DETERMINING A RISK OF DEVELOPMENT OF MALIGNANT TUMORS

Field of Art

The present invention relates to use of a known compoumd as medicament, particularly for treatment and/or prevention of disturbances in cellular growth and regeneration, and for lowering of sugar level in blood. The invention is further related to pharmaceutical preparation containing the compound, and to a method for assessment of the risk of malignant tumors formation. Potential use of the invention is aimed in the field of medicine and pharmacology.

Background Art

Basic research on the growth promoting, antirachitic vitamin D, took place in 1930-ies of the last centuy. Extensive investigations pointed out that the chemical structure of vitamin D could be related to 7-dehydrocholesterol. Further studies revealed the secosterol compounds named vitamin D2 (ergocalciferol) and D3 (cholecalciferol). The biologiccal activity of these compounds was very low and required activation by ultraviolet radiation. Chemical structure of vitamin D1 has never been identified, it remained to be known only as the UV-radiation product of vitamin D2. Similarly like in case of other derivatives of cholesterol, the vitamins D2 and D3 were considered to be the purely lipid soluble compounds. Their research was centered to the purely lipid soluble animal lipids such as butter or cod fish oils. With exception of the hydroxylated vitamin D3 in liver (vitamin D3 triol), our knowledge on the antirachitic vitamin D remained unchanged for the whole 20th century until the present time (Wolf, G. 2004. J. Nutr. UA , 1299-1302).

About 30 years later, a german chemist P. Karlson discovered accidentally in the search for an insect moulting hormone, for the first time chemical structure of the polyhydroxylated derivative of 7-dehydrocholesterol, named ecdysone (Karlson, P., 1966: Naturwissenschaften 53, 445-453). The compound was generally believed to be the hormone of the insect prothoracic gland, stimulating developmental process associated with moulting cycles (ecdysis). Ironically, numerous phytochemists of various countries in the world reported almost immediately the presence of these "insect moullting hormones" in different species of plaants. Certain species, such as a fern Polypodium vulgare, contained just in 1 gram of its rhizomes the same amount of ecdysone as did 500 kg of silkworm pupae used in the work of P. Karlson (Jizba, J. et ak, 1967: Tetrahedron Letters 18, 1689-1691). Extensive investigations during 1970-1985 revealed that the polyhydroxylated deivatives of 6-keto, 7-dehydrochol sterol (ecdysteroids), contained in numerous species of lower and higher plants, exhibited strong anabolic, growth stimulating effects in various invertebrate animals, vertebrates, mammals and also in the human body (review by Slama, K. and R. Lafont, 1995: European Journal of Entomology 92, 355-378). The results of my investigations revealed that prothoracic glands of insects and its hormone was not engaged in regulation of insect development and ecdysone did not stimulate insect ecdysis at all. Moreover, it appeared that ecdysone was not an animal hormone, because its biological status breached the definition of an animal hormone (Slama, K. 1998: Ann. Ent. Soc. Amer. 91, 168-174). All these extensive findings pointed out that the compound related to ecdysone, that is the polyhydroxylated derivatives of 6-keto, 7-dehydrovholesterol, have no biological status of an animal hormone. They constitute a very important class of essential factors of cellular growth in the majority of living organisms, including microorganisms, fungi, lower and higher plants, invetebrate and vertebraate animals, being important for growth of muscles and bones in the humans (Slama, K. &. A. Zhylitskaya, 2016: Comparative Biochemistry and Physiology, Part C 181-182, 56-67).

