Combination for correction of neurologic and psychoemotional status in case of organic cns disorders

FIELD: chemistry.

SUBSTANCE: invention represents pharmaceutical composition for correction and therapy of manifestations of amyloid intoxication in patients with brain pathologies, which are characterised by the fact that it contains melatonin 3-10 mg and memantine 5-300 mg.

EFFECT: effective treatment of patients, including cases of moderate cognitive disorders.

4 cl, 2 ex, 6 tbl, 7 dwg

 

BACKGROUND OF THE INVENTION.

Derivative of adamantane, memantine, in the modern view, is a non-competitive antagonist subtype of glutamate receptors that are sensitive to P-methyl-D-aspartate (NMDA receptor).

The structural formula of memantine (3,5-dimethyladamantane-1-amine):

Memantine was introduced into clinical practice in Europe in 2002 as a medicine for the treatment of Alzheimer's disease. Soon memantine for the same reasons was also registered in the United States. In numerous experiments on animals have been shown to improve cognitive functions under the influence of memantine. First of all, this action of memantine was shown on models of Alzheimer's disease [Filali M, Lalonde R, Riivest S. Subchronic memantine administration on spatial learning, exploratory activity, and nest building in an APP/PS1 mouse model of Alzheimer's disease. Neuropharmacology, 2011, v.60, pp.930-936.; Van Dam D, De Deyn PP. Cgnitive evaluation of disease modifying efficacy of galantamine and memantine in the APP23 model. Eur. Neuropsychopharmacol., 2006, v.16, pp.59-69 (in Russian)].

In the modern view, the effect of memantine on education is associated with its neuroprotective effect, i.e. the ability to protect nerve cells from death Kutzing MK, Luo V, Firestein BL. Protection from glutamate-induced excitotoxicity by memantine. BMES, 2011, DOI:10.107/s10439011-094-z]. Numerous experiments show that memantine protects nerve cells from the toxic effects of the neurotransmitter glutamate, which is hard visualaid what is in the brain in various diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy, brain ischemia, encephalopathy, caused by HIV infection (Kutzing MK, Luo V, Firestein BL. Protection from glutamate-induced excitotoxicity by memantine. BMES, 2011, DOI:10.107/s10439011-094-z., Van Dam D, De Deyn PP. Cgnitive evaluation of disease modifying efficacy of galantamine and memantine in the APP23 model. Eur. Neuropsychopharmacol., 2006, v.16, pp.59-69 (in Russian)).

Melatonin is a epiphyseal hormone, which is synthesized from serotonin. The structural formula of melatonin (N-acetyl-5-methoxytryptamine):

Melatonin exerts its biological effects through melatoninbuy receptors and is involved in regulation of circadian rhythms. In addition to impacts on biological rhythms, melatonin has strong antioxidant effect, which, apparently, is not related to its stimulating effect on melatoninbuy receptors. Antioxidant effect of melatonin was clearly shown in cell-free models [Cagnoli, Atabay CM, Kharlamova E, Manev H. Melatonin protects neurns from singlrt oxygen-induced apoptosis. J. Pineal Res., 1995, v.18, 222-226]. On the basis of these experiments it has been suggested that melatonin may exert a neuroprotective effect by suppressing oxidative stress. Currently, it is proved that in biological systems, oxidative stress causes cell death by direct damage to the DNA molecules, which leads to its fragmentation and the subsequent cell death is by apoptosis [Cagnoli, Atabay CM, Kharlamova E, Manev H. Melatonin protects neurns from singlrt oxygen-induced apoptosis. J. Pineal Res., 1995, v.18, 222-226.; 6.; Giusti P, Gusella M, Lipartiti M, Milani D, Zhu W, Vicini S, Manev H. Melatonin protects primary cultures of cerebellar granule neurons from kainate but not from N-methyl-D-aspartate excitotoxicity. Exp. Neurol., 1995, v.131, pp.39-46]. Special pathogenetic role oxidative stress plays in neuronal cell death in different pathologies, including the postischemic reperfusion of the brain and traumatic brain injury [Yamamoto H, Tang H-W. Melatonin attenuates L-cysteine-induced seizures and lipid peroxidation in the brain of mice. J. Pineal Res., 1996, v.21, pp.108-113.]. Increased production of free radicals is also observed in the aging brain, epilepsy and in patients with various neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's [Yamamoto H, Tang H-W. Melatonin attenuates L-cysteine-induced seizures and lipid peroxidation in the brain of mice. J. Pineal Res., 1996, v.21, pp.108-113].

In experiments on cultures of neurons in the brain, melatonin reduces the toxic effects of many toxins, including quinolinol and kainic acid. In animal experiments it was shown the protective effect of melatonin when convulsions caused by cyanide and l-cysteine [Yamamoto H, Tang H-W. Melatonin attenuates L-cysteine-induced seizures and lipid peroxidation in the brain of mice. J. Pineal Res., 1996, v.21, pp.108-113]. In experiments on mice, which have impaired learning in the passive avoidance test were caused by the introduction of the cholinergic antagonist of scopolamine, melatonin at a dose of 20 the g/kg in the/b introduction had a distinct antiamnesic action [Agrawal R, Tyagi E, Shukla R, Nath S. Effect of insulin and melatonin on acetylcholinesterase activity in the brain of amnestic mice. Behav. Brain. Res. 2008, v.189, pp.381-386]. Chronic administration of melatonin at a dose of 10 mg/kg eliminated memory impairment in mice, caused by aging or chronic introduction of alcohol.

