RU2542445C2 - Medication for treating alzheimer's disease and method of treating alzheimer's disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: claimed group of inventions relates to medicine, namely to neurology, and deals with treatment of Alzheimer's disease. For this purpose pharmaceutical composition, which contains activated potentiated form of antibodies to brain-specific protein S-100 and activated potentiated form of antibodies to endothelial NO-synthase, is introduced.

EFFECT: introduction of claimed composition provides efficient treatment of Alzheimer's disease due to synergic neuroprotective, anti-ischemic and anxiolytic action of composition components.

9 cl, 5 tbl, 3 ex

Description

The invention relates to medicine and can be used to treat Alzheimer's disease.

A neurotropic drug based on antiserum to the brain-specific protein S-100 is known in the art (RU2156621 C1, A61K 39/395, 09/27/2000).

There is also a study of a drug based on ultra-low doses of antibodies to the brain-specific protein S-100 in case of cognitive impairment, emotional and neurological status in an experimental model of Alzheimer's disease, which showed that the drug has the ability to restore impaired cognitive function, improves emotional state, reduces anxiety and may be promising for the prevention and treatment of Alzheimer's disease (Voronina T.A., Belopolskaya M.V. et al. The effect of ultra-low doses of anti ate to S-100 in violation of cognitive functions, emotional and neurological status in an experimental model of Alzheimer's disease. Bulletin of experimental biology and medicine. 2009, No. 8, appendix, p.174-176).

The invention aims to create an effective tool for the prevention and treatment of Alzheimer's disease.

The solution to this problem is provided by the fact that the drug for the treatment of Alzheimer's disease, containing the activated potentiated form of antibodies to the brain-specific protein S-100, according to the invention, is made in the form of a pharmaceutical composition and contains an activated potentiated form of antibodies to endothelial NO- as an additional reinforcing component synthase.

In this case, the activated potentiated form of antibodies to the brain-specific protein S-100 and the activated potentiated form of antibodies to endothelial NO synthase are used in the form of an activated potentiated aqueous or hydroalcoholic solution, the activity of which is due to the process of multiple repeated dilutions in an aqueous or hydroalcoholic solvent in combination with external mechanical action - vertical shaking of each dilution.

The pharmaceutical composition can be in solid dosage form and contain an effective amount of particles of a neutral carrier saturated with a mixture of aqueous or aqueous-alcoholic solutions of an activated potentiated form of antibodies to the brain-specific protein S-100 and an activated potentiated form of antibodies to endothelial NO synthase, and pharmaceutically acceptable additives including lactose, microcrystalline cellulose and magnesium stearate.

Moreover, aqueous or aqueous-alcoholic solutions of activated potentiated forms of antibodies to the brain-specific protein S-100 and to endothelial NO synthase are obtained by multiple sequential dilutions in combination with an external action - vertical shaking of each dilution from matrix solutions of affinity-purified antibodies to the brain-specific protein S-100 and to endothelial NO synthase with a concentration of 0.5 ÷ 5.0 mg / ml.

Preferably, each of the components of the ultra-low doses of affinity-purified antibodies is used as a mixture of various, mainly hundred, dilutions prepared by homeopathic technology.

The solution to this problem is also ensured by the fact that in the method of treating Alzheimer's disease by introducing into the body an activated potentiated form of antibodies to the brain-specific protein S-100, according to the invention, an additional (simultaneously combined) additionally introduced activated potentiated form of ultra-low doses of affinity-purified antibodies to endothelial NO- synthase.

In this case, the activated potentiated form of antibodies to the brain-specific protein S-100 and the activated potentiated form of antibodies to endothelial NO synthase are used in the form of an activated potentiated aqueous or aqueous-alcoholic solution of each component, the activity of which is due to the process of multiple sequential dilutions in an aqueous or aqueous-alcoholic solvent in combination with external mechanical action - vertical shaking of each dilution.

A mixture of different dilutions of antibodies to the brain-specific protein S-100 in combination with a mixture of different dilutions of antibodies to endothelial NO synthase prepared using homeopathic technology is preferably used as a single drug.

In addition, the drug contains active ingredients in a volume ratio of 1: 1, with each component being used as a mixture of three appropriate matrix solutions, diluted, respectively, 100 ¹² , 100 ³⁰ , and 100 ²⁰⁰ times, which is equivalent to hundreds of dilutions of C12, C30, C200 prepared according to homeopathic technology.

The claimed pharmaceutical composition is recommended to be taken, preferably, 1-2 tablets 2-6 times a day.

In the treatment of Alzheimer's disease, separate, but combined and simultaneous use (introduction into the body) of the claimed pharmaceutical composition in the form of two separately prepared preparations, both in the form of solutions and in solid dosage forms (tablets), each of which contains an activated potentiated form of ultra-low doses affinity-purified antibodies to the brain-specific protein S-100 and, accordingly, the activated potentiated form of ultra-low doses of affinity-purified antibodies to endothelial NO synthase.

The proposed combination of activated potentiated forms of antibodies to the brain-specific protein S-100 and to endothelial NO synthase in a pharmaceutical composition (i.e., forms of antibodies to the brain-specific protein S-100 and to endothelial NO synthase prepared according to the homeopathic potentiation technique by repeated serial dilution and external effects - vertical shaking (see, for example, V. Schwabe "Homeopathic medicines", M., 1967, p.14-29), which have activity due to technology p validation, in pharmacological models and / or clinical methods of treatment of Alzheimer's disease) provides an unexpected synergistic therapeutic effect, confirmed by adequate (valid) experimental models and clinical studies, which consists in increasing the effectiveness of treatment of Alzheimer's disease. The specified technical result is due to an increase in the neuroprotective activity of antibodies to the S-100 protein, an increase in the vegetative stabilizing effect, normalization of the vegetative status, the synergistic effect of both components on neuronal plasticity, and, as a result, increased brain resistance to toxic effects, which improves integrative activity and restores interhemispheric connections the brain, helps to eliminate cognitive impairment, stimulates reparative processes and accelerates recovery These functions of the central nervous system increase mental performance, restore learning and memory processes, normalize somatovegetative manifestations, increase cerebral blood flow, and, accordingly, increase the effectiveness of treatment of Alzheimer's disease.

