Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/24—Heavy metals; Compounds thereof
  • A61K33/30—Zinc; Compounds thereof

Definitions

• This disclosure relates to oncology, pharmacology and veterinary medicine and relates specifically to treating or preventing malignant skin diseases such as melanoma.
• Melanoma is a malignant tumor that develops from melanocytes (pigment-containing cells that produce melanin), which are predominantly located in the basal layer of the skin’s epidermis and the middle layer of the eye (Hurst E A et ah, Archives of Dermatology Research , 2003, 139: 1067-1073). This type of pathology accounts for 10 percent of all malignant skin lesions. Its annual incidence rate is 5%. Starting from 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 in women.
• the average incidence rates for skin melanoma vary from 3-5 cases per 100,000 people per year in the Mediterranean countries to 12-20 cases per 100 thousand people per year in the Nordic countries and continue to grow.
• the death rate is 2-3 cases per 100 thousand people every year with slight changes depending on the geographical location, and has remained relatively stable over the past decade.
• An increased exposure to ultraviolet radiation of a genetically predisposed population results in a steady increase in melanoma incidence over the past decades (Oncology Clinical Practice Guidelines of the European Society for Medical Oncology (ESMO), 2010, p. 294-300).
• Malignant melanoma is responsible for 60-80% of deaths from skin cancers and its five-year survival rate is 14%. In the United States, 2% of the population was diagnosed with this type of skin cancer, which is a cause of approximately 10,000 deaths every year. At the same time melanoma is a tumor with an extremely high potential of systemic metastases.
• a method is provided of preventing the development of melanoma comprising intratumoral and/or intravenous administration to a subject of 64 Zn e ( Asp)2, containing 2 molecules of aspartic acid for each molecule of zinc, at a therapeutically effective dose.
• the non- ⁇ Zn-cnrichcd form is known as zinc di-aspartate and has a molecular formula Zn(C 4 H 6 N0 4 ) 2 .
• the aspartate of 64 Zn e (Asp)2 is enriched for the L- enantiomer. In further embodiments, it is at least 90% L-enantiomer, at least 95% L-enantiomer, or least 98% L-enantiomer.
• a method is provided of preventing melanoma metastasis comprising intratumoral and/or intravenous administration to a subject of 64 Zn e (Asp)2, containing 2 molecules of aspartic acid for each molecule of zinc, at a therapeutically effective dose.
• compositions for use in preventing the development of melanoma and/or in preventing melanoma metastasis comprising 64 Zn e (Asp)2 in a therapeutically effective amount and at least one carrier or excipient.
• the ⁇ Zn-enriched zinc of the 64 Zn e (Asp)2 is at least 80% ⁇ Zn, at least 85% ⁇ Zn, at least 90% 64 Zn, at least 95% 64 Zn, or at least 99% 64 Zn.
• suitable levels of 64 Zn enrichment include any specific value within the recited ranges, such as 80%, 85%, 90%, 95%, 99%, and 99.8% 64 Zn.
• X% >4 Zn means that, out of 100 zinc atoms, X is >4 Zn .
• ⁇ Zn e is used herein as shorthand for “ 64 Zn-enriched zinc”.
• FIG. 1 shows survival data for experimental animals (% vs. control) that were given intratumoral (“i/t”) injections of 64 Zn e (Asp)2 comprising 2 molecules of aspartic acid/atom of zinc (written in shorthand herein as “ 64 Zn e (Asp)2”, unless otherwise indicated) at day 5 after transplantation of B 16 melanoma cells into mice.
• 64 Zn e (Asp)2 Intra-tumor administration of 64 Zn e (Asp)2 at 5th day after tumor transplantation
• FIG. 2 shows data on the antitumor activity of 64 Zn e (Asp)2 in mice (mean tumor volume in mm 3 ) that were given intratumoral injections of 64 Zn e (Asp)2 at day 5 after transplantation of B 16 melanoma cells compared to the control group.