The share of psychoorganic syndrome, not reaching the degree of dementia, vascular Genesis 25% of the diagnosed cases of mental disorders in persons over 60 years of age, attending the General outpatient clinic [Mikhailova N. M., 1996]. Such patients can be defined organic mental disorder with symptoms of carpeneti, slowness of psychomotor reactions, light gimnasticheskie disorders, disorders of attention and largely meet the criteria for a "mild cognitive disorder" (ICD-10, heading F06.7 "Mild cognitive disorder"). Others are more pronounced personality changes, accompanied by passivity and a significant decrease of the range of interests, with a strong tinge of complacency, or irritability with a propensity to psychopathic behavior (ICD-10, heading F07.0 "Organic personality disorder").

From the list of disorders included in the organic psychosyndrome derives proximity, if not identity, of these States with Unsharp pronounced, which is in its infancy, symptoms deme the Nations.

There is evidence that acute and long-term effects of sports traumatic brain injury), damage to the axon causes both regenerative and degenerative reactions in the brain and repeated shocks can trigger long-term neurodegenerative process, the calling, the so-called Boxing dementia and chronic traumatic encephalopathy (CTE). Chronic traumatic encephalopathy is much like other neurodegenerative diseases such as Alzheimer's disease, in particular in relation to metabolism and aggregation of Tau, β-amyloid and TDP-43 (TAR DNA binding protein) [Neuron. 2012 Dec 6; 76 (5): 886-99. doi: 10.1016/j.the neuron.2012.11.021. The neuropathology and neurobiology of traumatic brain injury. Blennow K, Hardy J, Zetterberg H.].

It is known that patients with hyperproduction of β-amyloid stabilize the state promoted the use of NSAIDs, especially indomethacin (Rogers J, et al., Clinical trial of indomethacin in Alzheimer's disease Neurology, 1993 Aug; 43 (8): 1609-11).

It was also studied the role of cyclooxygenase-1 in β-amyloid-induced neuronopathy and attempted use of inhibitors of COX (cyclooxygenase) as protectors against the development of Alzheimer's disease (A role for cyclooxygenase-1 in β-amyloid-induced neuroinflammation, Eduardo Candelario-Jalil AGING, Vol 1, No 4, pp 350-353).

As in neurodegenerative processes and cerebral ischemia plays an important pathogenetic role g is the dynamics of glutaminergic system, for the treatment of lung cognitive disorders, researchers have suggested the use of antagonists of the NMDA receptor.

As the closest analogue may be specified the use of memantine as a neuroprotector in amyloid toxicity (Hidalgo JJM, Alvarez XA, Cacabelos R, Quack G. Neuroprotection by memantine against neurodegeneration induced by beta-amyloid (1-40). Brain Research, 2002, v.958, 210-221). However, clinically the effects of memantine monotherapy moderate cognitive disorders (UKR) insignificant.

The present invention is the creation of an effective combination for elimination disorders in patients with pathologies of the brain, for the prevention, correction and treatment of the manifestations of amyloid toxicity.

This problem is solved by the use of a composition, sachetana containing Melatonin and Memantine for prevention, correction and treatment of the manifestations of amyloid toxicity.

This composition is administered to a mammal, including man.

Including amyloid toxicity in cases of RBM, cognitive impairment in vascular lesions and post-traumatic conditions.

Active ingredients formulations of the combination of Melatonin and Memantine are contained in therapeutically effective amounts. Preferred dosages range from 5 to 300 mg of memantine and from 3 to 10 mg of melatonin.

The form of the composition, the content of Asa Melatonin and Memantine, can be represented in the form: tablets, including sublingual form, capsules, dosage forms with modified release, injectable form, candles, powder for preparation of a drink, drops, including nose drops, transdermal, transbukkalno, aerosol form.

Pharmaceutically acceptable excipients are chosen to deliver a therapeutically effective dose amount of the Memantine and Melatonin in the conventional unit dosage form and to optimize the cost, ease, and stability of the production process. A necessary condition for the auxiliary substances are inert, chemical-physical compatibility with Memantine and Melatonin. Excipients used in solid dosage forms like tablets and capsules, may additionally include dyes and pigments, substances that mask the taste, flavors, sweeteners and adsorbents.

Thinners help to increase size pills with small amounts of active drug. The diluents include lactose, in the form of alpha-lactose or beta-lactose. Different types of lactose may consist of monohydrate lactose monohydrate alpha-lactose, anhydrous alpha-lactose, anhydrous beta-lactose and agglomerated lactose. Other diluents mo is ut include sugar, such as sucrose, investirovanie sugar, dextrose and dexterity. The preferred diluent is lactose monohydrate. Another diluent may be microcrystalline cellulose, including micronized.