In this case, the claimed drug, as well as its constituent components, does not have a sedative and muscle relaxant effect, does not cause addiction and addiction.

In addition, the claimed drug expands the arsenal of drugs intended for the treatment and prevention of Alzheimer's disease.

The drug is prepared mainly as follows.

To prepare the activated potentiated form of the active components, monoclonal or, mainly, polyclonal antibodies are used, which can be obtained by known technologies - the techniques described, for example, in the book: Immunological methods, ed. G. Fremel, M., "Medicine", 1987, S. 9-33; or, for example, in an article by Laffly E., Sodoyer R. Hum. Antibodies. Monoclonal and recombinant antibodies, 30 years after. - 2005 - Vol.14. - N 1-2. P.33-55.

Monoclonal antibodies are obtained, for example, using hybridoma technology. Moreover, the initial stage of the process includes immunization, based on the principles already developed in the preparation of polyclonal antisera. Further stages of the work include obtaining hybrid cells producing clones of antibodies of the same specificity. Their isolation in an individual form is carried out by the same methods as in the case of polyclonal antisera.

Polyclonal antibodies can be obtained by active immunization of animals. For this purpose, according to a specially developed scheme, animals are given a series of injections with the antigens required by the invention: brain-specific protein S-100 and endothelial NO synthase. As a result of this procedure, monospecific antisera with a high antibody content are obtained, which are used to obtain activated potentiated forms of the components. If necessary, the antibodies present in the antiserum are purified, for example, by affinity chromatography, using fractionation by salt precipitation or ion exchange chromatography.

Preferred for the preparation of the claimed pharmaceutical composition is the use of polyclonal antibodies to the brain-specific protein S-100 and to endothelial NO synthase, which are used as a matrix (primary) solution with a concentration of 0.5 ÷ 5.0 mg / ml for the subsequent preparation of activated potentiated forms.

Preferred for the preparation of each component is the use of a mixture of three water-alcohol dilutions of the primary matrix solution of antibodies diluted, respectively, 100 ¹² , 100 ³⁰ and 100 ²⁰⁰ times, which corresponds to hundreds of dilutions C 12, C30 and C 200 prepared according to homeopathic technology. When performing the claimed drug in solid dosage form, a mixture of these components is applied to lactose.

Preferred for the preparation of the claimed drug is the use of polyclonal antibodies to the brain-specific protein S - 100 and endothelial NO synthase, which can be obtained by immunization of rabbits as follows.

For experimental studies, antibodies were used, prepared by order of a specialized biotechnological company.

Polyclonal antibodies to endothelial NO synthase are prepared using the adjuvant and the whole endothelial NO synthase molecule of the following sequence as immunogen (antigen) for rabbit immunization:

1 MGNLKSVGQE PGPPCGLGLG LGLGLCGKQG PASPAPEPSR ARARATRNAR DHSPAPNSPT

61 LTRPPEGPKF PRVKNWELGS ITYDTLCAQS QQDGPCTPRR CLGSLVLPRK LQTRPSPGPP

121 PAEQLLSQAR DFINQYYSSI KRSGSQAHEE RLQEVEAEVA STGTIHLRES ELVFGAKQAW

181 RNAPRCVGRI QWGKLQVFDA RDCSSAQEMF TYICNHIKYA TNRGNLRSAI TVFPQRAPGR

241 GDFRIWNSQL VRYAGYRQQD GSVRGDPANV EITELCIQHG WTPGNGRFDV LPLLLQAPDE

301 APELFVLPPE LVLEVPLGAP HTGWRGPGL RWYALPAVSN MLLEIGGLEF SAAPFSGWYM

361 STEIGTRNLC DPHRYNILED VAVCMDLDTR TTSSLWKDKA AVEINLAVLH SFQLAKVTIV

421 DHHAATVSFM KHLDNEQKAR GGCPADWAWI VPPIYGSLPP VFHQEMVNYI LSPAFRYQPD

481 PWKGSATKGA GITRKKTFKE VANAVKISAS LMGTLMAKRV KATILYASET GRAQSYAQQL

541 GRLFRKAFDP RVLCMDEYDV VSLEHEALVL VVTSTFGNGD PPENGESFAA ALMEMSGPYN

601 SSPRPEQHKS YKIRFNSVSC SDPLVSSWRRKRKESSNTDS AGALGTLRFC VFGLGSRAYP

661 HFCAFARAVD TRLEELGGER LLQLGQGDEL CGQEEAFRGW AKAAFQASCE TFCVGEEAKA

721 AAQDIFSPKR SWKRQRYRLS AQAEGLQLLP GLIHVHRRKM FQATVLSVEN LQSSKSTRAT

781 ILVRLDTAGQ EGLQYQPGDH IGISAPNRPG LVEALLSRVE DPPPPTESVA VEQLEKGSPG

841 GPPPSWVRDP RLPPCTVRQA LTFFLDITSP PSPRLLRLLS TLAEEPSEQQ ELETLSQDPR

901 RYEEWKLVRC PTLLEVLEQF PSVALPAPLL LTQLPLLQPR YYSVSSAPNA HPGEVHLTVA

961 VLAYRTQDGL GPLHYGVCST WLSQLKTGDP VPCFIRGAPS FRLPPDPYVP CILVGPGTGI

1021 APFRGFWQERLHDIESKGLQ PHPMTLVFGC RCSQLDHLYR DEVQDAQERG VFGRVLTAFS

1081 REPDSPKTYV QDILRTELAA EVHRVLCLER GHMFVCGDVT MATSVLQTVQ RILATEGDME

1141 LDEAGDVIGV LRDQQRYHED IFGLTLRTQE VTSRIRTQSF SLQERHLRGA VPWAFDPPGP

1201 DTPGP

It is possible to obtain polyclonal antibodies to endothelial NO synthase using the following sequence as an immunogen (antigen) of a whole molecule of endothelial NO synthase:

1 MGNLKSVAQE PGPPCGLGLG LGLGLCGKQG PATPAPEPSR APASLLPPAP EHSPPSSPLT

61 QPPEGPKFPR VKNWEVGSIT YDTLSAQAQQ DGPCTPRRCL GSLVFPRKLQ GRPSPGPPAP

121 EQLLSQARDF INQYYSSIKR SGSQAHEQRL QEVEAEVAAT GTYQLRESEL VFGAKQAWRN

181 APRCVGRIQW GKLQVFDARD CRSAQEMFTY ICNHIKYATN RGNLRSAITV FPQRCPGRGD

241 FRIWNSQLVRYAGYRQQDGS VRGDPANVEI TELCIQHGWT PGNGRFDVLP LLLQAPDDPP

301 ELFLLPPELV LEVPLEHPTL EWFAALGLRW YALPAVSNML LEIGGLEFPA APFSGWYMST

361 EIGTRNLCDP HRYNILEDVA VCMDLDTRTT SSLWKDKAAV EINVAVLHSY QLAKVTIVDH

421 HAATASFMKH LENEQKARGG CPADWAWIVP PISGSLTPVF HQEMVNYFLS PAFRYQPDPW

481 KGSAAKGTGI TRKKTFKEVA NAVKISASLM GTVMAKRVKA TILYGSETGR AQSYAQQLGR

541 LFRKAFDPRV LCMDEYDVVS LEHETLWLVV TSTFGNGDPP ENGESFAAAL MEMSGPYNSS

601 PRPEQHKSYK IRFNSISCSD PLVSSWRRKRKESSNTDSAG ALGTLRFCVF GLGSRAYPHF

661 CAFARAVDTRLEELGGERLL QLGQGDELCG QEEAFRGWAQ AAFQAACETF CVGEDAKAAA

721 RDIFSPKRSW KRQRYRLSAQ AEGLQLLPGL IHVHRRKMFQ ATIRSVENLQ SSKSTRATIL

781 VRLDTGGQEG LQYQPGDHIG VCPPNRPGLV EALLSRVEDP PAPTEPVAVE QLEKGSPGGP

841 PPGWVRDPRL PPCTLRQALT FFLDITSPPS PQLLRLLSTL AEEPREQQEL EALSQDPRRY

901 EEWKWFRCPT LLEVLEQFPS VALPAPLLLT QLPLLQPRYY SVSSAPSTHP GEIHLTVAVL

961 AYRTQDGLGP LHYGVCSTWL SQLKPGDPVP CFIRGAPSFR LPPDPSLPCI LVGPGTGIAP

1021 FRGFWQERLHDIESKGLQPTPMTLVFGCRC SQLDHLYRDE VQNAQQRGVF GRVLTAFSRE

1081 PDNPKTYVQD ILRTELAAEV HRVLCLERGH MFVCGDVTMA TNVLQTVQRI LATEGDMELD

1141 EAGDVIGVLRDQQRYHEDIF GLTLRTQEVT SRIRTQSFSL QERQLRGAVP WAFEPPGSDT

1201 NSP

It is possible to obtain polyclonal antibodies to endothelial NO synthase using, as an immunogen (antigen), a synthetic peptide of endothelial NO synthase, selected, for example, from the following amino acid sequences:

1192-1195

Pwaf

1189-1192:

Gavp

1185-1205:

RHLRGAVPWAF DPPGPDTPGP

1194-1205:

AF DPPGPDTPGP

1186-1196:

HLRGAVPWAF D

1186-1205:

HLRGAVPWAF DPPGPDTPGP

Before blood sampling for 1-3 days, 1-3 intravenous injections are performed to increase the level of antibodies. During immunization, small blood samples are taken from rabbits to evaluate the amount of antibody. The maximum level of the immune response to the administration of most soluble antigens is achieved 40-60 days after the first injection. After the end of the first cycle of immunization of rabbits for 30 days, they restore health and re-immunization, including 1-3 intravenous injections. To obtain antisera from immunized rabbits, blood is collected in a 50 ml centrifuge tube. Using a wooden spatula, the formed clots are removed from the walls of the tube and the wand is placed in the clot formed in the center of the tube. Blood is placed in a refrigerator (temperature 4 ° C) at night. The next day, the clot attached to the spatula is removed and the remaining liquid is centrifuged at 13000g for 10 minutes. The supernatant (supernatant) is an antiserum.

The resulting antiserum should be yellow. Add to the antiserum 20% (weight concentration) NaNs to a final concentration of 0.02% and store until use in a frozen state at a temperature of -20 ° C (or without adding NaNs at a temperature of -70 ° C). To isolate antibodies to endothelial NO synthase from antiserum, solid phase absorption is performed in the following sequence:

1. 10 ml of rabbit antiserum is diluted 2 times with 0.15 M Nad, 6.26 g of Na ₂ SO ₄ are added, mixed and incubated for 12-16 hours at 4 ° C;

2. The precipitate formed is removed by centrifugation, dissolved in 10 ml of phosphate buffer and then dialyzed against the same buffer overnight at room temperature;

3. After removing the precipitate by centrifugation, the solution is applied to a column with DEAE-cellulose, balanced with phosphate buffer;

4. The antibody fraction is determined by measuring the optical density of the eluate at 280 nm.

The antibodies are then purified by affinity chromatography by attaching the obtained antibodies to endothelial NO synthase, which is located on an insoluble matrix, followed by elution with concentrated salt solutions.