• FIG. 3 shows data on the kinetics of B 16 melanoma growth in mice (mean tumor volume in mm 3 ) that were given intratumoral injections of 64 Zn e (Asp)2 at day 5 after transplantation of B 16 melanoma cells compared to the control group.
• FIGS. 4A-4C shows data on the inhibition of metastatic process in the lungs in C57B1 mice transplanted with B 16 melanoma after intravenous administration of 64 Zn e (Asp)245 minutes and 24 hours after transplantation of the tumor cells.
• FIG. 4C intravenous administration of 64 Zn e (Asp)245 minutes after transplantation of tumor cells.
• FIG. 5A - FIG. 5B show data on the quantitative evaluation of metastatic activity of melanoma cells after intravenous administration of 64 Zn e (Asp)245 minutes and 24 hours after transplantation of tumor cells, represented as the mean number of metastases (FIG. 5A) and the percentage of inhibitions of metastatic activity (FIG. 5B).
• the word “a” or “plurality” before a noun represents one or more of the particular noun.
• treating as used herein with respect to a medical condition such as melanoma means diminishing the severity and/or consequences of the condition, slowing the progression of the condition, preventing the spread of the condition in a patient who has the condition, preventing metastasis of the condition, at least substantially, and/or curing the condition.
• preventing as used herein with respect to a medical condition such as melanoma means preventing the development of the condition, at least substantially, diminishing the severity and/or consequences of the condition, slowing the progression of the condition, preventing the spread of the condition in a patient who has the condition, and/or preventing metastasis of the condition, at least substantially.
• “Effective amount,” “prophylactically effective amount,” or “therapeutically effective amount” refers to an amount of an agent or composition that provides a beneficial effect or favorable result to a subject, or alternatively, an amount of an agent or composition that exhibits the desired in vivo or in vitro activity. “Effective amount,” “prophylactically effective amount,” or “therapeutically effective amount” refers to an amount of an agent or composition that provides the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, prevention, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, disorder or condition in a patient/subject, or any other desired alteration of a biological system.
• a favorable result includes reducing impacts or severity of symptoms associated with a disease or disorder and/or increasing life expectancy compared to that in the absence of treatment.
• An effective amount can be administered in one or more administrations.
• the relationship between the dose levels in animals and humans is described, for example, in Freireich et al., (1966) Cancer Chemother Rep 50: 219.
• an effective amount can be first estimated either in accordance with cell culture assays or using animal models, typically mice, rats, guinea pigs, rabbits, dogs or pigs.
• An animal model may be used to determine an appropriate concentration range and route of administration. Such information can then be used to determine appropriate doses and routes of administration for humans.
• An effective daily dose is generally 0.01 mg/kg patient weight to 2000 mg/kg patient weight of an active agent, preferably 0.05 mg/kg patient weight to 500 mg/kg patient weight of an active agent.
• an exact effective dose will depend on the severity of the disease, patient’ s general state of health, age, body weight and sex, nutrition, time and frequency of administration, combination(s) of medicines, response sensitivity and tolerance/response to administration and other factors that will be taken into account by a person skilled in the art when determining the dosage and route of administration for a particular patient based on his/her knowledge of the art. Such dose may be determined by conducting routine experiments and at the physician's discretion. Effective doses will also vary depending on the possibility of their combined use with other therapeutic procedures, such as the use of other agents.
• a “patient” and a “subject” are interchangeable terms and may refer to a human patient/subject, a dog, a cat, a non-human primate, etc.
• Melanoma is a malignant tumor that develops from melanocytes (pigment-containing cells that produce melanin), which are predominantly located in the basal layer of the skin’s epidermis and the middle layer of the eye (Hurst E A et al., Archives of Dermatology Research , 2003, 139: 1067-1073). This type of pathology accounts for 10 percent of all malignant skin lesions. Its annual incidence rate is 5%. Starting from 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 in women.
