Instant diagnostic test for alzheimer's disease

FIELD: medicine.

SUBSTANCE: CD4+ lymphocyte or CD8+ lymphocyte expressing superficial marker CD69 are counted prior to and after mitogenetic stimulation. The related value reaching a 10-fold or maximum 30-fold limit is a sign of Alzheimer's disease (AD). A kit containing necessary components for diagnosing Alzheimer's disease (AD) is presented.

EFFECT: invention allows presenting the instant diagnostic test for Alzheimer's disease.

2 cl, 2 tbl, 1 ex

 

The present invention relates to a method of diagnosing Alzheimer's disease (AD), or its early stage, or predisposition to this disease, characterized in that the specified method is based on determining the number metagenetics expressed markers cell surface, preferably CD69, perifericheskie accessible cells, such as skin cells or lymphocytes, (a) before and (b) after mitogeneticheskoe stimulation, and the stimulation coefficient a:b, is a sign of Alzheimer's, or early, or predisposition to this disease. Also the present invention relates to a kit suitable for the implementation of the diagnostic method according to the present invention.

Through clinical methods, and available laboratory diagnostic methods, and methods based on the equipment and technology as such, Alzheimer's disease cannot be reliably diagnosed. Always need to control opening. When the diagnosis is often difficult to distinguish BA from other causes of dementia, particularly in the early stages of the disease. However, at these very early stages of the disease is accurately diagnosed is important for two reasons. On the one hand, this allows diagnostically to distinguish potentially treatable form of dementia, which thus is can be effectively treated, and, on the other hand, it is a precondition for any form of therapeutic intervention in neurodegenerative process of Alzheimer's disease, which can only be successful in the early stages. Such diagnostic certainty can only be achieved on the basis of biological markers (biomarkers) of Alzheimer's disease, that is easily identifiable biological changes with relevant this disease sensitivity and specificity.

Biomarkers of Alzheimer's disease are, therefore, the diagnostic value and, in particular, will help to safely identify risk groups and patients in preclinical stages and early clinical stages of ad. Biomarkers can also be used to follow up patients and, thus, to contribute to the prediction and control of responsiveness to therapeutic intervention. An ideal biomarker should meet certain theoretical and practical requirements. In particular, these requirements include high specificity and sensitivity, the ability to determine the preclinical stage and the reliability of the positive and negative result. If possible biomarkers should identify non-invasive way, not to burden and not to frighten the patient. The tests should be inexpensive and easy to implement, if possible, in the practice of the family doctor. Unfortunately, none of the known biomarkers of Alzheimer's disease does not meet the above requirements. In particular, due to the small sensitivity and specificity of known biomarkers, they do not meet the diagnostic needs. Other diagnostic studies, with considerable sensitivity and specificity, require complex additional technical conditions and, thus, are not suitable for local use significant groups of patients.

Thus, the invention is mainly based on the technical problem of creating a simple method for diagnosing Alzheimer's disease, which will help to establish the diagnosis of Alzheimer's disease, to determine the preclinical stage of the disease and to establish diagnostic differences between Alzheimer's disease and other types of dementia with sufficient sensitivity and specificity.

This technical issue has been resolved in the methods of the invention described in the claims.

It has become possible to develop a diagnostic method based on the determination mutageneticheskogo factor (factor activation), when using perifericheskie available cells of the patient, such as to EDI skin or blood lymphocytes, with and without mitogeneticheskoe stimulation, for example, after cell isolation. Activation of these cells is accompanied by surface representation of the activation markers that can be quantified, preferably through interactions "antigen-antibody"using magnetic particles, preferably coated with an antibody that allows the magnetic sorting of cells and then quantify the number of cells bearing this surface marker before and after mitogeneticheskoe stimulation. This characteristic shows the characteristic of the disease deviations from standard values. Thus, the method according to the present invention allows to diagnose Alzheimer's disease, to determine the preclinical stage of the disease and to establish diagnostic differences between Alzheimer's disease and other forms of dementia.

Thus, the present invention relates to a method of diagnosing Alzheimer's disease or early stage, or predisposition to the disease based on the sample from the patient, this method involves the following stages:

(a) mitogenic stimulation perifericheskie available cells in the sample;

(b) determining the number metagenetics stimulated cells in the cell population before and after stages (a) on the basis of one or more surface the local markers, expressionand after mitogeneticheskoe stimulation, the cells bearing surface markers, separated from cells not bearing surface markers by antibodies directed against the indicated surface markers, and

(C) determination of coefficient of stimulation as the ratio of the number of cells bearing surface marker or markers before and after stages (a),

thus the factor stimulation, reaching values at least 10 times as a maximum of 100 times, compared to estimulando control sample, is indicative of Alzheimer's disease, or early, or predisposition to the disease.

