Combination in the form of dietary supplements or medications and the treatment of humans and animals with its use (options)

 

(57) Abstract:

The invention can be used for the treatment of humans and animals. Combination in the form of dietary supplements or medicine contains as active ingredients only carotenoid substance nicotinamide substance and source of zinc in the form of their separate dosage forms. The specified combination selectively complements these substances are contained in a normal diet that provides increased resistance to DNA damage, increased reparative ability of DNA and stimulation of immune function. Methods of treatment of the human or animal to obtain the above effect are in the daily introduction of a daily dose of the claimed combination, regardless of the presence or absence of the same active substance combination in a normal diet. The second variant of the method consists in daily selective introduction of the dosage forms of the active substances of the composition. The use of the invention makes it possible to treat the manifestation of various illnesses, manifested, for example, during aging, cancer, cardiovascular diseases and autoimmune disorders such as diabetes, rheumatoid arthritis and ulcerative colitis.treatment and to methods of treatment of humans and animals to reduce DNA damage, enhance the reparative ability of DNA and stimulate the immune function of cells. In particular the invention relates to the introduction of people (or other animals) of a combination of carotenoid substances nicotinamide matter, which is the source of zinc (all terms have the meanings given below), for example, in the form of medication intended for treatment, or in the form of daily supplements, and to compositions containing this combination of substances.

The term "carotenoid substance" in the context of this description understand carotenoids, for example, a substance selected from the group comprising-carotene, -carotene, -carotene, lycopene, and mixtures thereof. The term "nicotinamide substance" in the context of the present description understand a substance selected from the group comprising nicotinamide, Niacin, tryptophan (an amino acid that is a precursor in the synthesis of Niacin and mixtures thereof. The term "substance, which is the source of zinc, in the context of the present description see the corresponding source of zinc, which is intended for administration to humans and/or other animals, for example, one or more with the onin war or aspartate, dipeptides, gluconate, halides, nitrates, oxides or acetates.

According to the invention proposes a new combination occurring in natural conditions carotenoid substances nicotinamide matter, which is the source of the zinc used for the combined treatment of patients in need of such treatment designed to combat damage to a cell's DNA, such as oxidative damage, to enhance the reparative ability of the cell's DNA and stimulation of the immune function of cells. The invention relates further to the use of this combination of chemicals as food additives or drugs intended to treat, to prevent (or increase the resistance of the individual) DNA damage, to enhance the reparative ability of DNA and to stimulate immune function in diseases in which these processes are crucial for the manifestation of the disease condition; for example, during aging, cancer, cardiovascular disease and autoimmune disorders such as diabetes, rheumatoid arthritis and ulcerative colitis (Cross and others, Ann. Int. Med. 107: 526-545, 1987; Harris, C. C., Cancer Res. 51 (Suppl.): 5023s-5044s, 1991; Olin K. L., and others, Proc. Soc. Expt. Biol. andnn. N. Y. Acad. Sci., 691: 262-263, 1993; Chew, B. P. J. Dairy Sci., 76(9): 2804-2811, 1993; Santamaria L., and others , J. Nutri. Sci. Vit. Spec., No: 321-326, 1992; Machlin L. J., Crit. Rev. Food Sci. and Nutri., 35(1-2): 41-50, 1995; Murakoshi, M. and others, Cancer Res., 52: 6583-6587, 1992; Okuzumi J. and others, Oncology, 49: 492-497, 1992), nicotinamide/Niacin (Mandrup-now T. Diabetes and other Metabolism Rev., 9(4): 295-309, 1993; Pero, R. W., and others, Biochimie, 77: 385-393, 1995; Shockett, P. J., Immunol. , 151(12): 6962-6976, 1993; Boulikas T., AntiCancer Res., 12(3): 885-898, 1992; Brown R. R., Adv. Exp. Med. Biol., 294: 425-435, 1991; Henkin Y. and others , Amer. J. Med., 91(3): 239-246, 1991; Jacobsen E. L., J. Am. Col. Nutri. , 12(4): 412-416, 1993) and zinc (Singh A. and others, J. Appl. Discrimination, 76(6): 2298-2303, 1994; Walsh, S. T., and others, " Environmental Health Perspectives, 102 (Suppl. 2): 5-46, 1994; Mocchegiani E., and others, Blood, 83(3): 749-757, 1994; Singh, K. R. and others, Immunopharmacol. Immunotoxicol., 14(4): 813-840, 1992; Mei W. and others, Biol. Trace Element Res., 28(1): 11-19, 1991; Chandra R. K., and others, Clin. Lab. Med., 13(2): 455-461, 1993) each one individually has the ability to prevent disease and has immunostimulant properties, but previously they were never United with each other as a combined drug which could prevent or delay the development of diseases in humans and to stimulate immune function (Compendium of Nonpresciption Products, Canadian Pharmaceutical Association, 1994; Canadian Drug Identification Code, June, 1995; The Extra Pharmacopeia, Martindale, 30th edition; U. S. Pharmacopeia Dispensing Information, 15th edition, 1995). Therefore, analysis of the known scientific data or access the different mechanisms of action, controlling one and the same type of diseases, in doses exceeding their normal levels in food, for carotenoids, such as vitamin A, 14671213 kcal, nicotinamide 33,126,7 mg and zinc 6,88,4 mg (Payette N., Am. J. Clin. Nutr. 52: 927-932, 1990), can be obtained a composition having distinct properties improve the ability to resist damage the DNA of cells, enhance the reparative ability of DNA cells and stimulate immune function cells from the individual.