• the average incidence rates for skin melanoma vary from 3-5 cases per 100,000 people per year in the Mediterranean countries to 12-20 cases per 100 thousand people per year in the Nordic countries and continue to grow.
• the death rate is 2-3 cases per 100 thousand people every year with slight changes depending on the geographical location, and has remained relatively stable over the past decade.
• An increased exposure to ultraviolet radiation of a genetically predisposed population results in a steady increase in melanoma incidence over the past decades (Oncology Clinical Practice Guidelines of the European Society for Medical Oncology (ESMO), 2010, p. 294-300).
• Malignant melanoma is responsible for 60-80% of deaths from skin cancers and its five-year survival rate is 14%. In the United States, 2% of the population was diagnosed with this type of skin cancer, which is a cause of approximately 10,000 deaths every year. At the same time melanoma is a tumor with an extremely high potential of systemic metastases.
• Primary melanoma is resistant to chemotherapy and radiation therapy.
• the main method of treatment of patients with primary melanoma is surgical removal of the tumor and some normal tissue around it.
• the amount of tissue removed with surgical excision depends on the tumor thickness (Breslow thickness) and the depth of the tumor invasion.
• surgical treatment of melanoma has a number of significant disadvantages associated with prolonged wound healing and occurrence of implantation metastases within 2 years of the surgery. This method of treatment is used only at early stages of the disease (stage 1-2). At the advanced stages, radiation therapy, chemotherapy, and immunotherapy are additionally used.
• Antitumor activity is exhibited by various compounds, such as alkylating compounds, antimetabolites, antibiotics, substances of plant or animal origin, hormonal preparations, and enzymes, which differ in their mechanism of action and chemical structure.
• these chemotherapeutic drugs produce severe adverse effects on normal tissues or organs and thus limiting their usefulness.
• Zinc isotopic composition in breast tissue can help in the diagnosis of breast cancer (F. Lamer el al, Zinc isotopic compositions of breast cancer tissue, Metallomics 2015, 7: 112-117). Further, certain patents and patent applications discuss the use of isotopically enriched compositions for therapeutic use. See, e.g., U.S. Patent Nos. 9,861,659; 10,183,041, and 10,226,484.
• W02007/140280 suggests using an anti-cancer composition for topical administration comprising a cesium ion source and/or a rubidium ion source as pharmaceutically acceptable salts to be used for melanoma treatment.
• the feasibility of using this therapy is based on an approach that involves changing the acidic pH of cancer cells to slightly alkaline, whereby the survival of cancer cells is compromised, and the formation of acidic and toxic materials, usually formed in cancer cells, is neutralized and eliminated (Sartori HE. Nutrients and cancer: an introduction to cesium therapy, Pharmacol. Biochem. Behav. 1984; 21, Suppl. 1: 7-10).
• a method for preventing the development of melanoma.
• the method effectively suppresses the development of a malignant tumor without surgical intervention and damage to surrounding normal tissues, and additionally exhibits a high anti metastatic effect.
• the use of the claimed method makes it possible to achieve effective inhibition of the development of melanoma without producing adverse effects on the body, as is characteristic of chemotherapeutic drugs.
• the method comprises administering to a subject in need thereof a light isotope of zinc in the form of aspartic acid salt.
• a pharmaceutical composition is provided to be used in the disclosed method for preventing the development of melanoma, which comprises 64 Zn e (Asp)2 in a therapeutically effective amount.
• a method of treating melanoma or preventing the development of melanoma comprises intratumoral and/or intravenous administration of a composition that contains 64 Zn e (Asp)2 in a therapeutically effective amount.
• the administration of the composition may be single or multiple.
• the treatment regimen comprises 5 to 10 injections of 64 Zn e (Asp)2.
• the aspartate present in 64 Zn e (Asp)2 is at least 90% the L-enantiomer, at least 95%, at least 98%, or all L-isomer. In some embodiments, it may be the D-enantiomer and in other embodiments it may be a mixture of the two enantiomers.