A qualified specialist in this field known appropriate methods suitable for obtaining applicable in the method according to the present invention samples from the patient, which contain enough metagenetics stimulated cells. For example, suitable samples are samples of skin cells, blood samples, preferably of venous blood, cells from the cerebrospinal fluid and cells from the urine.

In a preferred method of implementation of the diagnostic method according to the present invention, for example, using a blood sample before the other steps of the method add an anticoagulant, such as citrate or heparin sodium, with the purpose of stabilization of the promotion.

Used herein, the term "diagnosis of Alzheimer's disease also involves the monitoring of patients after treatment and, thus, prognosis, monitoring the effectiveness of therapeutic intervention and the establishment of diagnostic differences between this disease and other forms of dementia.

Used herein, the term "perifericheskie available cells" refers to cells that can be removed without surgery or (minimally) invasive form of the human body, and they include, for example, skin cells and peripheral blood lymphocytes, the latter is preferable for the method according to the present invention.

Mitogenic stimulation to obtain the expression of surface markers can be carried out in a known stimulants, such as phytohemagglutinin (RNA), protein A, PWM (mitogen, Phytolacca) or other compounds with trophic or metagenetics action. Stimulation can be done by adding individual compounds or combination of compounds.

A qualified specialist in this field is known the appropriate experimental conditions for such stimulation, for example, in relation to the concentration used mitogens, duration of stimulation, and other incubation conditions. The stimulation should be coming in their vessels, allow the appropriate gas exchange. The concentration of specific agents for the stimulation must be in the physiological limit of 1 µg/ml to 20 μg/ml for RNA, 1 μg/ml to 50 μg/ml for PWM, 10 μg/ml to 200 μg/ml for protein A. the duration of the stimulation depends on the rate of expression of the studied molecules. However, for accurate research may be necessary the time of stimulation from 2 to 24 hours. In the case of CD69 the optimal duration of stimulation is 4 hours. The stimulation should be carried out under physiological conditions, and it can be done, for example, in the gas incubator at 37°C and 5% CO2.

A qualified specialist in this field is also known suitable surface markers, which are the manifestation of mitogeneticheskoe stimulation, such as CD69, CD25, CD45RO, CD63 and HLA-Dr, the preferred surface marker CD69. For the purposes of this invention it is also possible to carry out determination of the combinations of surface markers or additional characterization of cells allocated on the basis of specific surface markers, such as CD69, for further separation into subpopulations, such as CD4+and/or CD8+and/or CD19+and/or CD56+subpopulations.

The coefficient of stimulation (factor activation) based on the ratio of the number of cells, not the current surface marker or markers before and after stimulation. The coefficient of stimulation that reaches at least 10 times as high as 100 times relative to estimulando control sample, is a sign of Alzheimer's, or early, or predisposition to a given disease. The coefficient of stimulation, reaching less than 10 times estimulando control sample, is not a sign of Alzheimer's disease, or early, or predisposition to this disease. Cells bearing surface markers can be determined by conventional methods such as Western blot, ELISA (method enzyme immunoassay), RIA (radioimmunoassay method of analysis), FACS (flow cytofluorimetry), LSC (liquid scintillation account), etc.

In order to identify cells carrying surface markers, their characteristic cellular features are preferably separated from cells not bearing surface marker or other bearing surface markers.

In the diagnostic method according to the present invention cells bearing surface markers, separated from the non-bearing surface markers of cells using antibodies directed against the desired surface marker(s). Suitable for this purpose, the antibodies may be monoclonal, polyclonal, synthetically and antibodies or their fragments. In this regard, the term "fragment" means all parts of monoclonal antibodies (e.g., Fab, Fv or single-chain Fv fragments), which are specific for the same epitope as the whole antibody. A qualified specialist in this field known to the receipt of such fragments, many antibodies directed against surface markers that are commercially available.

In the most preferred implementation of the diagnostic method according to the present invention specific for surface markers antibody or antibodies bound to the magnetic particles, for example, paramagnetic beads (e.g., available from DYNAL A.S., P.O.Box 158 Skoyen N-0212 Oslo, Norway), which allows you to highlight cells with appropriate surface markers by immunomagnetic allocation according to known methods.

Then you can set the coefficient of stimulation, determining the number of cells allocated on the basis of the desired surface marker, content-based coding of its nucleic acid and/or protein content, using known methods, for example by spectrophotometric method for determination of content of nucleic acid or protein after lysis of the cells or after detection of nucleic acids using specific dyes, such as ethidium bromide, propecia iodide, South Africa is in orange, DAPI, etc. using photometric quantification. The number of cells can be calculated based on the content of the sample protein and/or nucleic acids, using calibration curves.

