experimental animals treated with (Asp)2 was 4% of the corresponding value in the control group. 64 Zn e The antitumor effect of (Asp)2 was not very pronounced. Statistically significant suppression of tumor growth was observed only on days 21 and 24 of the experiment. Melanoma size was reduced by 85% (day 21) and 75.6% (day 24) compared to the control. Antitumor activity against melanoma was also observed in the percentage of surviving animals at day 31, particularly 5 days after inoculation of tumor cells. 64 Zn e The survival rate of mice injected with (Asp)2 was 80%, while only 20% of the control group survived.

[0061] Table 3 shows the results for Group 2 and individual drug administration regimens. 64 Zn e This shows the proliferation dynamics of B16 melanoma during (Asp)2 therapy. [Example 2]

[0062] In vivo studies conducted in mouse models supporting the effectiveness of the claimed method ( 64 Zn e (Asp)2 intravenous / intravenous + intratumor administration) The experimental model of hematogenous metastasis was used in experiments in which B16 melanoma cells could be inoculated. The characteristics of these cells are shown in Example 1. The cells were cultured in vitro under standard conditions. For transplantation, tumor cells were removed from the substrate with 0.02% bersen solution, and the cell density and viability of the suspension were evaluated in the presence of trypan blue using a hemocytometer. The suspension was then divided into 1 × 10⁻⁶ units. 6 The concentration was adjusted to cells / ml. Melanoma cells were placed in a 0.05 ml suspension (0.5 × 10⁶). 6 Cells (injected intradermally into the dorsal region of animals) were administered via IC injection. A portion of the fur on the back of the mice was removed with depilatory cream 24 hours prior to tumor cell inoculation. Cells isolated from B16 mouse melanoma were used in the experiment. The cells were cultured in vitro under standard conditions. For inoculation, tumor cells were removed from the substrate with 0.02% bercen solution. The cell density and viability of the resulting cell suspension were assessed using a hemocytometer in the presence of trypan blue. Cell concentration was measured in physiological saline at 1 × 10⁶ units. 6 The concentration was adjusted to cells / ml. Melanoma cells were placed in a 0.2 ml suspension (0.2 × 10⁶). 6 Intravenous injection was administered into the lateral tail vein (in cells / mouse).

[0063] 64 Zn e (Asp)2 was dissolved in deuterium-depleted water. The composition was administered using a microinjection syringe in a 60 μg dose. 64 Zn e / Intravenous injection at mouse dose: Two injections of 0.3 ml each (total 0.6 ml) at a mouse dose of 30 μg / mouse, 4 hours apart. Injections were given every other day for 10 days (total of 5 injections). The first injection of the composition of the present invention was given 45 minutes or 24 hours after inoculation of tumor cells. On day 26 after IV injection of tumor cells, the lungs were resected from all animals in each group, and the number and volume of metastases were then measured. In the experiment, mice were 12-14 weeks old and weighing 25-27 g. 57 BL mice (8 mice per group) were used. The conditions under which the animals were maintained are described in Example 1 above.

[0064] Prior to the experiment, all animals were healthy and possessed normal behavioral abilities. During the experiment, the animals were kept in plastic cages under natural light, provided with a standard diet, and allowed free access to food and water.

[0065] Using the Micro-Fine Plus microinjection syringe (Becton Dickinson), 64 Zn e The (Asp)2 composition was intravenously injected into the lateral tail vein of animals. The injection site was washed with 96% ethanol.

[0066] All animals in their cages were examined daily to determine mortality rates or any signs of abnormal health conditions. A thorough examination was conducted whenever any abnormality was detected. All abnormalities were documented.

[0067] The results were statistically analyzed using the STATISTICA6.0 software package, which is designed for the analysis of medical and biological statistics using Student's t-test.

[0068] The animals were divided into experimental groups as follows: Group 1: Control group, mice that received IV injections of B16 melanoma cells and IV injections of a solvent (deuterium-depleted water); Group No. 2: B16 melanoma cells were injected intravenously, and 24 hours after inoculation of tumor cells64 Zn e Mice injected IV with (Asp)2; Group No. 3: B16 melanoma cells were injected intravenously, and 45 minutes after inoculation of tumor cells... 64 Zn e Mice that received an IV injection of (Asp)2.