Thus obtained buffer solution of polyclonal rabbit antibodies to endothelial NO synthase purified on an antigen with a concentration of 0.5-5.0 mg / ml, preferably 2.0-3.0 mg / ml, is used as a matrix ( primary) solution for the subsequent preparation of the activated potentiated form.

To obtain polyclonal antibodies to the brain-specific protein S-100, the brain-specific protein S-100 is used, the physicochemical properties of which are described in the article by M.V. Starostin, S. M. Sviridov. Neurospecific protein S-100. Successes of modern biology, 1977, v.5, p.170-178; book of MB Stark, Brain-specific proteins / antigens / and neuron functions, M., "Medicine", 1985; p.12-14, isolated from tissue of the brain of a bull by the following method:

- fabric frozen in liquid nitrogen is ground into powder in a special mill;

- the extraction of proteins is carried out in a ratio of 1: 3 (weight / volume) in the extraction buffer during homogenization;

- the homogenate is heated for 10 min at 60 ° C and cooled to 4 ° C in an ice bath;

- thermolabile proteins are removed by centrifugation;

- carry out stepwise fractionation with ammonium sulfate, followed by removal of precipitated impurity proteins;

- the S-100 protein-containing fraction is precipitated with 100% saturated ammonium sulfate when the pH is reduced to 4.0 and collected by centrifugation;

- the precipitate is dissolved in a minimum volume of a buffer containing EDTA and mercaptoethanol, dialyzed against deionized water and freeze-dried;

- fractionation of acidic proteins is continued by chromatography on ion-exchange media — DEAE cellulose DE-52 and then DEAE-Sephadex A-50;

- collected and dialyzed fractions containing S-100 protein are separated by molecular weight by gel filtration on Sephadex G-100;

- purified S-100 protein is dialyzed and freeze-dried.

The molecular weight of the purified brain-specific protein S-100 is 21,000 d.

Due to the high content of aspartic and glutamic acids, the brain-specific protein S-100 is strongly acidic and occupies the extreme anode position during electrophoresis in a discontinuous buffer system in a polyacrylamide gel, which facilitates its identification.

To obtain a brain-specific antiserum, a purified S-100 protein (antigen) is prepared in combination with methylated bovine serum albumin as a carrier with Freund's complete adjuvant, which is administered subcutaneously to a laboratory rabbit animal in the amount of 1- to a selected brain-specific protein S-100. 2 ml On the 8th, 15th day, re-immunization is carried out. Blood is taken (for example, from an ear vein) on the 26th and 28th day.

The resulting antiserum has a titer of 1: 500-1: 1000, forms a single precipitation band with extract from nervous tissue, but does not react with extracts of heterologous organs, and forms a single precipitation peak with both pure S-100 protein and nervous tissue extract, which indicates the monospecificity of the obtained antiserum.

The activated potentiated form of each component is prepared by uniformly decreasing the concentration as a result of sequential dilution of 1 part of the above matrix solution in 9 parts (for decimal dilution D) or 99 parts (for hundredth dilution C) or 999 parts (for thousandth dilution M) of a neutral solvent in combination with multiple vertical (at least 10 times) shaking ("dynamization") of each dilution obtained and the use of separate containers for each subsequent dilution to p radiation required potency - the multiplicity of homeopathic dilution method (see, eg, W. Schwabe "Homeopathic medicines", M., 1967, s.14-29.).

External processing in the process of reducing the concentration can also be performed by ultrasound, electromagnetic or other physical effects.

For example, for the preparation of the 12th hundredth C12 dilution, one part of the above matrix solution of antibodies to the brain-specific protein S-100 (or to NO synthase) with a concentration of 2.5 mg / ml is diluted in 99 parts of a neutral aqueous or aqueous-alcoholic solvent ( mostly 15% ethyl alcohol) and repeatedly (10 or more times) vertically shake - potentiate the obtained 1st hundredth C1 dilution. From the 1st hundredth C1 dilution prepare the 2nd hundredth dilution C2. This operation is repeated 11 times, obtaining a 12th hundredth dilution of C 12. Thus, the 12th hundredth dilution of C12 is a solution obtained by diluting successively in different containers 12 times of the first part of the initial matrix solution of antibodies to the brain-specific protein S- 100 with a concentration of 2.5 mg / ml in 99 parts of a neutral solvent, i.e. a solution prepared by diluting the matrix solution in 100 ¹² times. Similar operations with the appropriate dilution ratio are carried out to obtain dilutions of C 30 and C 200.

When using as a biologically active liquid component a mixture of various, mainly hundred, dilutions of the active substance obtained by homeopathic technology, each component of the composition (for example, C12, C30, C0) is prepared separately according to the technology described above to their penultimate dilution (respectively, to obtain C11, C29, C199) and then, in accordance with the composition of the mixture, make one part of each component in one container and mix with the required amount of solvent (respectively, with 97 parts for Autun dilution). In this case, an activated potentiated form of antibodies to the brain-specific protein S-100 is obtained in an ultra-low dose obtained by super-dilution of the matrix solution, respectively, 100 ¹² , 100 ³⁰ and 100 ²⁰⁰ times, equivalent to a mixture of hundreds of homeopathic dilutions C12, C30 and C200.

It is possible to use each component in the form of a mixture of other various dilutions according to homeopathic technology, for example, decimal and or hundredths (C12, C30, C100; D20, C30, C100 or D20, C30, M100, etc.), the effectiveness of which is determined experimentally .

To obtain the claimed pharmaceutical composition, aqueous or aqueous-alcoholic solutions of the active ingredients are mixed, mainly in a volume ratio of 1: 1 and used in liquid dosage form.