• a method is provided of treating melanoma or preventing the development of melanoma comprising the administration of a composition comprising a therapeutically effective amount of 64 Zn e (Asp)2 to a patient in need thereof.
• the composition is an aqueous solution.
• the composition is administered intratumorally or intravenously.
• the 64 Zn e (Asp)2 comprises 2 molecules of aspartic acid. In some embodiments, from 0.2 pg/kg patient weight/day to 2000 mg/kg patient weight/day of 64 Zn e (Asp)2 is administered to the patient.
• the composition is administered once a day. In other embodiments, the composition is administered more than once a day. In some embodiments, the composition further comprises deuterium-depleted water as a solvent. In some embodiments, the method of treating/preventing melanoma is a method that prevents, delays, or ameliorates melanoma metastasis.
• Enantiomeric purity of the aspartate in 64 Zn e (Asp)2 may be determined by methods known in the art, such as, for example, chiral chromatography.
• the presence of zinc in the 64 Zn e (Asp)2 compound or other zinc -containing compounds may be confirmed by methods known in the art, such as, for example, atomic emission spectroscopy with an inductively coupled plasma.
• the sample Prior to subjecting the sample to atomic emission spectroscopy with an inductively coupled plasma, the sample may be treated with a mixture of mineral acids in a Teflon autoclave under the action of microwave radiation.
• Elemental impurities or impurities of sulfate ion in the 64 Zn e (Asp)2 compound or other zinc-containing compounds may be determined by methods known in the art, such as, for example, atomic emission spectroscopy with an inductively coupled plasma. Prior to subjecting the sample to atomic emission spectroscopy with an inductively coupled plasma, the sample may be treated with a mixture of mineral acids in a Teflon autoclave under the action of microwave radiation.
• Light isotopes may be purchased.
• Zn-64 oxide with the necessary degree of enrichment may be purchased from, for example, Oak Ridge National laboratory, Oak Ridge, TN, USA.
• an effective amount of 64 Zn e administered to a subject in need thereof may be from 0.2 pg/kg patient weight /day to 2000 mg/kg patient weight /day. This range of from 0.2 pg/kg/day to 2000 mg/kg/day corresponds to the amount of zinc present in the composition as part of aspartate.
• the range of 64 Zn e administered is from 0.01 mg/kg/day to 5 mg/kg/day, more preferably 0.1 mg/kg/day to 1 mg/kg/day.
• compositions for use in the disclosed methods contain corresponding amounts.
• a composition for use of a disclosed method contains an amount of 64 Zn e such that a single dose of the composition contains from 1 to 100 mg M Zn c in the form of ⁇ ZndAspL, such as 1, 5, 10, 20, 30, 40, 50, or 100 mg ⁇ Zn e .
• An exemplary composition is a solution of ⁇ ZndAspL that contains 1 mg 64 Zn e per ml of solution.
• the solvent is deuterium-depleted water.
• the solution is formulated for oral or parenteral administration, such as administration by injection, such as by intratumoral or intravenous administration.
• compositions for injection may be aqueous solutions, such as solutions with a salinity and pH optimized for the route of injection.
• the solution may contain excipients such as DMSO.
• the DMSO may be present at a concentration of 1%.
• Another exemplary composition for used in the disclosed method is a tablet or other solid composition for oral administration that contains 30 mg (A Zn c .
• the composition may also be a liquid for oral administration, such as an aqueous composition, for example, a syrup.
• a treatment regimen of the disclosed method can include either intratumoral administration or intravenous administration of a composition for use in a disclosed method, or both routes of administration. When both routes are used in a patient, the same composition may be administered via both routes, or different compositions may be administered. In some embodiments, combined intratumoral and intravenous administration routes are used.