Diluents may include starch and derivatives of starch. Starches include natural starches derived from different grains and/or other agricultural crops. The starches may also include pre-starch and starch-modified glycolate sodium. Starch and derivatives of starch are also in it dezintegriruetsja substances. Many also act as diluents dezintegriruetsja substance and a binder, and these additional properties should be considered when manufacturing technology pharmaceutical composition. Dezintegriruetsja substances added to destroy the tablets on the particles of the active pharmaceutical component and auxiliary, to facilitate dissolution and increase the bioavailability of a therapeutically active ingredients. Starch and starch derivatives, including sodium salt carboxymethylated ether of starch, such as starch, modified glycolate sodium, are used dezinfeciruyuhimi substances. Preferred, but not exclusive, dezintegriraat substance can b the th gelatinising starch. Other preferred dezintegriraat substance is carboxymethylcellulose sodium.

Binders are used as pharmaceutically acceptable auxiliary substances for the wet granulation to improve the concentration of therapeutically active substances, and other supporting ingredients in forming the granules. The binder is added to improve the fluidity of the powder and to improve compaction. Binders include cellulose derivatives such as microcrystalline cellulose, methylcellulose, carboxymethyl cellulose, hypromellose, hydroxyethylcellulose and hydroxypropylcellulose. Other binder ingredients selected from substances such as povidone, polyvinylpyrrolidone, gelatin, a natural gum, namely acacia, tragacanth, guar and pectin gum, starch paste, pre-gelatinising starch, polyethylene glycols, and sodium alginate.

Antifriction agents are lubricating and sliding substances that are used in the manufacturing of solid dosage forms to inhibit bonding tablets with technological surfaces and to reduce the buildup during the stages of pressing. Such substances include stearic acid, salts of stearic acid, such as stearate cal the Oia, magnesium stearate and stearyl fumarate sodium, talc, sodium benzoate, sodium acetate and sodium oleate.

Dosage forms for rectal application can be solutions or suspensions, or they can be obtained in the form of suppositories containing the active substance in a mixture with a neutral fat base, or gelatin rectal capsules that contain the active substance in a mixture with vegetable oil or paraffin oil.

Solutions for parenteral administration by injection can be obtained in the form of an aqueous solution of water-soluble pharmaceutically acceptable salts of the active substances, preferably in a concentration of from about 0.5% to about 2% by weight. These solutions may also contain stabilizers and/or buffer substances, and they may for convenience be obtained in capsules with different dosage units.

Effect: higher efficiency, including mild cognitive disorders.

The possibility of carrying out the invention may be illustrated below by examples.

Example 1 study of the pharmacological activity of the claimed combination

The study was conducted in two areas

1) study of the effect of the combination of memantine + melatonin in comparison with memantine violations motor is aktivnosti and episodic memory caused by the introduction of the peptide beta-amyloid in the lateral ventricle of the mouse brain;

2) Study the effect of the combination of memantine + melatonin in comparison with memantine on neurodegeneration caused by the introduction of the peptide beta-amyloid in the lateral ventricle of the mouse brain.

The study was conducted at the Institute of Pharmacology of the University of Tartu at the address biomedical Center, University of Tartu, Ul. of Law 19, of 51,014 Tartu, Estonia.

Research methods

All the experiments were carried out in accordance with the principles of laboratory animals (Principles of Animal Care Directive 86/609/EEC) according to the Declaration of Helsinki. The purchase of animals and the experiments were made on the basis of a license from the ethics Committee (Ministry of Agriculture of the Republic of Estonia). All persons involved in the care of animals and conducted the experiments have a personal license, which allows to carry out experiments on animals. In our experiments, every effort has been made to minimize the number of animals and their suffering.

All experiments were performed on mice male C57BL/6. Animals were purchased in the firm Harlan (England). The average age of the animal at the time of arrival in the animal facility of the Biomedical center at the University of Tartu was 5,5 (40 mice) and 6 weeks (20 mice). Ave the Genesis animals were placed for 2 weeks in quarantine at the animal facility of the Biomedical center at the University of Tartu. Then the animals were transferred to the vivarium of the Institute of Pharmacology (room room 3028). Animals were kept in plastic cages (5 mice in a cage) dimensions 25 cm×45 cm×12 cm (W×L×C) without restrictions on food and water with a 12-hour light cycle (light turns on automatically at 8.00). The cells of animals were in special climatic containers (Scanbur, Denmark). Containers equipped with HEPA filters to clean the air and have 24-hour monitoring of humidity and temperature. The relative humidity in the container is maintained in the range of 50±2%, temperature 22±1°C. the Access to the animals is allowed only to personnel who have the appropriate license. Cleaning the cells and ensuring food and water are carried out once per day. Animal feed: granular R70 (Lactamin, Stockholm, Sweden). The animals were housed in a climate containers for 1 week before experiments. Thus, the age of the animals to the beginning of the experiments was 8.5-9 weeks. Body weight of mice to the beginning of the experiments constituted 25.8±0,30 g (n=60). Before the experiments the animals were divided into 6 groups (table 1), 10 animals in each, using randomization.