The claimed pharmaceutical composition can also be used in solid dosage form, which contains an effective amount of particles of a neutral carrier - lactose, saturated by impregnation of an activated potentiated form of antibodies to the brain-specific protein S-100 and an activated potentiated form of antibodies to endothelial NO synthase, and pharmaceutically acceptable additives, including mainly lactose, microcrystalline cellulose and magnesium stearate.

To obtain a solid oral form of the claimed medicinal product, a fluidized bed is installed (for example, type “Hiittim Pilotlab” manufactured by Huttlin GmbH) irrigation until saturation of particles of a neutral substance - lactose (milk sugar) with a particle size of 150 ÷ 300 μm, a pre-prepared aqueous or aqueous-alcoholic solution of activated potentiated forms of antibodies to C brain-specific protein S-100 and to endothelial synthase of nitric oxide (NO synthase), mainly in the ratio and 1 kg of antibody solution to 5 or 10 kg of lactose (1: 5-1: 10) with simultaneous drying in a heated air stream supplied under the grate at a temperature of not higher than 40 ° C. The estimated amount of 0.17 ÷ 0.34 by weight of the solid oral form of the dried particles saturated with the activated potentiated form of antibodies is loaded into the mixer and mixed with microcrystalline cellulose introduced in an amount of 3-8 mass. parts of the total mass of the load - from the mass of solid oral form. Then 25 ÷ 45 masses are added to this mixture. parts of the total mass of the load of "unsaturated" pure lactose (to reduce the cost and some simplification and acceleration of the process without reducing the effectiveness of the therapeutic effect) and magnesium stearate in an amount of 0.1 ÷ 0.3 mass. parts of the total mass of the load. The resulting tablet mass is uniformly mixed and tabletted by direct dry pressing (for example, in a Korsch tablet press - XL 400) with the formation of round tablets weighing 150-500 mg. After tabletting, 300 mg tablets are obtained, impregnated with an aqueous-alcoholic solution of an activated potentiated form of antibodies to the brain-specific protein S-100 and NO synthase in an ultra-low dose, each component prepared from a matrix solution diluted, respectively, in 100 ¹² , in 100 ^{30 100,200} , which is equivalent to a mixture of hundred dilutions of C 12, C30 and C200 prepared according to homeopathic technology.

Preferably, the claimed pharmaceutical composition is recommended to take 1-2 tablets 2-6 times a day.

Example 1

Investigation of the effect of a complex preparation, which includes activated potentiated forms of polyclonal affinity purified rabbit antibodies to brain-specific protein S-100 (anti-S100) and to endothelial NO synthase (anti-eNOS) in ultra-low doses (SMD) obtained by super-dilution of the original matrix a solution (concentration of 2.5 mg / ml) in 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundred homeopathic dilutions C 12, C30, C200, in a 1: 1 ratio (SMD anti-S100 + anti-eNOS), and also the components included in its composition - activated potenti bath forms of polyclonal rabbit antibodies to brain-specific protein S-100, purified on antigen, in an ultra-low dose (SMD anti-S100) obtained by super-dilution of the initial matrix solution 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundred-percent homeopathic dilutions C 12, C30, C200 and the activated potentiated form of polyclonal rabbit antibodies to endothelial NO-synthase, purified on an antigen, in ultralow dose (SMD anti-eNOS), obtained sverhrazvedeniem initial matrix solution 100 ^12, 100 ^30, 100 ^{to 200} times the equivalent cell mixture GOVERNMENTAL homeopathic dilutions C12, C30, C200, performed in vitro on the binding of standard ligand [3H] pentazocine with recombinant human sigma 1 receptor was evaluated by radioligand.

Sigma-1 receptor is an intracellular receptor localized in the cells of the central nervous system, the cells of most peripheral tissues and immunocompetent cells. Receptors demonstrate a unique ability to translocate, which is caused by many psychotropic drugs. The dynamics of sigma-1 receptors is directly related to the various effects carried out by the drugs when they act on sigma-1 receptors. These effects include regulation of activity channels, ecocytosis, signaling, plasma membrane remodeling (raft formation), and lipid transport / metabolism. All this can contribute to the development of plasticity of neurons in the brain. There is evidence that sigma-1 receptors have a modulating effect on all major neurotransmitter systems: noradrenergic, serotonergic, dopaminergic, cholinergic systems and NMDA-regulated glutamate effects. Sigma-1 receptor plays an important role in the pathophysiology of neurodegenerative diseases, including Alzheimer's, and is involved in learning and memory. In this regard, the ability of drugs to influence the effectiveness of the interaction of ligands with the sigma-1 receptor indicates the presence of neuroprotective, anti-ischemic, anxiolytic, antidepressant, anti-asthenic components in the spectrum of their pharmacological activity, which allows us to consider these drugs as effective drugs, including including, for the treatment of cerebrovascular disease.

Potentiated distilled water (a mixture of homeopathic dilutions C12 + C30 + C200) was tested as a control of the tested drugs.

In the experiment (for measuring total binding), 20 μl of the complex preparation of SMD anti-S100 + anti-eNOS or 10 μl of SMD anti-S100 or 10 μl of SMD anti-eNOS was added to the incubation medium. Thus, the amount of SMD anti-S100 + anti-eNOS introduced into the experimental well when testing the complex preparation was identical to the number of SMD anti-S100 and SMD anti-eNOS tested as single drugs, which allows us to compare the effectiveness of the complex drug with its individual the components that make up its composition. Potentiated distilled water was introduced into the incubation medium in a volume of 20 μl and 10 μl.

Then 160 μl (~ 200 (-ig protein)) of the homogenate of the cell membranes of the Jurkat line (human leukemic T-lymphocyte line), and lastly 20 μl of the tritium-labeled [ ³ H] pentazocine radioligand (15 nM) were added.

To measure nonspecific binding, instead of preparations or potentized water, 20 μl of unlabeled ligand, haloperidol (10 μM), was added to the incubation medium.