• melanoma model systems are used to study the efficacy of a composition comprising 64 Zn e (Asp)2 to simulate in vivo a number of processes of tumor dissemination in warm-blooded animals, including operative/ surgical intervention in case of a possible spread of tumor cells to remote sites and/or local niches.
• Postoperative administration of the composition and/or its administration to prevent a possible melanoma metastatic process (and hence tumor progression) provides suppression of the metastatic process and has significant advantages.
• Cancer tissues are largely enriched with heavy isotopes (such as Zn 70 ) and depleted of light isotopes (such as M Zn c) of the basic elements.
• Substitution of M Zn c by 70 Zn e ( 70 Zn-enriched zinc) may result in an isotope-induced change in the chirality of one or more amino acids in the protein structure and, as a result, may affect the conformation of receptors, ligands, and signal molecules.
• Loss of the structure correctness, depletion and degradation of proteins may lead to disruption in intra- and intercellular homeostasis, and, as a consequence, to various pathologies.
• the rate of development of the disease and symptom expression may be slow or rapid, depending on the characteristics of pathological chiral amplification of amino acids due to autocatalytic reactions in living cells.
• the reactions of asymmetric autocatalysis have a nonlinear character of the relationship between yield and time. The most dramatic consequences occur when the above changes take place in the p53 protein, known as the “protector of the genome”, two-thirds of which consist of zinc fingers. There are five stable isotopes of zinc.
• “wrong” conformation of p53 causes failures in stopping the cell cycle, malfunctions of differentiation, apoptosis, metabolism, genomic stability, angiogenesis, DNA repair, aging, and other processes.
• the very onset of pathological changes is due to chirality induced by isotope substitution, leading to changes in the conformation of proteins. These changes are apparently reversible, and therefore a return to the normal state can be achieved by using light isotopes. With regard to p53, this may be the >4 Zn isotope.
• the disclosed method results in isotopic selective protein modulation, which makes it possible not only to restore damaged negative feedback in the cell communication system by restoring transmitting and receiving receptors and signal molecules but, more importantly, opens a possibility for eliminating mutations in biomolecules by reactivating the normal functions of p53 protein and associated pathways.
• the mass spectrometric study shown in the Examples provides confirmation.
• Example 1 In vivo study supporting the efficacy of the claimed method carried out in mouse models (intratumoral administration of 64 Zn e (Asp)2)
• B16 melanoma cells having the following characteristic were used in the experiment: [0044] Origin: Mus musculus skin (C57BL/6 mouse)
• Tumor inoculability is 100%.
• the minimum dose of cells that causes tumor growth for this melanoma is only 100 to 1000 per subcutaneous injection in the mouse.
• An average life expectancy of animals is 21-31 days.
• the tumor cell population is heterogeneous and includes both highly pigmented regions and fragments with a low content of melanin.
• a characteristic feature of B 16 melanoma cells is a very low mRNA expression level of c-myc, c-jun, and c-fos oncogenes and the absence of c-ras, c-abl, c-erb-B2, B-lym, c-sis and c - myb oncogene expression.
• Another feature inherent in the cells of this melanoma is that they produce in vitro a large amount of the factor exhibiting procoagulant activity.
• mice All studies that involved using mice were conducted in compliance with the rules of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes [Commission of the European Communities: Council Directive of 18 December 1986 on the Lows, regulating the Application of Principles of Good Laboratory Practice and the Verification of Their Applications for Tests on Chemical Substances (87/18/EEC). The Rules Governing Medicinal Products in the European Community. - 1991. - V. 1. - P. 145-146]
• mice 15 female C57BI/J6 mice (5 mice per group) at the age of 10-12 weeks, weighing 18-22g were used in the experiment.