[0069] The proliferation of metastases was evaluated as follows:

[0070] Twenty-six days after IV injection of tumor cells, the lungs were resected from all animals in each group, and the number and volume of metastases were then measured.

[0071] The transfer capacity was calculated using the following formula for the volume of the sphere: V = 4 / 3 × πr 3 In the formula, V is the transfer capacity (mm 3 ) and r is the transition radius (mm).

[0072] In experimental models of hematogenous B16 melanoma metastasis 64 Zn e To evaluate the in vivo antimetastatic activity of (Asp)2, data on the number and volume of metastases in the lungs of mice were used. The experimental results are shown in Table 4 and Figures 1, 2, 3, 4A, 4B, 4C, 5A, and 5B.

[0073] [Table 5]

[0074] The results above suggest that suppressing the onset of the melanoma metastatic process is important. 64 Zn e The efficacy of (Asp)2 has been confirmed. A statistically significant decrease in the number and volume of metastases after use of the selected administration route has been demonstrated. The effect was more pronounced in both the volume and number of metastatic lesions in lung tissue when the injection was given within a shorter time (45 minutes) after inoculation of tumor cells, which indicates a good outlook and the initiation of tumor growth, expansion of malignant cells, and 64 Zn eThis indicates that the time between the start of therapy with (Asp)2 needs to be reduced.

[0075] 64 Zn e The potential for simultaneous intratumoral and intravenous administration of compositions containing (Asp)2 was evaluated in additional experiments (data not shown). These experiments showed that it could suppress the metastatic process in the body. 64 Zn e The effectiveness of (Asp)2 was confirmed, as demonstrated by a reduction in tumor size, an increase in the number of surviving animals, and a decrease in the number of metastases. [Example 3]

[0076] Study of the distribution of light and heavy isotopes of chemical elements in cutaneous melanoma samples. The distribution of isotopes of various chemical elements in samples of melanoma from experimental animals and corresponding samples taken from healthy animals was analyzed and compared experimentally. Glow discharge mass spectrometry was used to measure trace elements. Samples for analysis were prepared as follows:

[0077] 1. Prepared tissue samples weighing up to 1 g were immersed in liquid nitrogen. Ultra-rapid cooling at a rate of approximately 100°C per second caused most of the water to turn into amorphous ice, whose structure is almost identical to that of water and does not expand in volume. This fact indicates that the structure of the tissue remains unchanged at the cellular level after freezing.

[0078] 2. Amorphous ice was removed by sublimation at low temperature and under reduced pressure, while dry nitrogen was automatically supplied to the drying chamber to accelerate the sublimation process. The gas volume supplied to the drying chamber did not exceed 0.1 l / min. The drying time for the sample under these conditions was approximately 10 hours.

[0079] 3. Secondary drying was carried out under reduced pressure. For this purpose, in a stage of nearly complete (up to 99%) dehydration, the sample was heated to a temperature of 35-40°C under reduced pressure and held isothermally under these conditions for approximately 1 hour.

[0080] The completion of the drying process was determined by the amount of sample mass obtained until two identical mass values ​​were acquired. During the last three hours of the drying process, the biological sample was removed from the vacuum chamber every hour and weighed on an analytical balance. The drying process was stopped as soon as two identical mass values ​​were obtained.

[0081] After the sublimation process was complete, the dried sample was removed from the vacuum chamber and placed in thin layers (each layer less than 5 μm thick) between copper grids fixed to a cage. Fifty semicircular copper grids, 50-100 microns thick, were used and held together tightly with metal clamps. The "sandwich" thus prepared for mass spectrometry consisted of 50 copper grids with the test material sandwiched and tightly pressed between them.

[0082] The analysis area on the clamp surface was a circle with a diameter of 10 mm, and at its center was a copper grid in which the sample was clamped and pressed.

[0083] The experiment used a Finnigan ELEMENT GD glow discharge mass spectrometer with the following specifications: • Dynamic range > 10 12 Linear, automated cross-calibration (from matrix elements (100%) to trace amounts (ppt)); • Sensitivity (peak height, total ion current): >1 × 10⁻⁶ 10 cps, 1.6 * 10 -9 A; • Dark noise <0.2 cps ·Mass resolution>10000; ·Mass stability 25ppm / 8 hours The results of the study are shown in Table 5.