Radioactivity was measured on a scintillation counter (Topcount, Packard) using a scintillation mixture (Microscint 0, Packard) after incubation for 120 minutes at 22 ° C in 50 mM Tris-HCl buffer (pH = 7.4) and filtration on glass fiber filters (GF / B, Packard). Specific binding (in experiment or control) was calculated as the difference between total (in experiment or control) and non-specific binding.

The results are presented as percent inhibition of specific binding in the control (distilled water was used as a control) (Table 1).

Table 1 The effect of drugs and potentiated water on the binding of a standard radioligand [ ³ H] pentazocine to a recombinant human sigma 1 receptor Experimental group The amount introduced into the experimental well % of specific radioligand binding in the control % inhibition of radioligand binding in the control 1st dimension 2nd dimension Average value SMD anti-S100 + anti-eNOS 20 μl 48,4 35.5 42.0 58.0 SMD anti-S100 10 μl 67.3 63.1 65,2 34.8 SMD anti-eNOS 10 μl 147, 5 161.1 154.3 -54.3 Potentized water 20 μl 98.1 75.8 86.9 13.1 Potentized water 10 μl 140, 1 106,2 123,2 -23.2 Note: % specific binding in the control = (specific binding in the experiment specific binding in the control) * 100%; % inhibition of specific binding in the control = 100% - specific (binding in the experiment specific binding in the control) * 100%).

Results reflecting inhibitions above 50% represent significant effects of the test compounds;

inhibition from 25% to 50%, indicate effects from mild to moderate; inhibitions of less than 25% are considered minor effects of the test compound and are within the background level.

Thus, in the conditions of this experimental model it was shown that: the complex preparation of SMD anti-5100 + anti-eNOS is more effective than its individual components (SMD anti-S100 and SMD anti-eNOS) inhibits the binding of the standard radioligand [ ³ H] pentazocine to recombinant human sigma 1 receptor; SMD anti-S100, introduced into the experimental well in a volume of 10 μl, inhibits the binding of the standard radioligand [ ³ H] pentazocine to the recombinant human sigma 1 receptor, but the severity of the effect is less than that of the complex drug SMD anti-S100 + anti-eNOS; SMD anti-eNOS introduced into the experimental well in a volume of 10 μl did not affect the binding of the standard radioligand [ ³ H] pentazocine to the recombinant human sigma 1 receptor; potentiated water introduced into the experimental well in a volume of 10 μl or 20 μl did not affect the binding of the standard radioligand [ ³ H] pentazocine to the recombinant human sigma 1 receptor.

Example 2

The study included patients with an established diagnosis of Alzheimer's disease.

To study the properties of the claimed drug in the treatment of Alzheimer's disease, 300 mg tablets were used, impregnated with a pharmaceutical composition containing water-alcohol solutions (6 mg / tablet) of activated potentiated forms of polyclonal affinity-purified rabbit antibodies to brain-specific protein S-100 (anti- S100) and to endothelial NO-synthase (anti-eNOS) in ultra low doses (RMA) obtained sverhrazvedeniem initial matrix solution (2.5 mg / ml) 100 ^12, 100 ^30, 100 ^{to 200} times equivalent CME and centesimal homeopathic dilutions C12, C30, C200 (SMD anti-S100 + anti-eNOS).

As a control group, we used a group of patients receiving a drug in the form of tablets weighing 300 mg, impregnated with an aqueous-alcoholic solution (3 mg / tablet) of an activated potentiated form of polyclonal rabbit antibodies to brain-specific protein S-100, purified on antigen, in an ultra-low dose (SMD anti-S100) obtained by super-dilution of the initial matrix solution (concentration of 2.5 mg / ml) 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundred-percent homeopathic dilutions C 12, C30, C200.

The study was an open, randomized, comparative clinical trial of the efficacy and safety of the study therapy (SMD anti-S100 and SMD anti-S100 + anti-eNOS) in two parallel groups in the treatment of patients with mild to moderate Alzheimer's disease.

The study included 6 patients with a diagnosis of Alzheimer's disease of mild to moderate severity, aged 55 to 64 years. The mean age of the patients was 59.0 ± 3.58 years.

The study examined patients' compliance with the following inclusion and exclusion criteria.

Inclusion Criteria:

1. Patients with mild to moderate Alzheimer's disease, confirmed by medical history, neurological examination and medical documentation.

2. Constancy of concomitant therapy for at least a month before Visit 1.

3. The lack of prerequisites for a change in neurological concomitant therapy throughout the observation period.

4. Lack of prerequisites for the appointment of immunomodulating agents over the next 6 months.

5. The level of education and the degree of understanding of the patient are sufficient to adequately communicate with the researcher and the coordinator of the study.

6. Patients are evaluated by the researcher as reliable, ready to perform all prescribed clinical visits, tests and procedures provided for by the protocol.

7. Patients have a valid home address.

Exclusion Criteria:

1. A history of surgical intervention on the brain.

2. A history of stroke.

3. A history of psychosis, bipolar disorder, or schizoaffective disorder.

4. Major depressive disorder according to the criteria for the depression module of the international neuropsychiatric mini-interview (MINI).

5. Factors / conditions of a medical or other nature that, in the opinion of the researcher, may affect the patient's test results in this study.

6. Answers “2A”, “2B”, “2B” or “3” in the heading “I” of the Beck Depression Questionnaire (active suicidal thinking with some intention to act, without a specific plan or active suicidal thinking with a specific plan and intent).

7. A history of autoimmune disease.

8. Acute liver damage or severe cirrhosis (Child-Pugh class C).

9. Unregulated thyroid dysfunction.

10. An uncompensated history of hypertension.

11. Serious or decompensated cardiovascular disease, liver, kidney disease, metabolic, respiratory or hematological disease, symptomatic peripheral vascular disease, or other medical or psychiatric condition that, in the opinion of the researcher, may affect the patient’s participation in the study or may lead to to extend the hospitalization or re-hospitalization of the patient during the study.

