Patent Application
20150110769
The invention relates to medicine and veterinary and can be used for increasing longevity and retarding unhealthy manifestations brought by ageing.
Various methods for retarding ageing are known, said methods are based on the application of antioxidants, immunomodulators, hormones, metabolic modulators (Ann. N.Y. Acad. Sci., 2002; vol. 959); however, the only method for correction (prolongation) of the life span with proven effectiveness is the non-medicamental method of restriction of calorie intake with food (CALORIE RESTRICTION, AGEING, AND CANCER PREVENTION: Mechanisms of Action and Applicability to Humans; Stephen D. et al., Annual Review of Medicine, February 2003, Vol. 54, pp. 131-152).
According to present day notions, the underlying cause of ageing is the loss of the ability to replicate by the somatic cells of the organism during the ageing (Hayflick and Moorhead, 1961, Exp. Cell Res. 25: 585-621; Hayflick, 1965, Exp. Cell Res. 37: 614-636; and Hayflick, 5 1970, Exp. Geront. 5: 291-303) and the accompanying alteration of the genes expression's profile in ageing cells (West, 1994, Arch. Derm. 130: 87-95), which leads to disturbances of their characteristic functions. Accumulation of such cells in the organs and tissues of the organism that comes with ageing (above all this applies to highly-specialized cells) lo forms a basis for manifestation of diseases and pathological states typical for an ageing organism.
The reasons behind the loss or perversion of replicative activity of cells brought by ageing are not completely clear yet. According to present day notions the underlying mechanisms may be either those determining the limit of replicative activity of cells, like the exhaustion of telomeres (Harley, 1991, Telomere loss: Mitotic clock or genetic time bomb? Mut. Res. 256:271-282) or the probabilistic mechanisms, for example, the accumulation of somatic mutations in the genome of somatic cells related to ageing (Woodruff R C, et. al., J Anti ageing Med, 2003 Spring 6:pp. 29-39).
In accordance with these notions, the modem medicamental methods for increasing longevity influence the intracellular genetic apparatus of cells. Such are the method of inhibiting the poly enzyme (ADP-ribose) of polymerase (U.S. Pat. No. 5,874,444) and the method of the telomerase enzyme activation (WO2000/31238).
The common disadvantage of methods which utilize active pharmacological intervention on genetic apparatus of ageing cell is the risk of unpredictable side effects (Cellular senescence, ageing and cancer. Campisi J, Scientific World Journal, 2001, Jan. 1 1:1 Suppl 3 65.).
Currently no method exists which allows efficiently retard unhealthy manifestations brought by ageing of human beings. In this connection, it is impossible to choose any known technical solution as a prototype of the present invention.
It is an object of the present invention to provide a solution for creating an effective method for retarding unhealthy manifestations brought by ageing of human beings (in particular, but not limited to the reduction of sexual activity and fertility, climax, changes in glucose tolerance, reduction of cognitive and mnestic functions, reduction of stress resistance, development of organ and tissue sclerosis) without directly affecting the genetic apparatus of ageing cells.
According to the invention this task is solved by administration into the blood circulation of the agent which inactivates extracellular blood plasma DNA; the extracellular blood DNA inactivating agent can be embodied in the form of an extracellular blood plasma DNA destroying agent; said extracellular blood plasma DNA destroying agent can be embodied in the form of an DNase enzyme; the extracellular blood plasma DNA inactivating agent can also be embodied in the form of an extracellular blood plasma DNA binding agent; the extracellular blood plasma DNA binding agent can be embodied in the form of anti-DNA antibodies; the extracellular blood plasma DNA inactivating agent can be administered in the form of an enzyme modifying the chemical composition of extracellular blood plasma DNA; the extracellular blood plasma DNA inactivating agent can be embodied in the form of an agent that stimulates synthesis or activity of endogenous deoxyribonuclease, or an agent that stimulates the synthesis of antibodies which capable to bind extracellular blood plasma DNA.
The beginning and development of unhealthy manifestations brought by ageing entails qualitative and quantitative alterations of extracellular blood plasma DNA, however, the applicant hasn't found any source of information containing data about genetic repertoire of extracellular blood plasma DNA of patients during ageing, the biological role of the extracellular blood plasma DNA during the ageing or about possible therapeutic effect that its destruction may have on the increase of longevity. In applicant's opinion, this enables to conclude that the invention conforms to the criterion “Novelty” (N).
The applicants found that the extracellular blood plasma DNA of patients during ageing contains a repertoire of genes and regulatory genetic elements that has unique qualitative and quantitative composition that differs dramatically from the repertory of DNA defined in the human genome. Unlike the intracellular DNA, the extracellular blood DNA during the ageing mostly contains unique human genes.