People were subjected to natural selection over hundreds of thousands of years not a single chemical compound, and countless chemical substances entering the human body from the environment, mainly from food. It can be assumed that human physiology is extremely well balanced for absorption and conversion of these chemical mixtures with the purpose of obtaining from them with the greatest possible efficiency necessary for life substances, such as obtained from food sources of energy and chemicals needed to maintain cellular homeostasis and reproduction. This should be done without showing any Toxicological effects. Therefore, there is Bolshaya their normal levels, typical food of the diet or the environment, there is a pronounced interaction between excessive doses of natural medicines, resulting in one Supplement limits the absorption and metabolism of the other, which is a natural model selection, with the help of which people can be protected from Toxicological effects and overdose. For example, in the prior art it is known that carotenoids and vitamins E or C are recovering radicals (electrophilic) and that these natural products can be combined in the form of additives to obtain the additive biological impacts. However, current literature does not support this practice, based on the scientific assumption, because it was determined that these waste radicals can inhibit the absorption of each other and negate the desired induction of biological effects (Inform. 6(7): 778-783, 1995; Zhang and others, J. Clin. Nutr. , 62: 1477S-1482S, 1995; Niki and others, Am. J. Clin. Nutr., 62: 1322S-1326S, 1995).

Known marketed commercial products containing excessive doses (i.e., above normal levels in the diet) of carotenoids, nicotinamide and zinc in combination with each other, and, in addition, they have n the tion. Below are two examples.

A commercial product I

("Radical Fighters", Twin Laboratories, Inc., Ronkonkoma, NY 11779, USA)

-carotene (EQ. provitamin A) 12500 IU

Vitamin C 750 mg

Ascorbyl palmitate - 125 mg

Vitamin E (d-tocopherol) - 250 IU

L-cysteine 250 mg

L-glutathione (reduced) - 12.5 mg

Selenium (Selenite) - 100 mcg

Zinc (gluconate) 10 mg

Vitamin B175 mg

Vitamin B250 mg

Nicotinamide 50 mg

Niacin 25 mg

Vitamin B5250 mg

Vitamin B6- 62,5 mg

Vitamin B12- 150 mcg

Folic acid 200 mcg

Biotin - 75 mcg

PABK (para-aminobenzoic acid) 150 mg

Inositol 100 mg

Choline 100 mg

Commercial product II

(Vitamins for women, Bonne Forme, 4250 Hempstead Tpke, Suite 21, Bethpage, NY 11714, USA)

Vitamin A - 5,000 IU

- carotene 3 mg

Vitamin D3- 400 IU

Vitamin E - 200 IU

Vitamin K 10 mcg

Vitamin C 500 mg

Vitamin B110 mg

Vitamin B210 mg

Vitamin B650 mg

Niacin 50 mg

Folic acid - 400 mcg

Vitamin B12- 50 mcg

Biotin - 50 mcg

Pantothenic acid 20 mg

Calcium - 1000 mg

Magnesium 300 mg

Potassium - 40 mg

Molybdenum - 10 mcg

However, these commercial products are not installed or is not obvious that a certain combination of carotenoids, nicotinamide and zinc are effective in the reduction of the induction of DNA damage cells or enhancing DNA repair and immune function. In contrast, as described below, according to the invention it was found that the introduction of carotenoids, nicotinamide and zinc in combination with other natural medicines or nutrients, for example, in the form specified above commercial product I, does not reduce the induction of DNA damage cells or not increased DNA repair and immune function that was assumed, but not proved in the prior art. This fact is also consistent with the prior art (Inform 6(7): 778-783, 1995; Zhanf and others, Clin. Nutr., 62; 1477S-1482S, 1995; Nidi and others, Am. J. Clin. Nutr., 62: 1322S-1326S, 1995), confirming that natural products (for example, drugs or nutrients) that have similar biochemical mechanisms of action, as installed, have the ability to block the absorption and the absorption of each other, which leads to changes in biological functions. Hence, although it has not found practical volumereduction, added in combination, it cannot be assumed that the additive combination of natural products in quantities greater than their levels in the diet should lead to additive biological effects of each separately introduced product.