12. Diseases and conditions that, in the opinion of the researcher, may interfere with patient participation in the study.

13. Taking Impaz (SMD anti-eNOS) or Tenoten (SMD anti-S100) before taking the study.

14. Admission of antidepressants of any group, not excluding herbal and homeopathic medicines.

15. Reception of anxiolytics of any group, not excluding herbal and homeopathic preparations.

16. Reception of immunomodulators, not excluding herbal and homeopathic preparations.

17. Systemic steroid treatment for 1 month or less until

18. Visit 1.

19. Vaccinations, including against the flu.

20. Participation in the study of Impaz (SMD anti-eNOS) or Tenoten (SMD anti-S100), if patients have taken at least one dose of the drug.

21. Participation in other clinical trials for 1 month before inclusion in this study.

22. Pregnancy, breast-feeding, the inability to use adequate contraception during the study and within a month after the last administration of the study drug.

23. The presence of allergies / intolerances to any of the components of the drugs used in the treatment, including lactose intolerance.

24. The use of drugs, antipsychotics, alcoholism, mental illness of the patient.

25. Patients are staff of the center directly related to the study and / or are members of the family of the staff of the research center directly related to the study. The concept of "family members" includes spouse (s), parents, children, brothers (sisters) - related to both biological and legal kinship,

26. Participation in litigation, or alleged compensation or participation in litigation, according to the researcher.

After determining whether the patient met the criteria for inclusion and exclusion of the protocol, the patients were randomized into two study groups: the SMD anti-S100 group (3 people, women 100%, men 0%, average age 59 ± 3.6 years) and the SMD group anti-S100 + anti-eNOS (3 people, women 66.66%, men 33.33%, average age 59 ± 4.36 years).

During this study, 5 visits were made to the research center. The treatment phase lasted from Visit 1 to Visit 4 for 84 ± 5 days. Visit 4 (Day 84 ± 5) was the first endpoint of the study, after which the follow-up phase began. Follow-up phase continued until Visit 5 (Day 168 ± 5).

The safety analysis included data from all patients participating in the study (n = 6). During the entire period of observation of patients, good tolerability of the studied drugs was noted. Adverse events were absent. All patients of the study groups completed the treatment within the time period established by the study protocol; there were no prematurely retired patients.

When assessing the effect of the drug SMD anti-S100 + anti-eNOS on the main neuropsychiatric manifestations of Alzheimer's disease (NPI neuropsychic questionnaire, severity section), on the severity of concomitant distress of the caregiver of the patient (NPI neuropsychic questionnaire, distress section), the patient's daily life activity (questionnaire ADS-ADL), as well as the patient's cognitive functions (MMSE questionnaire), an improvement was found in the main neuropsychic manifestations of Alzheimer's disease, expressed in a statistically significant decrease in total ba la under "severity" neurodevelopmental questionnaire NPI (from 24.33 ± 4.73 to 12.0 ± 3.46, p <0.05) for the visit 4 (Table 2).

A tendency to a decrease in the distress of the caregiver, and an increase in the activity of the patient’s daily life, which, however, did not reach statistically significant values by the end of therapy, which was possibly associated with an insufficient number of patients, were also revealed.

Along with this, a tendency toward improvement in cognitive functions was revealed, which manifested itself in an increase in MMSE scores from 23.66 ± 3.21 points to 26.66 + 1.53 points, which, however, also did not reach statistically significant values by the end therapy, which may also be associated with an insufficient number of patients.

Similar indicators in the group of patients receiving anti-S100 SMD did not show improvement trends, with the exception of a statistically insignificant improvement in MMSE score from 22.66 ± 0.577 points to 23.33 ± 0.577 points.

Moreover, the difference between the groups according to the overall MMSE score by the end of therapy was significant with a probability of p <0.05.

Thus, a clinical study of the combined preparation of SMD anti-S100 + anti-eNOS showed a positive effect on the main neuropsychic manifestations of Alzheimer's disease and a possible effect on the cognitive functions of patients with Alzheimer's disease. In addition, high tolerance to the drug has been confirmed. Adverse events when taking the drug were absent.

Example 3

The efficacy of drugs for scopolamine amnesia in rats (model of Alzheimer's disease).

Alzheimer's disease (AD) is a neurodegenerative disease and is characterized by a decrease in cognitive functions, memory impairment, confusion, and changes in the emotional background. Although the main reason for the development of this pathology is currently considered to be the accumulation of amyloid beta, leading to the formation of amyloid beta plaques and neurofibrillary tangles in the brain tissue, AD is also accompanied by a deficiency of the cholinergic system. On this basis, one of the most common methods of modeling AD in animals using an antagonist of the cholinergic scopolamine system. Administration of scopolamine to experimental animals (usually rats or mice) impairs learning ability and leads to memory impairment.

Various methods are used to evaluate the cognitive functions of rats and mice, in particular the Morris water maze. The essence of this test is that in a vessel with opaque water, animals released into the water from different points are forced to look for a hidden fixed platform. The advantage of this method is that it allows you to observe the learning process of the animal (forming an idea of the spatial location of the platform, regardless of where it was lowered into the water), and to evaluate the strength of memorization (for this a test test is carried out, when the platform is taken out).

Example 3 below examined the efficacy in rat scopolamine amnesia of the claimed drug in the form of a composition containing activated potentiated forms of polyclonal affinity-purified rabbit antibodies to brain-specific protein S-100 (anti-S100) and to endothelial NO synthase (anti-eNOS ) in ultra-low doses (SMD) obtained by super-dilution of the initial matrix solution (with a concentration of 2.5 mg / ml) 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times equivalent to a mixture of hundred-percent homeopathic dilutions C 12, C30, C200 (SMD anti-S100 + anti-eNOS).