The applicants found that the extracellular blood DNA during the ageing exerts a negative influence on the viability of cells of the organism.
The applicants found that the inactivation of the extracellular blood DNA during the ageing slows down the ageing process.
The abovementioned novel features of the invention, which are based on the fundamentally new notions regarding the role of the extracellular blood DNA during the ageing, enable to conclude that the inventive method conforms to the criterion “Inventive Step” (IS).
This invention is further explained, by way of drawings.
The inventive method is realized in the following way:
Materials and Methods:
The following extracellular blood DNA destroying agents were used: the bovine pancreatic DNAse enzyme (manufactured by Sigma), the recombinant human DNAse I enzyme (manufactured by Genetech),
The anti-DNA antibodies extracted from the blood of patients ill with systemic lupus erythematosus according to the A. M. Shuster method (A. M. Shuster et. al., Science, v.256, 1992, pp. 665-667) were used as the extracellular blood DNA binding agent. Such anti-DNA antibodies are not only able to bind the DNA, but also to hydrolyze it.
The antiG-f actin antibodies (manufactured by Calbiochem) were used as the agent that stimulates the synthesis and/or activity of endogenous biopolymers that bind or destroy or alter the chemical composition and/or conformation and/or polymery of the extracellular blood DNA without destroying it. The G-Actin inhibits the activity of the endogenous DNAse I. The binding of actin by the antibodies increases the activity of the endogenous DNAse I.
The bacterial Sss I Methylase (CpG Methylase) (manufactured by NewEngland Biolabs) was used as the DNA-altering agent. During the experiments the Sss I Methylase was integrated with small unilamellar vesicles (SUV) in the proportion of 1u of the enzyme per 1 mkg of the lipids (enzymosomes).
The DNA of the blood plasma was extracted in the following way: fresh (no more than 3-4 hours after the sampling) blood plasma with added anticoagulant (sodium citrate) was centrifuged on Ficoll-PlaquePlus (manufactured by Amersham-Pharmacia) during 20 minutes at 1,500 g at room temperature. The plasma was carefully detached (½ of total amount), without affecting the rest of cells on the Ficoll pillow, and further centrifuged at 10,000 g during 30 minutes, in order to remove cell fragments and debris. The supernatant was detached without affecting the sediment, and was toped up to 1% of sarcosil, 50 mM tris-HCl, pH 7.6, 20 mM EDTA, 400 mM NaCl, and then mixed with equal volume of phenol-chloroform 1:1 mixture. The prepared emulsion was incubated during 2 hours at 65.degree. C., then phenol-chloroform mixture was separated by centrifuging at 5,000 g during 20 minutes at room temperature. The procedure of deproteinisation with phenol-chloroform mixture was repeated in the identical way three times, whereupon the aqueous phase was processed first with chloroform, then with diethyl ether. Separation from organic solvents was made by centrifugation at 5,000 g during 15 minutes. The resulting aqueous phase had an equal volume of izopropanol added therein and then the mixture was incubated overnight at the temperature of 0.degree. C. After sedimentation the nucleic acids were separated by centrifugation at 0.degree. C., 10,000 g during 30 minutes. The sediment of nucleic acids was dissolved in the buffer containing 10 mM of tris-HCl, pH 7.6, 5 mM of EDTA, and deposited on the pillow made of cesium chloride gradient (1 M, 2.5M, 5.7M) in a centrifuge tube for rotor SW60Ti. The volume of DNA solution was 2 ml, the volume of each step of CsCl was 1 ml. The ultracentrifugation was done in the L80-80 centrifuge (manufactured by Beckman) during 3 hours at 250,000 g. DNA was collected from the surface of the 5.7M step into fractions. These fractions were dialyzed during 12 hours at 4.degree. C. The presence of the DNA in the fractions was determined by means of agar electrophoresis, with visualization of the DNA by means of the bromide ethidium staining. The amount of the DNA was determined by spectrophotometer (Beckman DU70) in cuvet with volume of 100 mcl at wavelength of 200-320 nm.
The bovine pancreatic Dornase enzyme was used as the extracellular blood DNA inactivating agent. 10 clinically healthy men from 50 to 55 took part in the research. Before the beginning of the research the participants were randomly divided into the test group (5 men) and the reference group (5 men). The men of the test group received capsules with the bovine pancreatic Dornase, 900,000 Kuntz Units per day (5 capsules 100 mg each), every day during 6 months. The men of the reference group received 5 capsules with placebo every day during 6 months.