Also the present invention relates to a kit which is suitable for the implementation of the diagnostic method according to the present invention and contains at least the following components:

(a) connection for mitogeneticheskoe stimulation;

(b) at least one antibody directed against a surface marker expressed after mitogeneticheskoe stimulation, preferably an antibody associated with a magnetic particle.

Also set according to the present invention preferably contains:

(a) at least one reaction vessel;

(b) an anticoagulant and/or buffer for cell lysis;

(C) a buffer for fixation of cells;

(d) substances that are required to quantify the concentration of DNA and/or protein, and complete solutions for obtaining calibration curves;

(d) a magnet for separation of cells associated with magnetic particles included in the composition, if used antibody associated with magnetic particle)and

(e) a reagent for removal of the associated magnetic particles included in the composition, if used antibody associated with magnetic particle).

In a preferred is the procedure of realization set in accordance with the present invention the antibody is an anti-CD69 antibody. In addition, the kit may optionally contain or may contain, instead of anti-CD69 antibodies anti-CD4 and/or anti-CD8 antibody.

In conclusion, the set according to the present invention may contain, where necessary, in combination with one or more applicable additional agents detection, such as fluorochrome-conjugated primary antibodies, secondary antibodies, agents for the detection of proteins and/or nucleic acids, such as intercalating dye, etc.

Example

Determination of the coefficient of mitogeneticheskoe stimulation on the basis of CD69 in patients suffering from Alzheimer's disease

The determination of characteristics known to date for Alzheimer's disease, which can be performed in living patients (biomarkers), only shows a lack of sensitivity and specificity or is not suitable for studies with a large number of patients because of cost or highly complex mechanism studies. With clinical tools diagnostic accuracy is only 80%, 90% and there are difficulties, particularly in the early stages of the disease, to establish diagnostic differences. Currently, the definition of preclinical stages of the disease is impossible due to the lack of appropriate biomarkers.

Neurodegenerative changes osnovyvayas is on narushenie intracellular processes mediating trophic and mitogenic signals in the case of Alzheimer's disease. These dysfunction of intracellular signal transduction is not limited to the nervous system. They can also be found in skin cells and peripheral blood lymphocytes of these patients. Due to the specificity of the disease, this change is of diagnostic value and ideal as a biomarker.

In the following example investigated whether there is a dysfunction typical of intracellular mediation of trophic and mitogenic signals in Alzheimer's disease, using immunomagnetic isolation of lymphocytes presenting CD69 before and after mitogeneticheskoe stimulation.

Blood was taken by injection into a vein, using a system of blood collection company SARSTEDT. In the process of taking blood stabilized using anticoagulants entered into the system for taking blood, such as sodium citrate or heparin sodium. In this form, the blood can be stored at room temperature for 24 to 48 hours. The experiments on the stimulation is carried out in reaction vessels that can be good to aerate, for example in a 24-hole tablet company Greiner bio-one for kultivirovanija cells in suspension. For this purpose, the mitogen - phytohemagglutinin (RNA), protein a and mitogen, Phytolacca (PWM) used separately or in different combinations for each 400 μl of stabilized blood. In this example, the final concentration of the respective mitogens nah who were in the physiological range of values and was 12 µg/ml for RNA, 50 mcg/ml protein a and 4 µg/ml for PWM. Stimulation was performed under physiological conditions at 37°C and 5% CO2in the gas incubator for 4 hours. Each 100 μl of stimulated whole blood incubated with magnetic particles coated with different antibodies. In this example, the used magnetic particles coated with anti-CD4 and anti-CD8 antibodies from DYNAL. The corresponding magnetic particles were added to a single sample in excess (10 µl of a suspension of magnetic particles) to provide a full selection of relevant subpopulations of lymphocytes. After a 30 minute incubation period at 4°C the corresponding subpopulation of lymphocytes magnetically isolated and after washing stages were transferred into 100 μl of a particular environment, in this example, RPMI 1640, mixed with 1% fetal calf serum (FCS). In this example, the bound magnetic particles were removed using a 10 ál DETACHaBEAD company DYNAL. After a 45 minute incubation period at room temperature remote magnetic particles were isolated and after several stages of washing the cell suspension was transferred in the described environment, in this example, RPMI 1640. By adding specific lytic buffer destroyed cells, DNA was labelled with dyes that are specific for DNA, such as ethidium bromide, propecia iodide, acridine orange or DAPI, and then count the quality was determined photometrically. The protein content in the samples was compared by the method of protein determination according to Bradford. The number of cells was calculated based on the content in the sample DNA and/or protein, using calibration curves. This methodology has allowed to draw a direct conclusion as to the number of cells. The ratio calculation based on the number of CD69-presenting cells before and after mitogeneticheskoe stimulation (factor stimulation), gave information about changes mitogeneticheskoe stimuliruemoe these cells.