[0084] [Table 6]

[0085] As can be seen from the table above, the results confirm our hypothesis that chiral pathological changes can be induced by isotopic substitution. Therefore, according to the present invention 64 Zn e Administration of (Asp)2 can prevent and restore the normal isotope distribution necessary for proteins such as transcription factors or cell cycle-regulating proteins to function properly, thereby suppressing the formation of malignant tumors. [Example 4]

[0086] In the experiment, 64 Zn e (Asp)2 is 64 Zn e It was synthesized from an oxide and was in powder form after synthesis. The required concentration of solution for the experiment was obtained in the required amount immediately before administering it to the animal. 64 Zn e (Asp)2 powder was prepared by dissolving it in physiological saline or deuterium-depleted water.

[0087] Atomic emission spectroscopy using inductively coupled plasma was performed to confirm the presence of zinc cations in the sample. Prior to measurement, the sample was treated with a mixture of mineral acids in a Teflon autoclave under microwave radiation. The presence of zinc in the sample was confirmed as the main cation; its content was approximately 17.6%. Impurities of other elements were also identified.

[0088] The Zn content was also measured using a complex titration method with eriocrom black T as the endpoint indicator. The zinc content in the sample was found to be 17.98 ± 0.17%.

[0089] The presence of aspartate ions and the determination of their optical isomer morphology were performed by liquid chromatography using a Chiralcel OD-R chiral chromatography column, detecting at a wavelength of 254 nm. Conventionally, derivatization was carried out using isophenyl cyanate. Chromatograms of derivatization standards for racemic mixture D,L-aspartic acid and pure L-aspartic acid were also obtained. The results showed that the sample contained only the L-isomer.

[0090] The zinc content is 17.98±0.17%, which is equivalent to 90.8±0.9% of zinc L-aspartate.

[0091] in the sample 64 The relative Zn content was measured after dissolving sample pai1 in deionized water and diluting it to a concentration of 1.33 ppm based on zinc. 64 Zn, 66 Zn, 67 Zn, 68 Zn, and 70 The signal intensity ratios of Zn isotopes were measured for the sample solution using an Agilent 7500ce inductively coupled plasma mass spectrometer. The results are shown in Table 6. The sample contained, 64 The Zn content was concentrated and measured at 99.39%.

[0092] [Table 7]

[0093] The sulfate ion content in the sample solution was determined by turbidimetric analysis using barium chloride. The sulfate content in the sample was found to be 0.66 ± 0.032%.

[0094] Elemental impurities and sulfate ion impurities were measured in the sample. Elemental impurities were recovered by atomic emission spectroscopy using inductively coupled plasma. Prior to measurement, the sample was treated with a mixture of mineral acids in a Teflon autoclave under microwave radiation. As shown in Table 7, phosphorus, calcium, sodium, silver, aluminum, bismuth, copper, iron, potassium, magnesium, and lead impurities were detected.

[0095] [Table 8]

[0096] While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to describe, not limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims. Thus, although only certain features of the invention have been described and written, many modifications and changes will come to mind for those skilled in the art. Therefore, it should be understood that the appended claims are intended to encompass all such modifications and changes that fall within the true spirit of the invention.

Claims

1. A therapeutically effective dose administered intratumorally or intravenously to reduce the growth of primary melanoma tumors. ６４ Zn ｅ (Asp) ２ A pharmaceutical composition containing the following:

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is an aqueous solution.

3. ６４ Zn ｅ (Asp) ２ The pharmaceutical composition according to claim 1 or 2, wherein the aspartic acid comprises two molecules.

4. From 0.2 μg / kg patient body weight / day to 2000 mg / kg patient body weight / day of ６４ Zn ｅ (Asp) ２ is administered to the patient. The pharmaceutical composition according to any one of claims 1 to 3.

5. 0.01 mg / kg / day to 5 mg / kg / day ６４ Zn ｅ (Asp) ２ The pharmaceutical composition according to claim 4, wherein the patient is administered the composition to the patient.

6. 0.1 mg / kg / day to 1 mg / kg / day ６４ Zn ｅ (Asp) ２ The pharmaceutical composition according to claim 4 or claim 5, wherein the composition is administered to the patient.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the composition is administered once a day.

8. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition is administered twice or more times a day.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutical composition further comprises deuterium-depleted water as a solvent.

Images