Statement of experience and the introduction of the investigated substances

The scheme of experiments are presented to f is g 1. Table 1 shows the scheme of introduction of substances. The first introduction of the investigated substances was performed 24 hours after injection of beta-amyloid in the lateral ventricle of the mouse brain. Substances were administered for 8 days (once a day), at the same time from 9.00 to 11.00 hours. All solutions of the compounds under investigation were prepared each day just before the introduction and after their preparation was coded head and transmitted in coded form to the experimenter. On the 6th and 7th day of the introduction of a test substance with animals were conducted behavioral experiments (see Fig.1).

Groups of animals (n=10) scheme and the introduction of the investigated substances.

48 PG/3 ál
Table 1
no group
animals
Doses of the compounds under investigationThe concentration and the amount of beta-amyloid injected in the lateral ventricleConcentration and volume of the solutions of the investigated substances introduced inside
1ControlSterile water (3 μl)The control solution of 0.1 ml/10 g of body weight
2The control for the studied substancesThe control solution of 0.1 ml/10 g of body weight
3Memantine 5 mg/kg48 PG/3 álThe solution of memantine (0.5 mg/ml), 0.1 ml/10 g of body weight
4Memantine 10 mg/kg48 PG/3 álThe solution of memantine (1 mg/ml), 0.1 ml/10 g of body weight
5Memantine 5 mg/kg + melatonin 3 mg/kg48 PG/3 álThe solution of memantine (0.5 mg/ml) and melatonin (0.3 mg/ml), 0.1 ml/10 g of body weight
6Memantine 10 mg/kg + melatonin 6 mg/kg48 PG/3 álThe solution of memantine (1 mg/ml) + melatonin (0.6 mg/ml), 0.1 ml/10 g of body weight

Memantine was presented in the form of a salt of memantine hydrochloride), molecular weight=215,8, party room 80611. Has a quality certificate. All doses of memantine were calculated on salt. Melatonin: molecular weight 232.278, party room 20110915. Has a quality certificate.

Preparation of solutions of the compounds under investigation.

1) the Solution of memantine hydrochloride (0.5 mg/ml) + melatonin (0.3 mg/ml)

5 mg memantine is HCl was dissolved in 9 ml of water. Separately were given 3 mg of melatonin, was placed in a mortar and added 2 drops emulsifier tween-80, rubbed to a smooth paste, adding portions of 1 ml of water. The resulting emulsion was mixed with a solution of memantine (9 ml). Before the introduction of the emulsion was intensively shaken on a shaker. The resulting emulsion was injected in a volume of 0.1 ml per 10 g of body weight of the mouse, which corresponds to doses of memantine 5 mg/kg and melatonin 3 mg/kg

2) the Solution of memantine hydrochloride (1 mg/ml) + melatonin (0.6 mg/ml)

10 mg of memantine HCl was dissolved in 9 ml of water. Was separately weighed 6 mg of melatonin was placed in a mortar and added 2 drops emulsifier tween-80, rubbed to a smooth paste, adding portions of 1 ml of water. The resulting emulsion was mixed with a solution of memantine (9 ml). Before the introduction of the emulsion was intensively shaken on a shaker. The resulting emulsion was injected in a volume of 0.1 ml per 10 g of body weight of the mouse, which corresponds to doses of memantine 10 mg/kg melatonin 6 mg/kg

3) the Solution of memantine hydrochloride 0.5 mg/ml

5 mg of memantine HCl was dissolved in 10 ml of water, which was added 2 drops emulsifier tween-80 and mixed with vigorous shaking with a shaker. The resulting emulsion was injected in a volume of 0.1 ml per 10 g of body weight of the mouse, which corresponds to a dose of 5 mg/kg

4) the Solution of memantine hydrochloride 1 mg/ml

10 mg of memantine hydrochloride was dissolved in 0 ml of water, added 2 drops emulsifier tween-80 and mixed with vigorous shaking with a shaker. The resulting emulsion was injected in a volume of 0.1 ml per 10 g of body weight of the mouse, which corresponds to a dose of 10 mg/kg

5) Control solution

To 10 ml of water was added 2 drops emulsifier tween-80 and mixed with vigorous shaking with a shaker. The resulting emulsion was injected in a volume of 0.1 ml per 10 g body weight of mouse.

Solutions 1-5 was introduced inside of the stomach using a metal probe for mice (FTSS-20S-38) firm Salomon Scientific (USA).

6) the Solution of the peptide beta-amyloid (48 PG/3 µl) Peptide fragment of beta-amyloid, the corresponding amino acid sequence of beta-amyloid man (25-35) was obtained from the company Tocris (UK) (Batch No: 6, molecular weight: 1060,27, has a quality certificate).