The coefficient of stimulation that reaches at least 10 times as high as 100 times relative to estimulando control sample, is a sign of Alzheimer's, or early, or predisposition to this disease. The coefficient of stimulation, which is 10 times less than for restimulating control sample, is not a sign of Alzheimer's disease, or early, or predisposition to the disease.

In another experiment, the protein content was determined in the sample and determined the DNA content without adding detecting DNA compounds for the quantitative determination of CD69-presenting cells. In this case, the measured absorption of light having a specific wavelength (for example, 260 nm or 280 nm), DNA or protein.

Experimental data

Were investigated factors stimulation on the I 9 people with clinically diagnosed suspected Alzheimer's disease (table a) and for 9 people not suffering from dementia (table). Patients suffering from Alzheimer's disease and mentally healthy with age. The table also shows the corresponding values for a brief scale for assessing mental status (MMSE - mini-mental state examination). MMSE is usually applied in order to establish the degree of severity of the defeat of the consciousness of the individual. Typically, the reading on the scale above 27 is considered normal; 20-26 means a slight mental disorder; 10-19 - moderate mental disorder, and below 10 - very severe mental disorder.

Metagenetics stimulation was performed using mitogen of lacunosa (PWM).

Peripheral mononuclear blood cells (RVMS) was isolated from blood samples taken from individuals, and suspended in medium at a concentration of 2.5×106cells/ml of the Selected cells stimulated with 4 µg/ml mitogen of lacunosa. Samples kept at 37°C and 7% CO2for 4 hours, then stirred with FACS lyse solution. Antibodies contained in the samples were stained and the samples were analyzed using FACSCalibur flow cytometer and CellQuest software Pro. X.X. Number of CD69+/CD69 - cells CD19+ before and after stimulation are shown for each person in the tables a and b, respectively. The coefficients of stimulation was calculated according to the formula, listed under the tables.

With regard to patients suffering from Alzheimer's disease, in table 8 of the 9 samples showed the coefficient of stimulation above 10. Only the coefficient of stimulation to patient G slightly below 10. The table also shows that the coefficient of stimulation above 10 was obtained for patients with mild and moderate mental disorders (patients a, b, H, and I) as well as for patients with severe mental disorders (patients C, D, E and F).

On the contrary, for all test cases without mental disorders were obtained coefficients stimulation significantly below 10.

Thus, the presented data confirm that by using the inventive method it is possible to distinguish a person suffering from Alzheimer's disease, from a person with intact consciousness. Given that the results largely correspond to the clinical diagnosis of Alzheimer's disease, the claimed method is considered as the secondary way investigation, providing a simple preliminary diagnosis of Alzheimer's disease. Thus, the implementation of the claimed method is confirmed.

Table a
The coefficients of the incentive is acii for patients suffering from Alzheimer's disease
A patient suffering from Alzheimer's diseaseAndInDEFGNI
MMSE251811659231714
Age838369767378795990
The number of CD69+/CD69 - in CD19+ cells
estimulando [CD6+] 162293101020212
estimulando [CD69-]25733520897175151202119155
PWM-stimulated [CD69+]2362541279217418318235139
PWM-stimulated [CD69-]486037421232364724
The coefficient of stimulation14,1813,3 18,6722,8917,3113,709,2725,8211.87 per

Table
The coefficients of stimulation for patients not suffering from mental disorders
Control123456789
MMSE282928293028292729
Age7685848578859184 85
The number of CD69+/CD69 - in CD19+ cells
estimulando [CD69+]15156349199310255235
estimulando [CD69-]49103267147271187421035317
PWM-stimulated [CD69+]134168127461413211241357314
PWM - stimulated [CD9-] 754983213105442746147
The coefficient of stimulation0,852,205,35is 3.088,752,651,162,786,85

Calculation of coefficient of stimulation:

n[CD69+]PWM-stim.

(n[CD69+]PWM-stim. + n[CD69-]PWM-stim.)

n[CD69+]non-stim.

(n[CD69+]non-stim. + n[CD69-]non-stim.)