In a study of the effectiveness of the drug SMD anti-S100 + anti-eNOS in scopolamine amnesia in rats (model of Alzheimer's disease), 48 male rats of the Rj: Wistar strain (Nap) (weight 180-280 g) were used. For 4 days, rats were injected subcutaneously with saline (n = 12, intact) or scopolamine at a dose of 0.5 mg / kg (n = 36) (scopolamine-induced amnesia). Rats with scopolamine-induced amnesia were divided into 3 groups and, respectively, distilled water (7.5 ml / kg, n = 12, control, group No. 1), SMD anti-S100 (7.5 ml / kg, n = 12, group No. 2) and SMD anti-S100 + anti-eNOS (7.5 ml / kg, n = 12, group No. 3) intragastrically for 9 days (4 days before the start of scopolamine injection, 4 days against the background of administration scopolamine and 1 day after the end of scopolamine administration).

Within 4 days of scopolamine administration, 60 minutes after administration of the test drugs and 30 minutes after administration of scopolamine, a training session was conducted in the Morris maze (4 consecutive tests with an interval of 60 s). The Morris Labyrinth is a round vessel (150 cm in diameter, 45 cm high), 30 cm filled with water (26-28 ° C). 18 cm from the edge of the vessel is a hidden platform (15 cm in diameter), recessed 1.5 cm below the water level. The water is made turbid by adding a non-toxic dye (for example, milk powder), which makes the platform invisible. For each test, the animal was placed in a maze at one of the starting points located at the same distance from the hidden platform, and allowed them to find it. If the animal could not find the platform in 120 seconds, it was placed on the platform for 60 seconds and then a new test was started. During 4 trials in random order, the animals began passing through the maze twice from each starting point. The tests were recorded on videotape, and then the distance traveled in search of the platform in each test and the latent period of the search for the platform were analyzed.

On day 5, a test was performed: the platform was removed from the maze, and the rat was allowed to swim freely for 60 seconds. The time spent in the place where the platform used to be was recorded.

The introduction of scopolamine significantly impaired the learning ability of animals: in the control group No. 1, the time spent by the animals on the search for the platform and the distance that the animals sailed in search of the platform increased significantly (Tables 3, 4). The test showed that the memory of the animals of the control group No. 1 deteriorated significantly: in the place where the platform used to be, they spent less time than intact rats (Table 5). The introduction of SMD anti-S100 in group No. 2 did not lead to an improvement in the studied parameters (Tables 3, 4, 5). The introduction of SMD aHTH-S100 + anti-eNOS in group No. 3 led to some improvement in training, which was expressed in shortening the latent search time of the platform (Table 3) and the distance covered (Table 4) during 4 days of training, and memory improvement, which was expressed in increasing the time spent in the place where the platform was (Table 5).

Thus, in the used model of Alzheimer's disease, the use of the anti-S100 + anti-eKHOZ SMD complex was more effective than the isolated administration of anti-S100 SMD.

Claims (9)

1. A pharmaceutical composition for the treatment of Alzheimer's disease, comprising an activated potentiated form of antibodies to the brain-specific protein S-100 and an activated potentiated form of antibodies to endothelial NO synthase. 2. The pharmaceutical composition according to claim 1, characterized in that the activated potentiated form of antibodies to the brain-specific protein S-100 and the activated potentiated form of antibodies to endothelial NO synthase are used in the form of an activated potentiated aqueous or aqueous-alcoholic solution, the activity of which is due to the process of sequential multiple dilutions in an aqueous or hydroalcoholic solvent in combination with an external mechanical action - shaking of each dilution. 3. The pharmaceutical composition according to claim 1 or 2, characterized in that it is made in solid dosage form and contains an effective amount of granules of a neutral carrier saturated with a mixture of aqueous or aqueous-alcoholic solutions of the activated potentiated form of antibodies to the brain-specific protein S-100 and the activated potentiated form antibodies to endothelial NO synthase; and pharmaceutically acceptable additives. 4. The pharmaceutical composition according to claim 1 or 2, characterized in that the aqueous or aqueous-alcoholic solutions of the activated potentiated forms of antibodies to the brain-specific protein S-100 and to endothelial NO synthase are obtained by repeated serial dilution in combination with external exposure - shaking each dilutions from matrix solutions of affinity-purified antibodies to the brain-specific protein S-100 and to endothelial NO synthase with a concentration of 0.5 ÷ 5.0 mg / ml. 5. The pharmaceutical composition according to claim 1 or 2, characterized in that each of the components based on ultra-low doses of affinity purified antibodies is used in the form of a mixture of various, mainly hundred, dilutions prepared by homeopathic technology. 6. The pharmaceutical composition according to claim 3, characterized in that the pharmaceutically acceptable additives include lactose, microcrystalline cellulose and magnesium stearate. 7. A method of treating Alzheimer's disease by introducing into the body an activated potentiated form of antibodies to the brain-specific protein S-100, characterized in that the activated potentiated form of ultra-low doses of affinity-purified antibodies to endothelial NO synthase is additionally simultaneously combined. 8. The method according to claim 7, characterized in that the activated potentiated form of antibodies to the brain-specific protein S-100 and the activated potentiated form of antibodies to endothelial NO synthase are used in the form of an activated potentiated aqueous or aqueous-alcoholic solution of each component, the activity of which is determined by the process multiple dilutions in an aqueous or hydroalcoholic solvent in combination with an external mechanical action - shaking of each dilution. 9. The treatment method according to claim 7, characterized in that a mixture of different dilutions of antibodies to the brain-specific protein S-100 in combination with a mixture of different dilutions of antibodies to endothelial NO synthase prepared according to homeopathic prepared in the form of a single drug is used technologies.