The exact mechanism of action of carotenoids such as - carotene, is not fully elucidated, but from a scientific point of view it is generally recognized that one of the main mechanisms is the utilization of oxygen originating from free radicals produced either as a by-product of metabolism, or having an exogenous origin (from the environment) (Lieber D. C., Ann. N. Y. Acad. Sci. 691: 20-31, 1993; Bohm F., and others, J. Photochem. Photobiol. 21(2-3): 219-221, 1993; Regnault C. and others, Ann. Pharmacotherapy 27(11): 1349-1350, 1993). It can be expected that, as a recovery of free radicals, carotenoids can reduce or protect cells from chemical damage to DNA, RNA and proteins of the cells in the toxic effects of environmental or endogenous metabolic abnormalities in the cell, which ultimately can lead to the painful condition. On the other hand, nicotinamide and zinc salts do not have this chemical property, which leads to improved biological functions of cells.

Zinc is different from carotenoids and nicotinamide in its mechanism of action is the fact that it has an impact on disease progression and immune function, as the major cofactor in several enzymatic functions involved in replication, DNA repair and antioxidant protection of cells. Zinc is needed for the replication of cells and the activity of DNA polymerase (Williams, R. O., and others , J. Cell Biol. , 58: 594-601, 1973). There are two zinc - "finger" DNA-binding domain of the gene poly-adenosyltransferase (ADPRT) and other proteins involved in DNA repair (Dawat P. and others, Environ. 141 (Pt 2): 411-417, 1995; Matsuda T., and others, J. Biol. Chem. 270(8): 4152-4157, 1995; Chiriccolo M. and others, Mutation Res., 295(3): 105-11, 1993), which contain OST the t to prevent binding of DNA and proteins involved in DNA repair (Mazen and others, Nucleic Acid. Res. 17: 4689-4698,1989; de Murcia, G., and others, BioEssays, 13(9): 455-462, 1989; Pero, R. W., and others, Biochimie, 77: 385-393, 1995; Althaus F., and others, Mol. Cell. Biochem. , 138(1-2): 53-59, 1994). In addition, superoxide dismutase, representing an antioxidant enzyme that protects cells from harmful superoxide anion, because this radical is a substrate for reaction with the participation of this enzyme also requires zinc as a cofactor (Brunori, M. and Rotilio G. , Methods in Enzymology, 105: 22-35, 1984).

In General though, it was established that carotenoids, nicotinamide/Niacin and zinc individually possess some useful properties in cells, and they are as individual agents primenenii models-animals to prevent certain diseases and to stimulate immune function, but no corresponding matching data against people (Bodgen J. D., and others, Amer. J. Clin. Nutri., 48: 655-663, 1988; Walsh, C. T., and others, " Environmental Health Perspectives, 102(Suppl. 2): 5-46, 1994). In addition, the specialist in the art it is impossible a priori to predict whether agents with different mechanisms of action to provide a synergistic, additive or inhibitory effect on the biological response that they will call when introduced in combination with each other.

A brief presentation is La introduction to people or other animals and consisting mainly of a combination of carotenoid substances, nicotinamide matter, which is the source of zinc, and practically devoid of other active ingredients. Under the concepts of "composed of" and "practically devoid of other active ingredients" means that the composition does not contain active nutrients in addition to the above carotenoid substances nicotinamide matter, which is the source of zinc. The concept of "active nutrients" in the context of the present description is used as a generic term for vitamins, minerals and other compounds that serve as antioxidants, cofactors antioxidants or other substances involved in the prevention of disease, in the inhibition of DNA damage, in enhancing the reparative ability of DNA and/or enhance immune function, such as previously received on sale in concentrated, isolated or combined form as food additives, etc., for consumption by humans and/or animals. In particular, the concept of "active nutrients" specifically includes the above-mentioned ingredients of the two products mentioned above as a commercial and commercial product I product II.

In other words the substance, nicotinamide substance and a substance which is the source of zinc, and containing no other active nutrients. The composition of the invention can be included in the formulation for oral administration or in another embodiment, in a formulation for parenteral administration.