1. Method of diagnosing Alzheimer's disease on the basis of the blood sample from the patient, while this method involves the following stages:
(a) stabilization of blood in the sample one or more anticoagulant;
(b) the Department of CD4+or CD8+lymphocytes bearing CD69, from blood cells by immunomagnetic separation by antibodies associated with magnetic particles, such as anti - CD69 antibody and anti - CD4 antibody for the determination of CD4+lymphocyte or anti - CD69 antibody and anti - CD8 antibody to determine CD8+lymphocytes in the sample;
(C) determining the number of CD4+or CD8 +lymphocytes expressing markers CD69, by destroying cells lyse solution in the sample;
(g) metagenetics stimulation of lymphocytes in the experimental sample using from 1 to 50 μg/ml of the mitogen Phytolacca (PWM) for from 2 to 24 h under physiological conditions;
(d) the Department of CD4+or CD8+lymphocytes bearing CD69, from blood cells by immunomagnetic separation by antibodies associated with magnetic particles, such as anti - CD69 antibody and anti - CD4 antibody for the determination of CD4+lymphocyte or anti - CD69 antibody and anti - CD8 antibody to determine CD8+lymphocytes in the prototype;
(e) determining the number of CD4+or CD8+lymphocytes expressing markers CD69, by destroying cells lyse solution in the prototype;
(g) determining stimulation index as the ratio of CD4+or CD8+lymphocytes bearing CD69 markers after mitogenic stimulation in the experimental sample to the number of lymphocytes bearing markers CD69, not subjected to mitogenic stimulation in the control sample;
the rate of stimulation, reaching at least 10-fold, or up to 30 times compared to estimulando control sample, is indicative of Alzheimer's disease.

2. A kit for diagnosing a disease Alzheimer is a, containing the following components:
(a) PWM for mitogeneticheskoe stimulation;
(b) antibodies associated with magnetic particles, such as anti - CD69 antibody and anti - CD4 antibody or anti - CD69 antibody and anti - CD8 antibody;
(C) an anticoagulant; and/or
(d) a buffer for lysis of cells;
(d) a magnet for separation of cells associated with magnetic particle;
(e) a reagent for the removal of associated magnetic particle;
(W) of the substance to quantify the concentration of DNA and/or protein and complete solutions for obtaining calibration curves.



 

Same patents:

FIELD: medicine.

SUBSTANCE: peripheral blood thrombocytes of women suffering gestosis of various severity levels on their 32-38 weeks of pregnancy are analysed for the activity of glutathione reductase (GY), NADF-dependent glutamate dehydrogenase (NADFGDG) and NADF-dependent isocitrate dehydrogenase (NADFICDG). A nicotine amide adenine dinucleotide phosphate transfer coefficient (NTC) represented by the relation of the GY activity to a product of the NADFGDG and NADFICDG activities is calculated. At the NTC value is equal to 1.3 and lower, the newborn's Apgar score is predicted to be equal to 6 and less, and the NTC value exceeding 1.3 provides the Apgar score being 7-10 points.

EFFECT: more accurate prediction of the newborn's state.

2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: clinical investigation is supplemented with simultaneously testing monoamine oxidase (MAO) activity in thrombocytes in nmole of benzaldehyde per 1 mg of protein an hour and semicarbazide-dependent amine oxidase (CAO) activity in blood serum in nmole of benzaldehyde per one ml of blood serum an hour. The blood serum CAO to thrombocyte MAO activity relation is calculated. If the value exceeds 0.6, a positive result of atypical antipsychotic drug treatment in schizophrenia is predicted.

EFFECT: more precise and objective prediction, reduced length of treatment in schizophrenics.

2 ex

FIELD: medicine.

SUBSTANCE: inventions concern methods and analyses for studying expression of one or more biomarkers in a mammal's tissue or cell sample; study kits and products are presented also. Identifying expression of GalNac-T14 molecules predicts sensitivity or specifies that the tissue or cell sample is supposed to be sensitive to apoptosis inducing agents, such as Apo2L/TRAIL.

EFFECT: information obtained by the analysis aiming at identifying GalNac-T14 expression in the tissue or the cell sample in a mammal can provide an attending physician with the information which can be used for prescribing an optimum treatment regimen for the patients suffering such diseases as pancreatic cancer, lymphoma, non-small cell carcinoma of lung, colon cancer, rectal cancer, melanoma or chondrosarcoma.

22 cl, 50 dwg, 6 tbl

FIELD: medicine.

SUBSTANCE: five-percent placental tissue extract is analysed for the concentration of arginine and histidine to be related, and if the relation is lower than 0.86, development of cerebral affection in a newborn is predicted.

EFFECT: more precise and specific prediction of perinatal CNS affection in the newborn and enabled well-timed pathogenetic therapy.

3 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: patient's blood serum is treated with 7% solution of polyethylene glycol-6000, incubated and with dye Sudan B at 40°C for 1 hour, separated electrophoretically in agarose gel. After that, additionally, before treatment of blood serum with 7% PEG-6000 to 0.6 ml of sample 0.2 ml of 0.1% tritone X-100 solution is added, incubated for 15 minutes at 20°C, after which mixture is mixed by shaking 120 times per 1 minute. Application of method makes it possible to detect additional intensive minor fraction of modified LP(a).

EFFECT: increase of diagnostics accuracy.

3 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: method of determination of triterpene saponins in vegetable raw material and medications includes dissolution of saponin-containing fraction in mixture water-ammonium buffer, determination of its optic density and calculation of saponin content in terms of oleanolic acid, under specified conditions.