Disclosure of the Invention

Based on a prolonged study on regeneration process in insects, I came to a controversial conclusion that vitamin D was incorrectly identified 100 years ago. Technical state of organic chemistry did not permit at that time structural elucidation of the partly water soluble polyhydroxylated derivatives of cholesterol. Results of new experiments revealed that the for a long time neglected and incorrectly identified antirachitic vitamin D1 actually represents the polyhydroxylated 6-keto, 7-dehydrocholesterol of the structual formula (I). This compound was discovered much later in the search for insect hormone. Recent pharmacology still recognizes secosterols as the antirachitic vitamins D2 and D3, which need to be activated by UV radiation and additionally hydroxylated in liver (vitamin D3 triol). From extensive investigations on tissue regeneration in insects I came to a conclusion that the so far unknown vitaminic nature of the "ecdysteroid insect hormones", actually the incorrectly asssigned vitamin D1 a 100 years ago, could stay behind some incurable diseases induced in fact by deficiency of the vitamin D1 in blood. Since avitaminosis cannot be sucessfully cured without knowledge of the responsible vitamin, the inability of recent medical science to explain the real cause of malignant cellular growth and regeneration depends on the notorious belief of professional chemists and biochemists in the concept that polyhydroxylated 6-keto, 7-dehydrocholesterol is an insect moulting hormone, thereby restrain from the research of its real vitamin-based biological nature (Kumpun, S. et al., 2011 : Food Chemistry 125,1126-1234; Lafont, R. a ostatni, 2011, In: Gilbert, L. I. (Editor). Insect Endocrinology. Elsevier B.V. Amsterdam, The Netherlands. 588 pp.).

The main contribution of my scientific work lies in supplying experimental evidence showing that ecdysteroids comprise in fact the animal vitamin Dl, which is required for sucessfull regeneration process. Insect cells contain 37 % of genes that are the same as also contained in the human genome (Devillers, I, 2013: CRC Press, Boca Raton, Florida, 387pp). It has been recently determined (Slama, K. a J. A. Santiago-Blay, 2017: Life: The Excitement of Biology 5, 4-70), that primordial structure and function of insect tissues and organs are orchestrated by similar sets of genes as found in the human tissues and organs. I feel fully authorised to conclude, therefore, that the presented evidence indicating that the effects of compounds of the general formula (I) - vitamin Dl- prevening malignant tumor growth in insects, is valid for prevention of malignant tumor growth in the human body as well.

The object of the present invention is a compound of general formula (I),

wherein X is independently H or OH, while at least one X is OH;

for use as a medicament.

The subject-matter of the invention is based on a surprising recognition that the regenerating animal cells require essential vitamins of the general formula (I) for the growth and successfull development of cytoplasmic memranes. This can be explained by the fact that living cells of an organism form a mutually integrated unit. Disturbance of the integrity, by natural cell death or artificial injury of some cells, induces regeneration process based on mitotic divisions in the marginal cells which have lost their contacts and the tissue integrity. These cells divide during the whole regeneration process until the mutual contacts become re-established, as can be observed in Figure 1. The animal, human and plant cells require the presence of compound of the general structural formula (I) for construction of the bipolar, hydro/lipophilic properties of their cytoplasmic membranes, which is responsible for recovering the tissue integrity. Deficiency of this compound in insect haemolymph or in the blood of animals and humans, which are unable to synthesize the compound, prevents the regenerating cells from establishment of the indicated tissue integrity needed for arresting further cell divisions. The animals are dependent on the dietary supply of the vitamin from organisms which are capable to biosynthesize the compound of general formula (I) (microorganisms, fungi, lower and higher plants, intestinal symbiotic flora).

The above described facts are also related to an important recognition that a deficiency of 20- hydroxyecdysone (compound of general structure (I)) in a diet, leads to malformed or defective regenerating cells, which are unable to arrest the cell divisions, which is associcated with the appearance of unusual, polynuclear syncytia of cells, characteristic for the malignant tumor structures.

The recent medical science considers the main growth-promoting vitamins to be the compounds related to vitamin D2 and D3. These vitamins are soluble only in the lipid solvents, occur only in the animals and are biologically inactive without UV-irradiation, and are biologically inactive in relation to cell regeneration. In contrast to this, the compounds of general formula (I) are partly soluble both in water and lipid solvents, occur in a number of plant species and are biologically effective without UV-radiation.

Another object of the present invention is the compound of general formula (I) for use in the treatment and/or prevention of cell growth and regeneration disorders.