In an illustrative or preferred variant of the invention, the carotenoid substance may be selected from the group comprising-carotene,- carotene, -carotene, lycopene and mixtures thereof, nicotinamide substance may be selected from the group comprising nicotinamide, Niacin, tryptophan and mixtures thereof, and a substance which is a source of zinc, may be one or more zinc salts.

For the introduction of human carotenoid substance nicotinamide substance and a substance which is the source of zinc can be present in combination and in proportions that provide increased resistance to DNA damage, increased reparative ability of DNA and stimulation of immune function in a patient man, which enter the composition in the form of a daily dose.

The invention also includes a method of treatment of a human or other animal, introducing the subject of carotenoid substances, N. the ion entity (i.e. without adding to the diet of any other active nutrients) and in the repeated introduction basically given a daily dose.

Thus, according to another variant of the invention relates to a method of treatment of a person providing a selective introduction to the subject the combination of carotenoid substances nicotinamide matter, which is the source of zinc, in the form of daily doses in an amount to provide increased resistance to DNA damage, increased reparative ability of DNA and stimulation of immune function. In particular, in one specific example implementation of this method people daily enter common preferred dose comprising about 100 mg of carotenoid substances, approximately 100 mg nicotinamide substances and approximately 10 mg of the substance, which is the source of zinc.

From a theoretical point of view, the present invention is based on the principle of pooling of chemical products, for which it is known that they individually possess properties or to prevent cancer, or to stimulate the immune system, in a single formulation, which contains only these and is different from the known mechanisms of action of other components. It is known from the prior art, this principle has not been previously used in this field.

The invention is based on the fact that natural products cannot be combined in the form of natural medicines until then, have not been studied, does each ingredient, an additive impact on the overall desired biological effect, and that one way to achieve this is the principle that you cannot combine natural products that have similar mechanisms of action and, therefore, competitive ways of absorption and excretion without testing combinations on the additivity of the action. It should be noted that the present invention allows to avoid inhibition of the uptake and absorption of natural products, allowing you to achieve additive biological effects by combining only natural products that have distinct different and, therefore, potentially noncompetitive mechanisms of action that have been observed, for example, in the case of an exclusive combination of carotenoids + nicotinamide + zinc.

Thus, for example, found that when a combination of carotenoids + nicotinamide + zinc lashes, enhancing the reparative ability of DNA and stimulation of immune function. The data obtained confirm that when combining these agents, which have different known mechanisms of action in respect of stimulate immune function, in concentrations exceeding those typical of normal diet, treatment with this combination leads to a more favorable character and additive effects on biological response.

According to the present invention provides for the introduction of more people or animals, for example, via oral, intraperitoneal, intravenous, subcutaneous or intramuscular methods of introduction, combinations of carotenoids + nicotinamide/Niacin + corresponding zinc salt, and the dose of this combination exceeds the characteristic of the Supplement to the normal diet. In the prior art it is known that a dietary Supplement containing this combination together with the simultaneous addition of other nutrients and/or natural products, can enhance immune function (Payette H. and others, Am. J. Clin. Nutr. , 52: 927-932, 1990; Zhang and others, J. Clin. Nutr., 62: 1477S-1482S, 1995), however, when used only carotenoids (Caloplaca, the firm of C. E. Jamieson, Ltd., ONT the levels in the diet, for example, 100 mg, 100 mg and 10 mg, respectively, by daily oral administration for 7 weeks, observed resistance to oxidative damage to cellular DNA and amplification of DNA repair and immune function.

Assessment in clinical conditions consisted in the comparison of each individual biological reactions before and after supplementation. Thus, each individual was his own control; for example, men conducted a baseline measurement of resistance to oxidative damage to cellular DNA and amplification of DNA repair and immune function once a week for 4 weeks, then they were daily injected a drug, and the same measurements were repeated once a week for the last 5 weeks 7 weeks of the intervention period. The first measurement (i.e., n = 4) served as the basic parameters of biological reactions, which were compared with the results of subsequent measurements (i.e., n = 5). One individual did not enter the additive, and he served as a control for individuals who have introduced additive. The data from this experimental study showed that resistance to oxidative damage to cellular DNA, and amplification of DNA repair, and stimulation of the immune F. the combination of drugs in doses above normal levels in the diet, but not when it is administered together with other additional nutrients or supplements natural product.

Other features and advantages of the invention are described in more detail below with reference to the accompanying drawings.

Brief description of drawings

In Fig. 1 presents a histogram characterizing single-strand DNA breaks, which caused 100 μm hydrogen peroxide and which were evaluated in CHOL (mononuclear leukocytes person) using the alkaline elution before and after adding natural products carotenoids + nicotinamide + zinc of the present invention. Data are given as mean values and standard deviations (CO)p<0.05 compared with the data before supplementation.