The DNA-hydrolytic activity in the daily urine and the content of the extracellular blood DNA were investigated. Before the beginning of the research and at its completion the biological age (BA) of the participants was determined according to the following method (see Gerontology, 1999, issue 2 “QUALITATIVE EVALUATION OF THE INDICES OF MORTALITY, AGEING, LONGEVITY AND BIOLOGICAL AGE” A. A. Podkolzin, V. N. Krutko, V. I. Dontsov: Institute of System Analysis of Russian Academy of Science, Moscow, National Center of Gerontology, Moscow, Moscow State Medico-Stomatological University of Ministry of Health of Russian federation):
BA=26.985+0.215*SBP-0.149*OC-0.151*BI+0.723*HI, where: SBP—systolic blood pressure;
OC—oxygen consumption (time interval between breaths, sec);
BI—time of static balancing, sec;
HI—index of subjective estimate of health (in the present research taken as 0.75).
The results of the research are shown in Table 1.
Thus, the application of the inventive method improves the parameters of biological age of human beings.
The stem cells of human umbilical blood (SCUB), transfected in vitro with the DNA of the gene coding for human deoxyribonuclease I, were used as an agent that stimulates synthesis of endogenous deoxyribonuclease.
The experiment was conducted on 40 white outbred rats. The rats in the test group 1 (10 animals), starting from the age of 24 months, were treated by intravenous administration of transfected SCUB, 50,000,000 cells per one animal per infusion, once a month, monthly, until the animal' death. The rats of the test group 2, starting from the age of 24 months, were treated by subcutaneous administration of the calf thymus DNA, once a month, 50 mg per one animal per injection, until the animal death. The rats in the reference group 1 (10 animals) were treated with not transfected SCUB. The rats in the reference group 2 (10 animals) were treated with sodium chloride. The longevity of rats from the reference group 1 averaged to 29 months. The longevity of rats from the reference group 2 (natural longevity) averaged to 27 months. The longevity of rats in the test group 1 averaged to 35 months, in the test group 2 it averaged to 33 months.
The bovine pancreatic DNAse enzyme (Sigma), conjugated with the polymer of sialic acid with molecular weight of 36 kDA, was used during the experiment. The experiment was conducted on 24-month old white randomly bred rats. The rats in the test group (15 animals), starting from the age of 24 months, were treated with above said preparation at 500 mg/kg per injection, intravenously, twice a week, during 2 months. The rats of the reference group (15 animals) were treated with phosphate buffer. The longevity of rats in the reference group to 27.8 months. The longevity of rats in the test group averaged to 30.1 months.
Therefore, the destruction of the extracellular blood DNA by the DNAse enzyme slows down the process of ageing according to the inventive method.
During the experiment a line of fibroblasts subcultivated from bioptic material of a healthy 73-year old man was used. The cells were cultivated without adding antibiotics in a minimal essential medium (MEM) with addition of 2 mM of glutamine and 10% of embryonic calves' serum. For the experiment the cells were plated in the amount of 10,000 of cells per cm.sup.2 of the surface in the Petri dish and grown in the atmosphere of 5% CO2 at 37.degree. C. The activation of [3H] thymidine was used for determining the intensity of the DNA synthesis during the initial stage of the growth. At the second day after the plating the cultures had [3H] thymidine added to them until reaching the final concentration of 0.1 mCi/ml and then the cells were incubated during 24 hours more, whereupon they were washed off and dissolved in formamide, and their radioactivity was measured.
In the first line (6 dishes) the cells were cultivated without adding the DNA.
In the second line (6 dishes) the extracellular DNA extracted from the blood of the donor was added to the incubating medium in the concentration of 0.005 mkg/ml.
In the third line (6 dishes) the extracellular DNA extracted from the blood of a young healthy volunteer 24 years old was added to the incubating medium in the concentration of 0.005 mkg/ml.
In the fourth line (6 dishes) the extracellular DNA extracted from the blood of the donor in the concentration of 0.005 mkg/ml and the recombinant Dornase-alpha (manufactured by Genetech) in the concentration of 0.5 mkg/ml were added to the incubating medium.
In the fifth line (6 dishes) the recombinant Dornase-alpha (manufactured by Genetech) in the concentration of 0.5 mkg/ml was added to the incubating medium.
The results of the experiment are shown in
The activation of the [3H] thymidine by the cells 48 hours after the passing.
Thus, the extracellular DNA of the blood plasma of an aged donor has a negative influence on the synthesis of the DNA in the cells. The destruction of the extracellular DNA of the patient's blood by means of the DNAse enzyme counteracts this influence according to the inventive method.
During the experiment a line of fibroblasts subcultivated from bioptic material of a healthy 73-year old man was used. The cells were cultivated without adding antibiotics in a minimal essential medium (MEM) with addition of 2 mM of glutamine and 10% of embryonic calves' serum. For the experiment the cells were plated in the amount of 10,000 cells per cm2 of the surface in the Petri dish and grown in the lo atmosphere of 5% CO2 at 37°. C. The activation of [3H] thymidine was used for determining the intensity of the DNA synthesis during the initial stage of the growth. At the second day after the plating the cultures had [3H] thymidine added to them until reaching the final concentration of 0.1 mCi/ml and then the cells were incubated during 24 hours more, whereupon they were washed and dissolved in formamide, and their radioactivity was measured.