Another object of the present invention is the compound of general formula (I) for use in lowering the blood sugar concentration, particularly in the treatment and /or prevention of diabetes, especially diabetes of type II.

In one preferred embodiment, the cell growth and regeneration disorders are selected from a group, comprising cancer, disorders of growth and regeneration in the somatic bone and muscle cells, in particular breast gland cancer, salivary gland cancer, prostate cancer, pancreatic cancer, intestinal cancer, bone cancer and skin cancer.

In one preferred embodiment, the compound according to the present invention has the structural formula (la).

This compound (20-hydroxy ecdysone) was used as a pilot compound in a majority of experimental procedures on insects and humans, related to the subject of the present invention.

Further object of the present invention is a pharmaceutical preparation containing the compound of general formula (I), and at least one pharmaceutically acceptable carrier, selected from the group comprising conservants, stabilizers, wetting agents, emulsifiers, solubilizers, salts, tasting agens (flavour agents), buffres.

In one preferred embodiment, the pharmaceutical preparation further comprises at least one cytostatics, preferably selected from the group of cytostatic compounds used in the treatment of cancer in the milk glands, salivary glands, prostate, pancreas, intestine, bones and skin. Therefore, the pharmaceutical preparation can be conveniently employed for the combined application of the compound of general formula (I) and chemotherapeutics in the treatment of cancer or for preventing formation of metastases.

The object of the present invention further comprises the use of the above described pharmaceutical preparation for treatment and/or prevention of cell growth and regeneration disorders, particularly for treatment and/or prevention of cancer. Another object of the present invention is a method of determining a risk of development of malignant tumors, wherein at first, the amount of the compound of general formula (I), preferably of compound of general formula (la), in a blood sample taken from the subject is determined,

followed by comparing the determined amount of the compound of general formula (I), preferably of compound of general formula (la), with a threshold value of the risk of development of malignant tumors, and

the risk of development of malignant tumors is evaluated.

The threshlod value of the risk of development of malignant tumors for compound of general formula (I) or (la), respectively, is determined by statistical evaluation of the contents of the compound of general formula (I) or (la), respectively, in healthy individuals (> 0.1 to 4 pg /ml of blood) with the content of compound of general formula (I) or (la), respectively, in persons suffering from malignant tumors (without supplementation with compound (I) or (la), respectively), determined to be less than 0.01 pg /ml of blood. The threshold interval was experimentally determined to be within the limits of from 0.001 to 0.01 pg/ml of blood.

Evaluation of the risk of development of malignant tumors is performed such that the amount of the compound of general formula (I) in a blood sample taken from a subject is determined and compared with the threshold interval of the risk of development of malignant tumors. If the content of the compound of general formula (I) in the blood of the subject is above the threshold range, the risk of tumor incidence is considered to be low. If the content of the compound of general formula (I) in the sample falls within the threshold range, the incidence of tumors is considered as intermediate. Finally, if the determined content of compound of general formula (I) in blood of the subject is lower than the treshold range, the risk of tumor formation is considered to be high.

The above described determinbation of the risk of tumor formation is convenient though not limited, for determination of the compound of general formula (I) or (la), respectively, in a blood sample of the subject using methods of enzymatic ELISA and radioimmunoassay (RIA) procedures, developed earlier for determination of ecdysteroid content in insects (Delbecque J - P. and K. Slama, 1980: Z. Naturforsch 35C: 1066-1080).

Brief description of figures Figure 1: Schematic outline of regeneration processes described in Example 1. Mechanical removal of an area of epidermal cells (B) regenerates by mitotic divisions in marginal cells (C), replacing the wound. The mitotic divisions take place until the regenerating cells unite and establish a tissue integrity (D). When the new and old cells unite, the mitotic divisions become arrested and the extracellular cuticular cover (E) is secreted. If there is a mechanical obstacle in the wound, the disconnected cells continue dividing until they establish the tissue integrity (F). Under deficiency of the compound of general formula (I) (less than 0.01 pg/g of body mass), the cells cannot successfully unite. The cells further divide and form polynuclear syncytia which are characteristic for malignant tumors (see Example 2). The described regeneration principle is valid for tissues of all animals, including humans. The most vulnerable against the deficiency of commpound of general formula (I) are the relatively large cells secreting proteinaceous products (milk glands, salivary glands, prostate cells, pamcreatic cells, intestinal cells).