In Fig. 2 presents a histogram characterizing the DNA repair through 30 minutes after the induction of single-strand DNA breaks 100 μm hydrogen peroxide in CHOL, which was assessed using the alkaline elution before and after adding natural products carotenoids + nicotinamide + zinc of the present invention. Data are given as mean values and standard deviations (CO)p<0.05 compared with the data Polaris 60 minutes after the induction of single-strand breaks in DNA by hydrogen peroxide in CHOL, which was assessed using the alkaline elution before and after adding natural products carotenoids + nicotinamide + zinc of the present invention. Data are given as mean values and standard deviations (CO)p<0.05 compared with the data before supplementation.

In Fig. 4 presents a histogram describing the activity of poly-ADPRT in CHOL before and after adding natural products carotenoids + nicotinamide + zinc of the present invention. Data are given as mean values and standard deviations (CO) p<0.05 compared with the data before supplementation.

In Fig. 5 presents a histogram characterizing the inclusion of [3H] -thymidine after due PHA mitogenic induction in CHOL before and after adding natural products carotenoids + nicotinamide + zinc of the present invention. Data are given as mean values and standard deviations (CO)p<0.05 compared with the data before supplementation.

In Fig. 6 presents a histogram characterizing concentrations ABOVE in human erythrocytes before and after adding natural products carotenoids + nicotinamide + zinc of the present invention. These predanie supplements.

In Fig. 7 shows a series of histograms to compare in vivo the effect of adding the composition of carotenoids + nicotinamide + zinc according to the present invention ("KSC"), and supplements specified above as a commercial product I ("KPI") on DNA damage in mononuclear leukocytes of man. DNA damage caused 100 μm hydrogen peroxide and evaluated using the alkaline elution. Data are in one column, representing the average value and interval uncertainty, characterizing the standard deviation (CO) (n= 4-5). indicates that the difference was significantly (p<0,05, in accordance with the student test) compared with the corresponding baseline. Comment: the patient is identified as an option And did not receive a commercial product I, as he had an attack of acute appendicitis just before the beginning of the course of the intervention, and it did not take blood samples during the 3 weeks after that.

In Fig. 8 shows a series of histograms to compare in vivo the effect of adding combinations of carotenoids + nicotinamide + zinc according to the present invention ("KSC") and supplements the above commercial product I ("KPI") for the repair of DNA in mononuclear leukocytes of man. The DNA repair center the data given in the form of a column, represents the average value and the error range, which characterizes the standard deviation (CO) (n=4-5 for each column). indicates that the difference was significantly (p<0,05, in accordance with the student test) compared with the corresponding baseline. Comment: option not received a commercial product I, as he had an attack of acute appendicitis just before the beginning of the course of the intervention, and it did not take blood samples during the 3 weeks after that.

In Fig. 9 presents a series of histograms to compare in vivo the effect of adding the composition of carotenoids + nicotinamide + zinc according to the present invention ("KSC") and supplements the above commercial product I ("KPI") on stimulation with PHA of human lymphocytes. The results are expressed as % of baseline (column represents the mean value, and the line error characterizes the standard deviation of the (CO)). indicates that the difference was significantly (p<0.05) as compared with the corresponding baseline. Comment: option not received a commercial product I, as he had an attack of acute appendicitis just before the beginning of the course of the intervention, and it did not take blood samples in tiante embodiment of the invention presents the use of a combination which basically consists of carotenoids + nicotinamide + zinc gluconate (and does not contain other active nutrients), as additives for oral administration, the levels of these components exceed the normal typical dietary intake levels or are within these levels, which is injected every day to improve the individual's resistance to damage a cell's DNA, amplification of DNA repair and stimulation of immune response cells in vivo. The main idea of the study to confirm the invention consisted in the Association of substances with known properties concerning the prevention of cancer and to stimulate immune function, but with different mechanisms of action; for example, carotenoids are electrophilic recovery radicals, produced by endogenous cells or exogenous in the environment, nicotinamide is a potent source of energy due to the increase in education OVER or ATP, and zinc is an essential cofactor for antioxidant enzymes involved in replication and DNA repair in cells. The hypothesis was that since none of these substances did not cause pronounced effects on people with the introduction of these agents in otdelnosti pronounced additive chemopreventive (chemoprophylactic) biological response due to non-competitive mechanisms of action, not inhibiting, for example, the actions of each other.