In the first line (6 dishes) the cells were cultivated without adding the DNA.
In the second line (6 dishes) the extracellular DNA extracted from the blood of the donor was added to the incubating medium in the concentration of 0.005 mkg/ml.
In the third line (6 dishes) the extracellular DNA extracted from the blood of a young healthy volunteer 24 years old was added to the incubating medium in the concentration of 0.005 mkg/ml.
In the fourth line (6 dishes) the extracellular DNA extracted from the blood of the donor in the concentration of 0.005 mkg/ml and the anti-DNA antibodies in the concentration of 5 mkg/ml were added to the incubating medium.
In the fifth line (6 dishes) the extracellular DNA extracted from the blood of the donor in the concentration of 0.005 mkg/ml and the enzymosomos in the concentration of 20 mkg/ml was added to the incubating medium.
The results of the experiment are shown in
The activation of the [3H] thymidine by the cells 48 hours after the passaging.
Thus, the extracellular DNA of the blood plasma of an aged donor has a negative influence on the synthesis of the DNA in the cells. The binding and alteration of the extracellular DNA of the patient's blood counteracts this influence according to the inventive method.
The experiment was conducted on 20-month old males of mice of the C57B1 line. The males in the test group (9 mice), from the age of 18 months, had intravenous injections of the fraction of mouse antiG-f antibodies (manufactured by Calbiochem) in the amount of 200 mkg per mouse administered to them once a week, every week during 8 weeks. The 9 mice of the reference group had intravenous injections of phosphate buffer. At the age of 20 months the males of the test and the reference groups were put into cells, 3 males per one cell, whereupon 2-month old female mice of the C57B1 were introduced (3 females per one cell). After one week the females were transferred to separate cells and after 23 days more the amount of viable progeny was evaluated for the test and the reference groups. Additional reference was provided in the form of 10 5-month old males of the C57B1 line that mated with 2-month old females of the same line.
The results are shown in Table 2.
Therefore, the introduction of an agent that suppresses the endogenous inhibitor of its own DNAse I, the biopolymer that destroys the extracellular blood DNA, slows down the reduction of reproductive ability brought by ageing.
The experiment was conducted on 20-month old males of mice of the C57B1 line. The males in the test group (10 mice), from the age of 18 months, had intravenous injections of the fraction of human anti-DNA antibodies (manufactured by IgG) in the amount of 200 mkg per mouse administered to them once a week, every week during 8 weeks. The males of the reference group (10 mice) had intravenous injections of phosphate buffer. At the age of 20 months the males of the test and the reference groups each had 30 mkg of virus protein (influenza, strain PR8) introduced subcutaneously, whereupon the antibody titer was evaluated, 15 days after the immunization. Additional reference was provided in the form of 10 5-month old males of the C57B1 line.
The results are shown in Table 3.
Therefore, the introduction of an agent that binds the extracellular blood DNA slows down the weakening of the immunity brought by ageing.
The experiment was conducted on 20-month old males of mice of the C57B1 line. The males in the test group (10 mice), from the age of 19 months, had intramuscular injections of the liposomal DNAse I (200.mu.g of DNAse I in 100 mkg of the mixture of soya phosphatidylcholine and cholesterol (7:3)) administered to them daily. The males of the reference group (10 mice) had intramuscular injections of “empty” liposomes of the same composition. At the age of 20 months the mobility of the animals was measured, by means of one video-recorded observation during 30 minutes with subsequent computer processing. Additional reference was provided in the form of 10 5-month old males of the C57B1 line. The 30-minute activity of 5-month old mice was taken as 100%.
The results are shown in Table 4.
Therefore, the introduction of an agent that destroys the extracellular blood DNA slows down the reduction of locomotor activity brought by ageing.
The inventive method is realized by means of easy-to-obtain components and known equipment. In applicant's opinion, this enables to conclude that the invention conforms to the criterion “Industrial Applicability” (IA).
This application is a Divisional of U.S. application Ser. No. 11/919,141 filed on Oct. 23, 2007, which is a U.S. National Phase Application under 35 U.S.C. §371 of International Patent Application PCT/RU2005/000236 filed Apr. 25, 2005. The International Application was published on Dec. 7, 2006 as International Publication No. WO 2006/130034 under PCT Article 21(2). The entire contents of these applications are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11919141 | Oct 2007 | US |
| Child | 14579041 | US |