1 mg of the substance of the peptide was dissolved in 1 ml of sterile water and placed in a sterile incubator at 37°C for 96 hours for the aggregation of the peptide (oligomeric form). Immediately before use the solution was diluted with sterile water to a concentration of peptide 16 nm and injected into the left lateral ventricle of the mouse brain at number 48 PG 3 microliters.

7) Monitoring for beta-amyloid

Sterile water in a volume of 3 μl was injected into the left lateral ventricle of the mouse brain.

The introduction of the peptide beta-amyloid in La is erally ventricle of the mouse brain.

The introduction of beta-amyloid in the lateral ventricle of the mouse brain was performed under General anesthesia. For anesthesia was used, a mixture of hypnorm (Hypnorm, VetaPharma; Lot P736/001; contains 0,315 mg of fentanyl and 10 mg fluanisone 1 ml), dormicum (Dormicum, Roche; Lot: F1038F71, 5 mg/ ml), and water in the ratio 1:1:2. The mixture was introduced in a/b in the amount of 0.1 ml per 10 g of body weight. After 2-3 min after the injection of anesthetic in animals evolved stage surgical anesthesia, after which the animal was fixed in a stereotaxic apparatus David Kopf (Leica Microsystems, Germany). On the skull of the mouse made the cut, cleaning the surface of the bones and was bregma. The coordinates for the injection of beta-amyloid in the lateral ventricle were using a computer system (Computer-assisted Stereotaxic system), using the parameters of the Atlas of the mouse brain (The Mouse Brain in Stereotaxic Coordinates, Franklin KBJ and G. Paxinos, 2012). The coordinates for the injection of beta-amyloid in relation to bregma were as follows: forward - 0.5 mm, lateral 1 mm, ventral - 2 mm.

The skull was powerlevels hole using boron and into the left lateral ventricle of the brain was introduced needle connected to a programmable micronesica (Syringe pump SP101IZ, Gentaur, Germany) applying a solution of beta-amyloid. The solution of the substance was administered at a speed of 0.75 μl/min and after infusion, the needle was left in the brain for 1 min. Control group received an infusion of sterile water the volume of 3 μl. After that, the wound was treated with an antiseptic solution was sutured and animals were placed on a heated table to exit from anesthesia.

Surgery caused the death of four mice, one animal in the group, intended for the introduction of the investigated substances. Thus, group of animals, which had to be introduced memantine or memantine + melatonin consisted of 9 mice.

The definition of motor activity and episodic memory models, for detecting a new object in mice.

On the 6th day of the experience, after 1 hour after administration of the substances under study animals were taken in behavioral experiments to study cognitive functions.

To evaluate the cognitive function test used to recognize a new object. This test is widely used to assess episodic memory in animals and its violation is characteristic for patients with Alzheimer's disease. The test is based on the fact that a healthy animal explores a significantly longer time a new object than the old one.

The experience consisted of three phases: habituation phase, training phase and phase retention.

1) Phase of addiction.

During this phase, mice were individually placed in a wooden box with dimensions 50 cm×50 cm×50 cm (length×width×height), located in the experimental room, dimly lit by incandescent lamps, with constant light 60 Lux. The floor of the box is was b is divided into 16 equal squares with a side length of 12.5 see The animal was in the box for 5 min and the experimenter recorded the number of crossed squares. This figure, in the future, were used to assess locomotor activity of mice. After 5 min, the animal was removed from the box and the floor of the box, rubbed a 5% solution of ethanol to eliminate the smell.

2) Phase of the exercise.

2 hours after the end of the habituation phase, conducted the training phase. To do this, the animal was again placed in the centre of the box on the floor which were installed two of the same object. The objects were two wooden cube, located in opposite corners of the box (Fig.2).

The animal was given the opportunity to explore the objects for 5 min and recorded the time during which the animal examined each of the objects. These data are needed to assess the degree of motivation and exploratory activity of animals. After each animal the floor of the box, rubbed a 5% solution of ethanol. At the end of the experience, the animal was placed in the home cage.

3) Phase retention.

After 24 hours after the training phase mice were injected analyte (seventh introduction) and after 1 hour after administration of the substances, the animals were again placed in the box for studies in which one object was replaced by a new object with a different shape and color (Fig. 4).

The animal is Nova was given the opportunity to explore old and new objects for five minutes and study each object was recorded by the experimenter.

The preference study of the new object was presented as the ratio of research time a new object in relation to the total time of the study of the old and new objects by the formula (Tnow×100)/(TST+Tnow) where TST and Tow - the study of the old and new objects.

Preparation of histological specimens and determination of cell death.

24 hours after completion of the behavioral tests, animals were introduced last time of the analyte and, later, 1 hour after injection, the mice were injected anesthetic solution of chloral hydrate (350 mg/kg/b). Within 5 min the animals developed deep anesthesia. To achieve anesthesia of the animals were fixed, opened the chest and was introduced perfusion needle into the left ventricle. At the same time opened right atrium and held transcardially perfusion of the circulatory system, first physiological solution (0.9% NaCl) followed by 4% solution of paraformaldehyde in 0.1 M phosphate buffer. Perfusion was carried out using a peristaltic pump Biorad Econo Pump (Biorad, Sweden). At the end of perfusion were extracted from the brain and placed for 24 hours in 4% solution of paraformaldehyde (postfixation).