Figure 2. An impact of the compound of general formula (I) on the regeneration of excised epidermal cells in larvae and pupae of tobacco homworm (Manduca sexta ) in Example 1.

Figure 3. Regeneration patches produced by mitotic divisions in the 3 x 3 mm excised area of epidermal cells in a tobacco homworm larva (Example 2).

Figure 4: The effect of the compound (la) and vitamins D ₂ and D ₃ on growth of the body mass in chicken broilers (Example 3).

Figure 5. The effect of compound of formula (la) on the level of blood cholesterol (upper portion) and blood glucose (lower portion) in clinical patients with slightly elevated cholesterolemia and glycemia.

Figure 6. The effect of compound of formula (la) on mitotic divisions and tissue regeneration after excision of 3 x 3 mm area of pupal epidermal cells in Manduca sexta.

Examples

Example 1. The effext of compound (la) on regeneration of excised epidermal cells in larvae and pupae of the tobacco homworm (Manduca sexta)

The effect ot he compound (la) on regeneration of excised epidermal cells in larvae and pupae of Manduca sexta is shown in Figure 2. The excised epidermal window was covered by gelatine containing 2 mg/ml of compound (la) (20-hydroxyecdysone). In larvae, the preparation was overlayed by a drop of polymerizing cyanoacryllic glue (Figure 2A , 2C). The regenerating cells started to divide, replaced the missing tissue and, after connecing with cells of the original epithelium, the cell divisions were arrested (Figures 2B, 2D). Example 2. Result of investigations concerning a mixture of regeneration structures of different developmental stages on the body of adult tobacco hornworm.

Regeneration of excised 3 x 3 mm patches of epidermal cells in larvae of the tobacco hornworm, removed during the time of deficiency in enfogenous concentration of compound (la) in the body (less than 0.01 pg/g of body mass), is depicted in Figure 3. This period of temporary deficiency of the compound (la) in the body takes place during the period in between the prepupal and pupal maximum tissue proliferation (histolysis/histogenesis), between endogenous peaks in concentration of the compound (la) in the body (larger than 4 pg/g body mass). By combined use of compound (la) with bioanalogues of insect juveniale hormone (JH-I; methyl, 10,11-epoxy, 7-ethyl-3,l l-dimethyl-2,6-tridecadienoate; Paroulek M. a K. Slama, 2014: Life: The Excitement of Biology 2, 102-123) it was possible to induce a so far unknown mixture of regeneration structures of different developmental stages on the body of a single specimen. It is possible to resolve epidermis of the adult host (a), adult epidermal structure without scales and hair (b), pupal epidermis (c), second pupal cuticle (d) and, finally, the malignant tumor-like larval epidermis (e) formed during the deficiency period of compouind (la) in the body. Specimens containing the tumor-like structures of of this type (e) prematurely perish due to moulting failures and developmental disorders.

Example 3. Effects of the compound (la) and vitamins D and D on somatic growth in chicken broilers

The investigated compounds (compound (la), indicated here as viamin Di, and vitamins D ₂ and D ₃ ) were used as supplements in the commercial dietary mixture for chicken broilers. Each experimental group contained 10 specimen (see Figure 4). In the group of 40-day-old chicken broilers we took samples of blood for determination of the content of the compound (la) in blood serum. The results were obtained by means of the previously describwed radioimmunoassay method (Slama, K. et al. 1995: Experientia 52: 702-706). The results revealed substantial anabolic effect of compound (la) on growth of bone and body mass of chickens, unlike to the effects of vitamins D ₂ and D 3, which were ineffective. It was revealed that deposition and storage of the compound (la) in blood of 40-day-old chicken broilers was directly proportional to concentration of this compound in the food. In Figure 4, the dashed line corresponds to the deposition and storage of the compound (la) in blood of 40-day-old chicken broilers, and the disclosed values correspond to concentration of this compound in their diet. Example 4. Determination of the effects of compound of structural formula (la) on cholesterolaemia and glycaemia in humans