In this experiment, the purpose of the study the present invention were four healthy male volunteers aged 30-40 years, working in the same conditions. Once a week for 4 consecutive weeks conducted a baseline assessment of the reactions of mononuclear leukocytes of man (CHOL) prior to the introduction of additives in vivo in relation to each of the following indicators: (I) the stability of the cell's DNA damage determined by alkaline elution DNA, (II) enhanced DNA repair, determined by the ability to repairbots after treatment with standard dose of hydrogen peroxide, and (III) stimulation of immune function, as defined by mitogenic stimulation phytohemagglutinin (PHA). Three volunteers, as described below, was administered orally carotenoids, nicotinamide and zinc gluconate daily for 7 consecutive weeks, and biological characteristics of the reaction was determined once a week for the last 5 weeks. One of the volunteers did not receive supplements during the 7-week intervention period, but he had to take blood samples and subjected them to biological research in relation to the analyzed characteristics of biological reactions poslednego control". Then the results of the experimental data for each individual before and after treatment were combined and conducted group statistical analysis to determine impacts during treatment and without treatment.

Used drugs were purchased from the firm of C. E. Jamieson, Ltd. (Ontario, Canada): carotenoids in the form of Caloplaca 100 mg in gelatin capsules, soft coated, nicotinamide 100 mg in the form of tablets and zinc gluconate 10 mg in tablet form, and proposed according to the present invention the combination of these three substances, sometimes in the context of the present description were identified as "KSC". Careplex is a patented natural source of carotinoides derived from palm oil containing 60% -carotene, 34% -carotene, 3% -carotene and 3% of lycopene (lwasaki R. and Murakoski M., Inform, 2(2):210-217, 1990). These three drugs are used together by oral administration in the same period of time from Monday to Saturday (every day) for 7 weeks. In all cases, each volunteer was allowed no more than 3 days without treatment during the entire period of the experiment.

To test the hypothesis that carotenoids, nicotinamide and zinc (KSC) when used in combination in prisuta stability of the individual to damage the cell's DNA or amplification of DNA repair and immune function, the same volunteers who received supplements KSC, was given a 13-week period without intervention, then they are within the 4-week period was re-defined the basic values of biological indicators used in KSC-experiment, then started oral introduction of the above commercial product I (sometimes referred to in this description as "KPI"), which was continued for 6 consecutive weeks along with control without intervention, and biological parameters were determined once a week during the last 4 weeks. KPI contain 20 ingredients, including carotenoids, nicotinamide and zinc, and it was introduced as part of the daily concentrations of each component listed in the table above under the heading "Commercial product I". In this experiment, in all cases for each volunteer was also permitted no more than 3 days without treatment throughout the treatment period, except for option A, which has been an attack of acute appendicitis just before the beginning of the KPI-intervention, and, thus, the volunteer was seen as a "control without interference."

Each week were selected approximately 20 ml of venous blood in 2 azzy plasma in accordance with the method, described in Pero and others , (J. Int. Med., 233:63-67, 1993). CHOL with a density of 2106cells/ml suspended in 10% fresh autologous plasma with the addition of medium RPMI 1640. This culture medium used in all experiments. Immediately after the taking of the blood sample were analyzed by single-strand DNA breaks using the alkaline elution, the activity of poly-ADPRT and induced phytohemagglutinin (PHA) mitogenic response in CHOL, and identified a pool of OVER using liquid chromatography high pressure (ghvd). Other unused samples were stored frozen at -70oC for further analyses.

Oxidative DNA damage and DNA repair were analyzed using alkaline elution by processing CHOL on ice standard dose of 100 μm H2ABOUT2within 60 minutes (or leaving CHEOL without processing) and then cells were incubated at 37oC for 0, 30 or 60 minutes for DNA repair. DNA damage and repair at different time points was assessed using the alkaline elution, as described by Kohn and others (edited by Friedberg, E. C. and P. Hanawalt, DNA Repair: A Laboratory Manual of Research Procedures, Marcel Dekker, New York, PP 379-402, 1981) with modifications for the evaluation of unlabeled DNA using microfluorometry (C. F. Cesarone, etc., Anal. Bioche the deposits permeability of cells, developed by Berger, with previously described modifications (Pero, etc., Carcinogensis, 10: 1657-1664, 1989). Activity of poly-ADPRT both normal and induced physiological condition, measured on CHOL at a density of 1106cells/ml at treatment or no treatment standard dose of 100 μm H2ABOUT2at 37oC for 30 minutes and then the cells were collected by centrifugation, increasing their permeability and determined the activity of poly-ADPRT radiometric method, as described in Pero and others, Carcinogensis, 10: 1657-1664, 1989).