• 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% Versene solution and the suspension cellularity and viability was evaluated in the presence of trypan blue in a hemocytometer and the suspension was adjusted to a concentration of 10 7 cells/ml by dilution with saline solution. Melanoma cells were injected intracutaneously (i.c.) in 0.05 ml of the suspension (0.5xl0 6 cells/mouse) into the animal’s back region. 24 hours before the injection of tumor cells, the wool cover on the back of each mouse was removed with depilation cream. [0060] Grouping. The animals were divided into groups as follows:
• Group No. 1 control group, mice injected i.c. with B16 melanoma cells;
• Group No. 2 mice injected i.c. with B16 melanoma cells + intratumoral injections of 64 Zn e (Asp)2 at day 5 after the tumor cells were inoculated.
• Group No. 3 mice injected i.c. with B16 melanoma cells + intratumoral injections of 64 Zn e (Asp)2 at day 11 after the tumor cells were inoculated
• the composition to be administered was prepared immediately prior to its administration.
• 64 Zn e (Asp)2 was dissolved in deuterium-depleted water with addition of 1% DMSO.
• Animals in group No. 2 were injected with the composition comprising 64 Zn e (Asp)2 intratumorally and around the area of tumor growth at a dose of 200 pg/mouse in a volume of 20 pl/mouse after the tumor reached 0.5 cm in diameter (at day 5 after i.c. administration of the tumor cells).
• the following injection of 64 Zn e (Asp)2 was given according to the above scheme in the event an experimental animal had a new tumor growth.
• group 3 mice received 300 mcg/mouse of 64 Zn e in the form of 4 Zn e aspartate on the 11th day and 200 mcg/mouse of 4 Zn e aspartate on the 13th, 15th, 17th and 19th days after tumor cell injection.
• group 2 mice received 200 mcg/mouse of 4 Zn e aspartate on the 5th day after tumor cell injection and then 4 Zn e aspartate was given in the event an experimental animal had a new tumor growth at a dose 200 mcg/ml.
• V 4/3 x abc; where V is the tumor volume (mm 3 ); a, b, c is the rumor radius (mm): a is the radius along the x axis, b is the radius along the y axis, c is the radius along the z axis.
• FIGS. 1-4 show the results of the efficacy of the claimed method in mice.
• the survival rates of the experimental animals that received intratumoral injections of the composition comprising 64 Zn e (Asp)2 were also determined during the experiment (see Table 2).
• Antitumor activity against melanoma also manifested itself in the percentage of alive animals on the 31st day.
• the survival rate for mice injected with 64 Zn e ( Asp)2 at day 5 after inoculation of tumor cells was 80%, while only 20% of animals survived in the control group.
• Table 3 shows growth kinetics of B16 melanoma during 64 Zn e (Asp) 2 therapy in group 2 and individual regimens of drug administration
• Example 2 In vivo study supporting the efficacy of the claimed method carried out in mouse models (intravenous / intravenous + intratumoral administration of 64 Zn e (Asp)2)
• Example 1 An experimental model of hematogenous metastasis was used in the experiment which allowed for the inoculation of B 16 melanoma cells. The characteristic of these cells is given in Example 1. The cells were cultured in vitro under standard conditions. For transplantation, tumor cells were removed from the substrate with 0.02% Versene solution and the suspension cellularity and viability were evaluated in the presence of trypan blue in a hemocytometer and the suspension was adjusted to a concentration of lxlO 6 cells/ml. Melanoma cells were injected intracutaneously (IC) in 0.05 ml of suspension (0.5xl0 6 cells/mouse) into the back area of the animal.
• IC intracutaneously
• ⁇ Zn e iAspji was dissolved in deuterium-depleted water.
• the composition was injected intravenously using a microinjection syringe at a dose of 60 pg of 64 Zn e /mousc: two injections at a dose of 30 pg/mouse, each in a volume of 0.3 ml (0.6 ml in total), for 4 hours.
• the injections were given every other day for 10 days (5 injections in total).
• the first injection of the inventive composition was given 45 minutes or 24 hours after inoculation of tumor cells.
• the lungs were excised from all animals in each group and the number and volume of metastases were then determined.