7he experiments were carried out in clinical patients, comprising assays and measurements of the effects of compound of structural formula (la) - vitamin Di, (20-hydroxyecdysone) on the level of cholesterolaemia and glycaemia (see Figure 5). The experiments were carried out on two groups, each comprising twenty 65- to 85- years old patients, exhibiting the iniitial values of 7.2 mmol cholesterol per 1 1 of blood and 8.2 mmol of glucose in 1 1 of blood. Each investigated person received a capsule with 5 mg of compound (la), 3- times a day for the period of 30 days. Control group of 15 persons received gelatine capsules filled with starch. The compouind (la) was represented by 98% pure natural product (20-hydroxyecdysone) isolated from the seeds of Leuzea carthamoides by conventional chromatographic methods in the pharmacological Company Galena in Komarov u Opavy (Czech Republic).

While the effects of compound (la) on decreasing the level of cholesterolemia has not been confirmed, curiously enough, the effects of this compound on the decrease of blood sugar in diabetic patients of the older age was evidently confirmed. The values of glucose blood concentration initially 8.7 mmol/1 decreased to 4.3 mmol/1, whereas the normal range of flood sugar concwentration is 2.9 to 5.2 mmol/1. These results provide evidence that the compound (la) evidently exhibits decreasing effect on the level of blood sugar, which can be used in medication of diabetes.

As can be observed in upper portion of Figure 5, dosages of 15 mg of the compound (la) per day had no remarkable effect on the level of blood cholesterol, the obtained values do not differ significanttly from the controls. By contrast, the blood sugar level decreased during the 30-day application allmost by half (from 8.7 to 4.5 mmol/1 (of glucose equivalent). This shows that the compound (la) favours the metabolic combustion of glucose, probably due to its enhancement of metabolic intensity. This is a very important effect with respect to medication of type II diabetes.

Example 5: Determination of the effect of compound (la) on the outcome of chemotherapy in patients suffering from breast tumors

In further experiments, the compound (la) was applied to women suffering from tumors of the milk glands. The medication was commenced from 2 to 5 weeks after termination of the first cycle of chemotherapy. The patients (15 women of 36 to 50 years) were selected from 4 principal county hospitals in Moravia. They voluntarily agreed to take the compound (la) (absolutely nontoxic natural product isolated from Leuzea), 3-times per day, in form of 5 mg gelatine capsulles for the period of 30 days (analogically to the glycemia experiments in Example 4). The results of anamnesis of these assays were astonishing: not a single woman from the 15 investigated women taking the compound (la) required the necessity to undergo the 2nd treatment of chemotherapy. After 5 years, all of them were alive without incidence of the malignant metastases. Such results are very unexpected and unusual. These results confirm antimalignant effect of compound (la) in humans, which is fully consistent with previous results obtained with the action of this compound in insects. These results support my presented claims for using the compounds (la) for reduction of malignant growth and for potentiation of the effects of chemotherapy, eventually for its substitution.

Example 6: Evidence for essentiality of vitamin D presence for successful mitotic divisions in animal cells.

Characteristic feature of malignant tumors is disturbance of the mitotic divisions, associated with the appearance of polynucleated metaplasia cells. Human body is distinguished by a closed circulatory system composed of arteries, veins and capillaries, which do not permit a direct observations of the mitotic divisions in vivo. The obstacle can be avoided by using insects possessing an open circulatory system, where individual tissues and organs are suspended in the haemolymph. This work was performed on developing and diapausing pupae of Manduca sexta, which are characteristic by different endogenous concentrations of vitamin Di (20- hydroxy ecdysone) in the body. The pupae were subjected to excision of a small, 3 x 3 mm window of epidermal tissue. The wound was covered by microscoopic cover slide, which was cemented on the margins by a drop of paraffine wax heated by a platinum wire loop of electrocautery. The progress of mitotic divisions, initiated in the margin of the wound, was evaluated in 12 h intervals under microscope, using side illumination in combination with UV- light and vital stains.