To determine the ABOVE method Ehud frozen erythrocyte sealed debris (500 μl) was subjected to thawing in 600 μl of 1.8 M perchloro acid (PHC) on ice. After homogenization and added as an internal standard 25 µl of 2.4 mm thymidine (dThd) samples were centrifuged at 14000 xg to remove undissolved material. Supernatant (0.5 ml) was neutralized by adding 150 μl of 2M solution of K2CO3. After re-centrifugation at 14000 xg supernatant was ready for analysis using ghvd. The chemicals used for preparation of buffer solutions were analytically pure and buffer for elution consisted of a 150 mm potassium phosphate, pH 6, s sterile polysulfonyl filter with a pore size of 0.2 μm (firm VacuCap, Gelman Sciences, Ann Arbor, MI). The analysis ABOVE was performed in column C18(3 µm) (h,3 mm (inner diameter), firm Perkin Elmer Corp., Norwalk, CT) with chetyrehmagnitnoy system type Perkin Elmer (410 LC) with UV detector with variable wavelength (LC - 95) and an integrator (LCI-100). Baseline separation was obtained in less than 12 min when analyzed aqueous solution containing ADP-ribose, AMP, NADP, US, OVER and dThd. General conditions of the analysis were as follows: flow rate = 1.0 ml/min; mobile phase - 1,4% methanol for 3.5 min and 4% for 10 min; the temperature 20-25oC; the second cycle between the shoulder-straps - 10 min; detection at 254 nm. A standard curve was obtained when using frozen samples of red blood cells, which were incubated for 1.5 h at 37oC before extraction PC then adding 0-40 µm ABOVE. Concentration OVER in the samples was determined as a function of the peak height ABOVE with respect to the peak height of internal standard (dThd).

Induced phytohemagglutinin (PHA) mitogenic response were analyzed using 2105CHOL, which were incubated in advance tablets for micro cultures containing 200 μl of RPMI medium 1640 with the addition of 10% autologous plasma, and 6 μl of PHA/ml (farmacii in the presence of [3H]-thymidine (final concentration 6 CI/mm, 1 µci/ml) cells were collected and analyzed for the content associated with3H] -thymidine radioactively labeled material present in 2105CHOL.

Below the present invention is illustrated in the examples.

Example 1

This example shows the processing efficiency of the volunteers carotenoids (100 mg in the form of careplex) + PP (100 mg) + zinc gluconate (10 mg) (KSC) during oral administration daily for 7 consecutive weeks and the results of further analysis of this intervention in relation to the protection of individuals against the harmful effects of oxidative damage (e.g., 100 μm H2ABOUT2DNA in mononuclear leukocytes of man (CHOL). These data show that all three volunteers subjected to the above processing, the detected significant (p<0.05) increase in the ability of CHOL to resist the induced H2ABOUT2DNA damage, while the volunteer acting as a control, which during the period of the interference of these substances are given only in the form of dietary supplements, there was no impact on the biological parameters of this reaction (Fig. 1).2ABOUT2). The results show that all tested volunteers with the passage of the process of DNA repair within 30 minutes and 60 minutes found significant (p<0.05) increase in DNA repair caused by damage (100 µm H2ABOUT2), while not subjected to the processing control of the individual during the period of intervention gave these substances only in doses corresponding to their levels in the diet, there was no significant change during the intervention period (Fig. 2-3).

Example 3

This example confirms the data already presented in examples 1-2, and it concerns the evaluation of (quantitative) enzyme involved in DNA repair, poly-ADPRT, before and after oral administration of volunteers in vivo carotenoids (100 mg in the form of careplex), nicotinamide (100 mg) and zinc gluconate (10 mg) (KSC) daily for 7 consecutive weeks. The data show that the activity of poly-ADPRT increased to a greater extent in the intervention compared with the control individuals who did not receive any supplements during the period of the intervention (Fig. 4).

Although these results were not statistically significant is the Finance of drugs has resulted in the reduction of oxidative damage to DNA cells and at the same time, cells could significantly better at restoring DNA after damage.

Example 4

This example shows the processing efficiency of the volunteers nicotinamid, when nicotinamide (100 mg) was administered orally daily for 7 consecutive weeks together with carotenoids (100 mg in the form of careplex) and zinc gluconate (10 mg) (KSC), which was assessed by the impact on energy pools ABOVE. The data presented in Fig. 5, clearly show that the addition of nicotinamide significantly increases concentration ABOVE the erythrocytes in the cell and, thus, the comparative analysis confirms the ability to reduce DNA damage and enhance DNA repair in CHOL, which was shown in examples 1-3 of the same study. It was also found that the red blood cells are a good General indicator of the status of the ABOVE in containing the nucleus of the cells (Jacobson, E. L., and others, in ADP-Ribosylation Reactions (Poirier G. G. and P. Moreau, editor), pages 153-162, Springer-Verlag, New York, 1992). This example also shows that the presence of the additive carotenoids and zinc are not blocked or does not inhibit the biological response increases pools ABOVE that, as is known from the literature, occurs when the introduction of one of nicotinamide.