• mice C 57 BI mice (8 mice per group) at the age of 12- 14 weeks, weighing 25-27g were used in the experiment. The conditions in which animals were maintained are described in Example 1 above. [0082] Before the experiment, all the animals were healthy, with normal behavioral performance. During the experiment, the animals were maintained in plastic cages under natural light illumination on a standard diet with free access to food and water.
• the 64 Zn e ( Asp)2 composition was injected intravenously into the animal’s lateral tail vein using a Micro-Fine Plus microinjection syringe (Becton Dickinson). The injection site was cleaned with 96% ethanol.
• Group No. 1 control group, mice injected IV with B16 melanoma cells + IV injection of the solvent (deuterium-depleted water);
• Group No. 2 mice injected IV with B16 melanoma cells + IV injection of 64 Zn e ( Asp)224 hours after the tumor cells were inoculated;
• Group No. 3 mice injected IV with B16 melanoma cells + IV injection of 64 Zn e ( Asp)245 minutes after the tumor cells were inoculated
• volume of metastases was calculated using the following formula for the volume of a sphere:
• V 4/3 x 7tr 3 , where V is the metastasis volume (mm 3 ); r is the metastasis radius (mm).
• V the metastasis volume (mm 3 ); r is the metastasis radius (mm).
• Example 3 Study of the distribution of light and heavy isotopes of chemical elements in samples of cutaneous melanoma
• Amorphous ice was removed by sublimation under vacuum at a low temperature with automatic supply of dry nitrogen to the drying chamber to accelerate the sublimation process.
• the volume of gas supplied to the drying chamber did not exceed 0.1 1/min.
• the drying time of the sample under these conditions was about 10 hours.
• a constant mass of the sample until two identical mass values were obtained was a criterion for the completion of the drying process.
• the biomaterial 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 values of the mass were obtained.
• the dry sample was removed from the vacuum chamber and laid in thin layers (not more than 5 pm each layer) between copper grids fixed in the cage.
• 50 semi-circular copper grids 50-100 microns thick were used which were tightly pressed together by a metal clamp holding them.
• the “sandwich” prepared in this way for mass spectrometric analysis consisted of 50 copper grids, between which the test material was firmly pressed.
• the analyzed area on the clamp surface was a circle 10 mm in diameter, in the center of which there were copper grids with the sample pressed in between them.
• Sensitivity peak height, total ion current: > 1 x 10 10 cps, 1.6* 10 9 A;
• Example 4 For the experiment, 64 Zn e (Asp)2 was synthesized from 64 Zn e oxide and was in a powdered form after the synthesis. A solution of the concentration required for the experiment was prepared immediately prior to its administration to the animals by dissolving the required amount of obtained 64 Zn e (Asp)2 powder in physiological saline or in deuterium-depleted water. [00111] The method of atomic emission spectroscopy with an inductively coupled plasma was endured to confirm the presence of Zinc cation in the sample. Prior to determination the sample was treated with a mixture of mineral acids in a Teflon autoclave under the action of microwave radiation. The presence of Zinc in the sample was confirmed as the main cation; its approximate content was 17,6%, The impurities of other element s was also revealed.
• a zinc content was found to be 17,98 ⁇ 0,17% corresponding to 90,8 ⁇ 0,9%; of Zinc L- aspartate.
• Sulfate ion content in the sample solution was determined by turbidimetry method using Barium chloride. The content of sulfates in the sample was found to be 0,66 ⁇ 0.032%.
• Elemental impurities and impurities of sulfate ion in the sample were determined. Elemental impurities were recovered by atomic emission spectroscopy with an inductively coupled plasma. Prior to determination, the sample was treated with a mixture of mineral acids in a Teflon autoclave under the action of microwave radiation. Impurities of Phosphorus, Calcium, Sodium, Silver, Aluminum, Bismuth, Copper, Ferum, Potassium, Magnesium and Plumbum were detected, as shown in Table 7.

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