The results obtained in these observations are presented in Figure 6. Each experimental group consisted of 16 to 20 specimens (=n). Vertical bars show standard deviation, the experiment was performed at room temperature of 25 to 27 °C. The first graph A shows the course of mitotic divisions in the excised window in the deveping pipae with endogenous concentration of vitamin Di close to 4 pg /g of haemolymph. Graph B exemplifies inability of epidermal cells to reconstruct the injured epidermal cells by regular mitotic divisions. In absence of endogenous vitamin Di in diapausing pupae, at the concentration lower than 0.05 pg/g of haemolymph. Only after several days, there appeared clusters of indifferent tissue (clusters of haemocytes with polynuclear syncytia). Graph C shows the regeneration in diapausing pupae in which the mitotic divisions were stimulated by injections of exogenous vitamin Di (20-hydroxy ecdysone, 2 pg/g pupal body mass). Determination of endogenous concentration of vitamin Di in pupae was made by the previously described method (Delbecque J.-P., Slama K. ,1980. Z. Naturforsch 35C: 1066-1080). According to these results, regeneratio of the excised epidermal cells proceeds by means of mitotic divisions within several hours, provided that vitamin Di (20-hydroxyesdysone) has been present in the body of the animal. The mitotic divisions become automatically arrested when the new and old cells unite together. Under deficiency of the endogenous vitamin Di (diapause), the wounded epidermal tissue is unable to regenerate by means of regular mitotic divisions. The divisions are not initiated even after several days. Later on, in absence of vitamin Di, mitotic divisions are replaced by a strange, indifferent regeneration tissues camouflaged by accumulated haemocytes. By injections of exogenous vitamin Di we managed to induce the mitotic divisions in the injured regenerating cells. Based on the data of comnparative physiology, which shows that regeneration in insect and human tissues is very similar (Slama K., Santiago- Blay J. 2017: Life Excit. Biol. 5: 4-70), it is possible to apply the data obtained here on the role of vitamin Di in insects, described in Figure 6, to conditions existing in the human body. So far, it was impossible to monitor mitotic divisions and the origin of tumors in the living human body. For this reason, the describd results on the mitotic divisions in insects in vivo could tentatively help to extrapolate the regeneration behaviour of human cells.

Example 7: Determination of 20-hydroxyecdysone concentration in human blood and assessment of the risk of malignant tumor formation

Determination of 20-hydroxyecdysone concentration in human blood was performed according to the previously described radioimmunoassay (RIA) method. The concentration of 20- hydroxyecdysone (vitamin Di) was determined in samples of blood taken from 20 anonymous human subjects. Individual measurements were performed in samples of 150 pi of blood according to the RIA method described by Slama et al. 1996, Experientia 52, 702-706. The content of 20-hydroxyecdysone was in the range of from 0.3 ng/ml to 3 ng/ml of blood, the average value was 0.7 ng/ml ±0.6 ng SEM (standard error of the mean). The critically low value for the deficiency of vitamin Di (a threshold value indicating the risk of tumor incidence) was in the range from 0.001 pg/ml to 0.01 pg/ml of blood. Assessment of the risk of malignant tumors was made by initial determination of the content of the compound (la) in the blood of tested subject and, consecutively, the determined content of the compound (la) was compared with the threshold value for the incidence of malignant tumors. Provided that the content of the compound (la) in the blood sample of the subject was higher than the above indicated threshold range, the risk of tumor appearance was evaluated as low. When the content of the compound (la) in the blood sample of a subject was within the threshold range, the risk of tumor formation was evaluated as intermediate. Provided that the content of the compound (la) in the blood of a subject was lower than the threshold range, the risk of tumor formation was evaluated as high.