Example 5

and on immune function before and after daily oral administration of carotenoids (100 mg in the form of careplex), nicotinamide (100 mg) and zinc gluconate (10 mg) (KSC) for 7 consecutive weeks. Data show that individuals subjected to medical intervention, found increased PHA induced mitogenic response, as seen by the increase in the inclusion of [3H]-thymidine in the CHOL compared with the reaction of one volunteer, who evaluated the immune function in the last 5 weeks of the intervention period, but which had not received any additive (control) (Fig. 6). Moreover, if these data are combined with data presented in examples 1-4, they suggest that when immune function is increased, this happens in parallel and mechanically connected with the improved ability of CHOL to resist oxidative DNA damage (examples 1, 4) and with increased reparative ability of DNA (examples 2, 3).

Example 6 this example shows the effect of in vivo to DNA damage in CHOL in oral introduction KSC (carotenoids + nicotinamide + zinc) in comparison with the impact of commercial product I (KPI containing carotenoids + nicotinamide + zinc + 17 other additives) in the study on the same subjects (Fig. 7). The presented data show that although additive KSC results in the second hydrogen peroxide solution for options B and C compared with control, additive KPI had no such effects (i.e., when comparing this parameter with those of the individuals designated by the control and option). This experiment shows that the presence in addition to other natural products in addition to carotenoids + nad + zinc limits biological effectiveness of these three compounds used in the form of an exclusive combination of sustainability, DNA cell damage.

Example 7

This example shows the effect of in vivo for DNA repair in CHEOL after oral administration KSC (carotenoids + nicotinamide + zinc) compared to the influence of KPI (carotenoids + nicotinamide + zinc + 17 other additives) in the study on the same subjects (Fig. 8). For option B it is established that while additive KSC was greatly enhanced DNA reparation, not identified appropriate action when the same individual was introduced additive with KPI or did not introduce additives (i.e., when comparing this parameter with those of the individuals designated by the control and option). This experiment shows that the presence in addition to other natural products in addition to carotenoids + nad + zinc ogranichyenii DNA repair.

Example 8

This example shows the effect in vivo on immune response CHOL in the processing of in vitro mitogen-phytohemagglutinin (PHA) after oral administration of KSC (carotenoids + nicotinamide + zinc) in comparison with the influence of KPI (carotenoids + nicotinamide + zinc + 17 other additives) in the study on the same subjects (Fig. 9). For option B is established that the addition Nicoplex greatly enhances the stimulation CHEOL-phytohemagglutinin. However, when the same individual was introduced additive with KPI or did not introduce additives (i.e., when comparing this parameter with those of the individuals designated by the control and option A), the appropriate action was not identified. This experiment shows that the presence in addition to other natural products in addition to carotenoids + nad + zinc limits biological effectiveness of these three compounds used in the form of exclusive combinations, in relation to immune responses of cells.

It should be noted that the present invention is not limited to the specific above characteristic features and examples of implementation and it can be implemented using other ways without deviating from its Sousa as active ingredients only carotenoid substance, nicotinamide substance and source of zinc in the form of their separate dosage forms for the selective introduction and additions by these same substances substances present in the normal diet of the subject and providing increased resistance to DNA damage, increased reparative ability of DNA and stimulation of immune function.

2. Combination in p. 1 made in the form of a formulation intended for oral administration.

3. Combination in p. 1 made in the form of a formulation intended for parenteral administration.

4. The combination of PP.1-3, in which the carotenoid substance chosen from the group comprising-carotene, -carotene, -carotene, lycopene, and mixtures thereof.

5. The combination of PP.1-4, in which nicotinamide substance selected from the group comprising nicotinamide, Niacin, tryptophan and mixtures thereof.

6. The combination of PP.1-5, in which zinc is one or more zinc salts.

7. The method of treatment of a human or animal, providing increased resistance to DNA damage, increased reparative ability of DNA and stimulation of immune function, which consists in the daily introduction of a daily dose combinations, oher is ivoti DNA damage, enhancing the reparative ability of DNA and stimulation of immune function, consisting in daily selective introduction of the dosage forms carotenoid substances nicotinamide substance and source of zinc in addition to the substances present in a normal diet.

9. The method according to p. 8, in which daily injected approximately 100 mg of carotenoid substances, 100 mg nicotinamide substances and 10 mg of the substance, which is the source of zinc.

 

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