EFFECT: claimed method represents express method, facilitates analysis and increases degree of reliability of obtained results.

2 ex

FIELD: medicine.

SUBSTANCE: in order to estimate efficiency of treating ischemic nephropathy in newborn babies in early neonatal period activity of gamma-glutamyltransferase and cholinesterase in child's urine is determined in dynamics of treatment. If activity of said enzymes decreases with respect to initial level, treatment is estimated as efficient, if activity increases or does not change - as inefficient.

EFFECT: application of method makes it possible to increase accuracy of estimation of ischemic nephropathy treatment in newborns, carry out correction of therapeutic measures in due time and improve disease outcome.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: in a first trimester of pregnancy, the microalbuminuria level is determined. If the value is 45 mg\l and more, placental insufficiency is predicted.

EFFECT: method enables early prediction of placental insufficiency by a simple quantitative estimation and thereby ensures early adequate preventive treatment.

3 ex

FIELD: medicine.

SUBSTANCE: detecting expression of GalNac-T14 molecules enables to predict sensitivity or indicates that a tissue or cell sample is sensitive to apoptosis inducing agents, such as DR4 or DR5 agonist antibodies. The information obtained by the analysis aimed at detecting GalNac-T14 expression in the mammal's tissue or cell sample can provide a hospital doctor with data which can be used for prescribing an optimal treatment schedule for patients suffering such diseases as pancreas cancer, lymphoma, non-small cell carcinoma of lung, colon cancer, rectal cancer, melanoma or chondrosarcoma.

EFFECT: expanded scope of the compounds.

16 cl, 53 dwg, 14 tbl

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular toxicology and resuscitation science and can be used for early prognosis of course of acute poisoning with psychotropic drugs. At the time of patient's admission to hospital albumin fraction of blood serum is isolated. After that, general level of reduced thiols is determined, and if its value is lower than 220 mcmol/l development of negative disease dynamics is predicted.

EFFECT: method allows to increase efficiency of performed treatment in said category of patients.

1 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine and pharmacology and deals with pharmaceutical composition, possessing antihypoxic, neoroprotective and antiamnestic activity and ability to increase physical performance capacity, intended, in particular, for treatment of acute and chronic disorders of brain blood circulation, which contains as active componets semax and choline alfoscerate, taken in weight ratio from 1:50 to 1: 8000 respectively and pharmaceutically acceptable auxiliary substances.

EFFECT: invention ensures extension of arsenal of medications, possessing expressed neuroprotective activity in combination with antihypoxic and antiamnestic activity and ability to increase physical performance capacity.

7 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to aryl-isoxazole-4-yl-imidazole derivatives of formula I and to their pharmaceutically acceptable acid addition salts. The compounds of the present invention exhibit GABA A α5 receptor binding site activity and selectivity. In general formula I

each of R1-R3 independently represents hydrogen atom or halogen atom; R4 represents hydrogen atom, lower alkyl, C3-C7cycloalkyl, -(CH2)n-O-lower alkyl or hydroxy substituted lowest alkyl; R5 represents -(CH2)m-phenyl or -(CH2)m-(5-6-members heteroaryl with 1-2 heteroatoms independently seected from N, O) which optionally substituted by one or more substitutes selected from a group consisting of halogen atom, cyano, nitro, lower alkyl, lower alkoxy, lower alkylsulphanyl, lower alkyl substituted by halogen atom, -C(O)-lower alkyl, -C(O)-O-lower alkyl, -NH-C(O)-O-lower alkyl or -C(O)-NH-R' where R' represents the lower alkynyl or hydroxy substituted lower alkyl, or represents -(CH2)n-C3-C7-cycloalkyl, -(CH2)n-(6-members heterocyclyl with 1-2 heteroatoms selected from N, O), -(CH2)n-(5-6-members heteroaryl with 1-2 heteroatoms selected from N, O) or -(CH2)n-phenyl optionally substituted by halogen atom; R6 represents hydrogen atom, -C(O)H, -(CH2)n-O-lower alkyl, -C(O)O-lower alkyl, lower alkyl substituted by hydroxy or halogen atom, or represents C3-C7-cycloalkyl, phenyl, or represents -(CH2)n-O-CH2-phenyl optionally substituted by halogen atom or lower alkyl, or represents -(CH2)n-O-CH2-(6-members heteroaryl with 1 heteroatom selected from N) optionally substituted by lower alkyl or lower alkyl substituted by halogen atoms, or represents -(CH2)n-NH-(CH2)o-(6-members heterocyclyl with 2 heteroatoms selected from N; n means 0, 1, 2 or 3; m means 0 or 1; o means 1, 2 or 3.

EFFECT: presented preparation of a drug containing one or more compound of formula I and application of the compounds for preparing the drug.