Sagittal brain slices (thickness 40 μm), passing through the dorsal hippocampus were prepared using a vibrating microtome (Leica, Germany). The sections were placed the camping in tablets (one slice in the hole), filled with 0.1 M phosphate buffer and stored at 4°C (shelf life 3-4 days). For each animal were selected 4 slice passing through the dorsal hippocampus. For this, all of the slices from each animal were distributed on series 6 slices each. For each animal sample sections were performed based on the Cavalieri principle: first, randomly chosen slice of the first series and then each subsequent series took a slice with the same sequence number.

Staining was performed on free-floating sections in the 24 local tablet. Sections were washed in 0.1 M phosphate buffer, then incubated in a solution of 0.025% trypsin (Sigma, USA) and 0.1% CaCl2 in phosphate buffer for 10 minutes After washing the sections in phosphate buffer, was added 0.25% solution of Triton x-100 (Sigma, USA) and the sections were incubated for 1 hour. After that, the sections were washed again and thereto was added a solution of hematoxylin-eosin for 2 min, and then sections were rinsed with running water and immersed for 5 seconds in alcoholic hydrochloric acid solution (1% HCl in 70% ethanol) and again rinsed with water. Stained sections were placed on glass slides, were filled environment Vectashild (Vector Laboratories, USA) and covered with cover glasses.

Analysis of cell death was carried out using a BX51 microscope (Olympus, Japan). The microscope is equipped with a video camera DFC495 (Leica, Germany). The work is a of the microscope is controlled by software NewCAST company Visiopharm, Denmark). First under low magnification lens UPlan APO 4×/0.16 and using Newcast constructed superimage, which was to determine the preferred structure (Fig.3) and the program recorded the coordinates of the structures, which should determine the number microtechnic cells.

Then, using the lens LCAch 20×/0.40 PhC ∞/1 was analyzed and calculated picotesla cells in the structures of the CA1 and CA3 region of the hippocampus and sensory-motor areas of the cortex. Picotesla cells were defined as cells containing condensed hyperchromic nuclei, fragments of nuclei and condensed cytoplasm. The count was carried out in each square, as shown in Fig. 7, 8 and 9. The area of the squares wondered program and NewCast, depending on size structure and density of cells, it ranged between 0.1-0.5 mm2. For each animal, the number microtechnic cells and the square on which they were determined on four selected slices were summed and calculated the average density microtechnic cells at 1 mm2the slice. The obtained average values of density microtechnic cells in areas CA1, CA3 of the hippocampus and sensory-motor areas of the cortex for each animal were later used for statistical analysis.

Statistical analysis

For each group of mice rasschitat who were mean values (M) ± standard error of the mean (m). Further, the data analizirovali using t-test t-test (control and beta-amyloid), a one-way ANOVA followed by the application of a retrospective criterion of Bonferroni (action of the substances under study, the influence of the dose). The comparative evaluation of the actions of combinations of substances used two-factor analysis of variance. Differences between groups were considered significant at p<0,05. Statistical data processing was performed using the statistical software GraphPad PRISM 5 (USA).

The results of the study and discussion of the results.

The rationale for the selection of doses of memantine and melatonin for research.

In clinical practice, therapeutic daily dose of memantine 20 mg/day provides a level of concentration of the substance in the plasma of 0.5-1 mmol/liter of plasma (12). In rodents (mice), concentrations of memantine (1 µmol/l plasma) was observed after injection of memantine at a dose of 30 mg/kg/day (9). Since the aim of the study was to examine whether melatonin potentiate the effects of memantine, the study of memantine has been used in doses lower than those that give the maximum therapeutic concentration in plasma. This study used doses of memantine 5 and 10 mg/kg, and, accordingly, the concentration of memantine in these doses should be below m is xymalos therapeutic (1 µmol/l plasma).

Doses of melatonin were selected from a ratio of memantine: melatonin = 5:3.

Thus, the investigated dose combination memantine + melatonin were 5+3 mg/kg and 10+6 mg/kg Separately Comparators, memantine was studied at doses of 5 and 10 mg/kg

Characterization of populations of animals used in experiments.

In this study, for the analysis of homogeneity of populations, we used the body mass index (table 2). As can be seen from table 2, group of animals did not differ in body weight (data ANOVA: F5,59=0,93; p=0,47; differences not statistically significant). This suggests that by this measure in our experiments we used a homogeneous group of animals.

The influence of the studied substances on the locomotor activity and the level of research motivation in mice after injection of beta-amyloid in the lateral ventricle of the brain.

Data on the influence of the studied substances on the locomotor activity in the habituation phase and the level of research motivation in the training phase are shown in table 2. As can be seen from table 2, after 6 days after injection of beta-amyloid in the lateral ventricle of the brain in animals there was an increased locomotor activity compared with the control group of mice. Re-introduction of memantine at doses of 5 and 10 mg/kg had no effect on increased locomotor activity. The introduction is combinatii substances memantine + melatonin in doses of 5+3 mg/kg and 10+6 mg/kg reduced locomotor activity to control level. Univariate analysis of variance showed a significant effect of the combination of substances (F2,27=16,97; p<0,0001), and test Bonferroni showed a highly reliable effect of both combinations: memantine 5 mg/kg + melatonin 3 mg/kg (p<0.01) and memantine 10 mg/kg + melatonin 6 mg/kg (p<0,01).

When assessing the motivational behavior of animals after injection of beta-amyloid and of the substances under study, statistically significant differences were not identified (table 2).

Numerous studies indicate that in patients with disease Allgamer, disturbed circadian rhythms, and during the night phase, there psychomotor activation. As in mice, there is a reverse rhythm, and the phase of sleep they coincide with the day time of the day, the soothing effect of the combination of memantine + melatonin on hyperactivity in mice daytime evidence of restoration of normal circadian rhythms, which may have therapeutic value in patients with Alzheimer's disease.

Body weight, locomotor activity (number of crossed sectors) in the habituation phase and the study of interest (motivation) in the phase of training in mice after injection of beta amyloid and the investigated substances. Presents mean values ± standard error of the mean values (M±M) for groups of 9-10 animals. ##p<0,0001 compared to control **p< in 0.01 in comparison with beta-amyloid.

Table 2
AnalyteThe number of animalsBody weight of animals, g (M±M)Locomotor activity (M±M)Time (s) of research interest (M±M)
Control1025,0±0,9to 100.7±6,27,0±1,1
Amyloid-beta1026,1±0,8156,0±8,7##8,1±0,9
Memantine 5 mg/kg924,2±0,9146,8±11,6##8,5±1,0
Memantine 10 mg/kg926,2±0,7142,2±13,4##7,6±1,2
Memantine 5 mg/kg + Melatonin 3 mg/kg926,0±0,7107,0±7,0**6,1±0,6
Meant the h 10 mg/kg + Melatonin 6 mg/kg 926,4±0,3100,0±5,5**5,5±0,9

The influence of the studied substances on memory impairment in mice, caused by the introduction of beta-amyloid, in the test of recognition of the new object.

The results of studying the influence of the investigated substances in violation of episodic memory in mice caused by intracerebral introduction of beta-amyloid, are presented in table 3 and figure 4.

Animals of the control group was observed a marked preference in the study of the new object: the preference index in the control group was 87.8±2,3%. After 7 days after injection of beta-amyloid in the lateral ventricle of the brain in mice there was a decrease of the preference index of the new object to 49,7±14%. Statistical analysis showed highly reliable (p<0,0001, t-test t-test) difference compared with the control group. Reduced preference index indicates impaired memory in mice after injection of beta-amyloid. Memantine and memantine in combination with melatonin increased the preference index, which indicates the enhancement of memory disturbed by the introduction of beta-amyloid. For statistical analysis of the action of the investigated substances were used univariate analysis of variance, where the control was used, the group introduced the receiving beta-amyloid. Analysis of the actions of memantine showed improved memory (F2,27=34,23; p<0,0001), and this action of memantine was shown in both doses of 5 mg/kg (p<0.01) and 10 mg/kg (p<0,01). Study of the effect of the combination of memantine + melatonin revealed more significant enhancing effect on memory: F2,27=71,90; p<0,0001, and test Bonferroni revealed a highly reliable effect of both combinations memantine 5 mg/kg + melatonin 3 mg/kg (p<0.01) and memantine 10 mg/kg + melatonin 6 mg/kg (p<0,01).

The influence of the studied substances on memory impairment in mice, caused by beta-amyloid, in the test of recognition of the new object. Presents the average values of the preference index of the new object ± standard error of the mean values (M±M) for groups of 9-10 animals.

Table 3
AnalyteThe number of animalsThe preference index of the new objectThe confidence level
Control10of 87.8±2,3
Amyloid-beta1049,7±1,4p<0.0001 as compared with the control
Memantine 5 mg/kg967,4±2,7p<0.01 as compared with beta-amyloid
Memantine 10 mg/kg971,0±1,8p<0.01 as compared with beta-amyloid
Memantine 5 mg/kg + Melatonin 3 mg/kg977,4±1,7p<0.0001 as compared with beta-amyloid
Memantine 10 mg/kg + Melatonin 6 mg/kg9an 80.2±2,8p<0.0001 as compared with beta-amyloid

The next step of the statistical analysis was to compare, differ among themselves on whether the antiamnesic effect of the combination of memantine + melatonin from the action of memantine. For comparison we used two-factor analysis of variance, where one factor was "substance" and other factors "dose" (table 4). The analysis showed the strong influence of the "substance" (F1,32=17,72; p=0.0002) and the lack of influence of the factor "dose" (F1,32=1,837; p=0,18), and no interaction between "substance" × "dose" (F1,32=0,04; p=0,8). From data analysis it follows that the antiamnesic effect of the combination of memantine + melatonin significantly exceeds the antiamnesic effect of a meme is ntina on the model of amnesia in mice caused by the introduction of beta-amyloid, in the test of recognition of the new object. In addition, the improving effect on memory impairment induced by beta amyloid, manifested already at doses of combination memantine 5 mg/kg + melatonin 3 mg/kg

Matrix for comparison antiamnesic activity of the combination of memantine + melatonin action of memantine on memory impairment in mice, caused by beta-amyloid, in the test of recognition of the new object. The table shows the average values of the preference index of the new object ± standard error of the mean of a group of 9 animals.