31 cl, 168 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine and pharmacology and deals with application of derivatives of aminospirits of formula

or their phosphate derivatives for manufacturing medication for treatment of various autoimmune diseases, such as disseminated sclerosis, peripheral nephritis, retrobulbar neuritis, amyotrophic lateral sclerosis and uveitis.

EFFECT: invention ensures high treatment efficiency.

7 cl, 2 dwg, 1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of the formula I

, where: m equals 0, 1 or 2, where if m=0, disappears such that an open ring or single bond forms, n equals 0, 1 or 2, wherein when n=0, disappears such that an open ring or single bond forms; m' and n' are independently equal to 0, 1 or 2; X denotes a carbon atom; Y denotes a carbon or sulphur atom; provided that m and n are not equal to 0 at the same time; denotes a single or double bond, if needed; --- absence of a bond or a single bond, if needed; R1 is selected from a group comprising CN, Hal, OAIk, OH, NRCN, C(CN)=C(OH)(OAlk), SR, NRR', (Alk)p-C(O)NRR', piperidine, wherein Alk is optionally substituted with Hal or OAlk, where p=0 or 1; R3, R4, R5 and R6 are identical or different and are independently selected from a group comprising H, OAIk, Alk, Hal, OH; R2 is selected from a group comprising H and O, and p'=0 or 1; R7 is selected from a group comprising H, O, OH, N-OH, N-aryl, N-OAlk, N-O-aryl, N-O-Alk-aryl, N-NR-CONRR', N-O-CO-Alk, or 2 R7, bonded with the same Y, together form lioksalan; wherein said Alk is optionally substituted with OAlk, -CO-(NR-Alk-CO)p'-OAlk, and p'=0 or 1; R and R', which are identical or different, are independently selected from a group comprising H, and Alk; or pharmaceutically acceptable salt or optical isomer or diastereomer thereof, except those compounds for which: R3, R4, R5, R6=H, R1=CN, denotes a single bond, and denotes -C(=N-(2,4,6-trimethylphenyl))-, -C(=N-(2,6- dimethylphenyl))-, -C(=N-(2,6-diethylphenyl))-, -C(=N(2-methylphenyl))-, -C(=N(2-ethylphenyl))-, -C(=N-(2-trifluoromethylphenyl))-, -C(=N-(2-isopropylphenyl))-, -C(=N-phenyl)-, -C(=N-(naphthyl)- or -C(=O)-, -CH2-, or R3, R5, R6=H, R4=OMe, R1=CN, denotes a single bond, and denotes -C(=O)-, or R3, R4, R5, R6=H, R1=NH2, denotes a single bond, and denotes -CH2- or -CH2-CH2-; or R3, R4, R5, R6=H, R,=NH2, denotes -CH2- or -CH2-CH2-, and denotes a single bond. The invention also relates to a cysteine protease based pharmaceutical composition based on compounds of formula I, use of the compound of formula I to prepare a drug for inhibiting cysteine protease, for treating and preventing cancer, as well as inflammatory diseases and others.

EFFECT: novel compounds which can be used in medicine are obtained and described.

38 cl, 43 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention describes a method of determining whether a compound is a neurotrypsin inhibitor, as well as a method of measuring catalytic activity of neurotrypsin, where the tested compound is incubated together with human or mouse neurotrypsin or a fragment of human or mouse neurotrypsin which contains a neurotrypsin protease domain, and with a complete length or soluble agrin, or agrin-C45, or a fluorescent protein, or a peptide containing agrin, agrin-C45, in an aqueous buffer solution at pH 7.5 and the number of agrin cleavages is measured.

EFFECT: improved properties.

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry, and deals with method of obtaining highly dispersed powders of organic medicinal substances and can be used in manufacturing novel medications. Method of micronosation of N-carbamoyl-methyl-4-phenyl-2-pyrrolidone is realised by the following method. Evapouration of N-carbamoyl-methyl-4-phenyl-2-pyrrolidone is realised in neutral gaseous medium with vacuum 950-10 Pa, and precipitation of its vapours is carried out at rate 1015 - 1017 molec/sec·cm2 on precipitation surface, which has temperature 120 - 30°C, precipitation surface being inclined at angle 15-80° relative to direction of precipitation rate vector, rate of evapouration of N-carbamoyl-methyl-4-phenyl-2-pyrrolidone exceeds rate of their precipitation in 1.2-2.5 times. Also claimed is medication, containing water-dispersive organic medicinal substance in form of micronised powder of N-carbamoyl-methyl-4-phenyl-2-pyrrolidone.

EFFECT: elaboration of efficient method of obtaining highly dispersive powders of organic medicinal substances.

2 cl, 2 ex, 2 dwg

FIELD: food industry.

SUBSTANCE: invention relates to food industry. A water-dispersible granulate has average weighted weight diameter at least 100 mcm, preferably at least from 200 mcm and up to 2000 mcm. The said granulate contains: 40-90 wt % of carbohydrates; 2-30 wt % of lipids containing unsaturated fatty acids in an amount of at least 1%, preferably no less than 3% of the granulate weight and at least 30 mg/kg - 800 mg/kg of a metal enhancing oxidation and selected from the group consisting of ferrum, cuprum and their combinations wherein at least 50 wt %, preferably at least 70 wt % of lipids are present in the form of undispersed lipids.

EFFECT: invention allows to produce a granulate resistant to oxidation the production method whereof is implemented under mild conditions.

18 cl, 6 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are described new N-cyclic sulphonamido-compounds and their pharmaceutically acceptable salts of formula : of formula 1a of formula 1b of formula 1c where the ring A means phenyl, thienyl which can be substituted by halogen, or pyridinyl; a value of the ring B is presented in the patent claim, and also a pharmaceutical composition containing them, and a method of treating Alzheimer's disease.

EFFECT: compounds are gamma-secretase inhibitors and can be used for treating Alzheimer's disease.

16 cl, 98 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel substituted 3-arylsulfonyl-pyrazolo[1,5-a]pyrimidine-2,6-diamines of general formula 1, their pharmaceutically acceptable salts and/or hydrates which have serotonin 5-HT6 receptor antagonist properties and can be used in treating or preventing development of various central nervous system diseases, whose pathogenesis is associated with 5-HT6 receptors, particularly Alzheimer's disease, Parkinson's disease, Huntington disease, schizophrenia, other neurodegenerative diseases, cognitive and anxiety disorders and obesity. In general formula (I): R1, R2 and R4, which are optionally identical, independently denote C1-C3 alkyl; R3 and R4, which are optionally identical, denote: a hydrogen atom, optionally substituted C1-C3 alkyl or acetyl; R5 denotes a hydrogen atom or halogen atom.

EFFECT: high treatment efficiency.

13 cl, 12 dwg, 4 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula I, which are HSP90 (heat-shock proteins) inhibitors and can be used to prepare a medicinal agent for treating tumorous diseases affected by HSP90 inhibition. In formula I R1 denotes Hal, H, OA or A, R2, R3 each independently denotes -O-(X)s-Q, -NHCO-(X)s-Q, -CONH-(X)s-Q, -NH(CO)NH-(X)s-Q, -NH(CO)O-(X)s-Q, -NHSOr(X)s-Q, NHCOA, Hal, Het or H, where, if R2=H, then R3≠H, or if R3=H, then R2≠H, R4 denotes H, R5 denotes H, Hal, A, OA, (CH2)nCOOH, (CH2)nCOOA, O(CH2)oCONH2, NHCOOA, NHCO(CH2)nNH2, NHCONHA or O(CH2)oHet1, A denotes a straight or branched alkyl containing 1-10 carbon atoms, in which 1-5 hydrogen atoms may be substituted with F, Cl and/or Br, X denotes a straight or branched C1-C10 alkylene which is unsubstituted or substituted once, twice or thrice by A, O A, OH, Hal, CN, COOH, COOA, CONH2, NH2, NHCOA, NHCOOA, Q denotes H, Ar or Het, Ar denotes phenyl which is unsubstituted or substituted once, twice or thrice with A, OA, OH, NO2, Hal, CN, (CH2)nCOOH, (CH2)nCOOA and/or tetrazole, Het denotes a cyclic saturated or aromatic 5-6-member heterocycle containing 1-2 N and/or O atoms, optionally condensed with a benzene ring which may be substituted once, twice or thrice with A, OA, OH and/or =O (carbonyl oxygen), Het1 denotes a monocyclic saturated, unsaturated or aromatic heterocycle containing 1-2 N and/or O atoms, which may be mono- or disubstituted with A, OA, OH, Hal and/or =O (carbonyl oxygen), Hal denotes F, Cl, Br or I, n equals , 1, 2, 3 or 4, o equals 1, 2 or 3, s equals 0, 1 or 2.

EFFECT: high efficiency of using said derivatives.

4 cl, 4 dwg, 1 tbl, 29 ex

FIELD: medicine.

SUBSTANCE: versions of the CD38 specific antibodies and their functional fragments are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a pharmaceutical and diagnostic compositions, a method of treating, a method of detecting CD38 in erythrocyte, a method of inducing specific CD38 expressing tumour cell killing with using the antibody. The offered new antibodies exhibit the unexpected properties: to bind minipig's CD38 and to cause cross-linked specific CD38 expressing cell killing.

EFFECT: use of the invention can find further application in therapy of the CD38 mediated disorders.

87 cl, 37 dwg, 4 tbl, 6 ex

Up!