Table 4
Factor (1): substancesFactor (2): dose mg/kg
510
Memantine + melatonin77,4±1,7an 80.2±2,8
Memantine67,4±2,771,0±1,8

The influence of the studied substances on the death of neurons caused by the introduction of beta-amyloid

Data on the influence of the studied substances on the death of neurons in areas CA1, CA3 of the hippocampus and sensory-motor areas of the cortex of th the injection of beta-amyloid are presented in table 5. As can be seen from the table, in the brain of mice of the control group in all the investigated structures were identified single picotesla cells, which appears to be due to mechanical damage to brain tissue, with the introduction of the control solution into the lateral ventricle. Introduction beta-amyloid caused significant neuronal loss in all investigated brain structures: CA1, CA3 of the hippocampus and the area of the sensory-motor cortex. Statistical analysis showed highly reliable (p<0,0001; t-test t-test) the action of beta-amyloid compared to control in all the studied structures (table 5). The introduction of memantine reduced cell death, and univariate analysis of variance showed a significant effect of the substance: measures analysis of variance for area CA1 were (F2,27=9,42; p<0,001). Subsequent analysis using the test Bonferroni showed a significant effect of dose of memantine 10 mg/kg (p<0.01) and revealed no significant effect of memantine at a dose of 5 mg/kg the effect of the combination of melatonin + memantine on the death of neurons caused by the introduction of beta-amyloid in area CA1 shown in Fig.5. The results of the statistical analysis are shown in table 5. The combination of memantine + melatonin vysokorentabelno reduced density microtechnic cells in the CA1 region of the hippocampus (F2,27=14,13; p<0,0001), and this effect was observed after administration of combinations of meranti is 5 mg/kg + melatonin 3 mg/kg (p< 0.001) and memantine 10 mg/kg + melatonin 6 mg/kg (p<0,001).

The introduction of memantine also reduced cell death in region CA3, and univariate analysis of variance showed a significant effect of the substance: measures analysis of variance for area CA3 were (F2,27=20,84; p<0,0001). Subsequent analysis using the test Bonferroni showed a significant effect of dose of memantine 5 mg/kg (p<0.001) and 10 mg/kg (p<0.0001) (table 5). The influence of the combination melatonin + memantine on the death of neurons caused by the introduction of beta-amyloid in the CA3 region of the hippocampus is shown in Fig.6.

The results of the statistical analysis are shown in table 5. The combination of memantine + melatonin vysokorentabelno reduced density microtechnic cells in the CA3 region of the hippocampus (F2,27=34,45; p<0,0001), and this effect was observed after the introduction of combination memantine 5 mg/kg + melatonin 3 mg/kg (p<0.001) and memantine 10 mg/kg + melatonin 6 mg/kg (p<0,001).

Memantine also significantly reduced the density microtechnic cells in the sensory-motor cortex (F2,27=48,08; p<0.0001) and both doses of 5 mg/kg and 10 mg/kg had a significant effect (table 5). The combination of memantine + melatonin also vysokorentabelno reduced density microtechnic cells in sensory-motor areas of the cortex (F2,27=34,45; p<0,0001), and this effect was observed after doses of the combination of memantine 5 mg/kg + melatonin 3 mg/kg (p<0.001) and memantine 10 mg/kg + m is latonin 6 mg/kg (p< 0,001) (Fig.7 9, table 5).

The effect of the analyte on the density microtechnic cells in the brain of mice after injection of beta-amyloid in the lateral ventricle of the brain. The average density values (N/mm2) ± standard error of the mean (M±M) on a group of 9-10 mice. ##p<0.001 (t-test t-test) compared with control, **p<0,01; ***p<0,0001 compared with the group receiving beta-amyloid.

Table 5
Structure of brainControlAmyloid-betaBeta-amyloid + Memantine 5 mg/kgBeta-amyloid + Memantine 10 mg/kgBeta-amyloid + Memantine 5 mg/kg) and melatonin-3 mg/kgBeta-amyloid + Memantine 10 mg/kg
and melatoni h 6 mg/kg
CA1 of the hippocampus8,6±2,135,7±4,9##24,3±3,213,7±1,6**12,6±3,2***11,3±2,2***
CA3 of the hippocampus13,7±3,143,9±3.1##26,1±1,9**18,4±3,4** 20,6±1,5***16,8±2,6***
Sensory motor region of the cerebral cortex7,8±1,770,0±4,5##42,2±4,0***19,9±1,4***29,4±2,3***18,6±1,2***

The next step of the statistical analysis was to compare the effect of combination of memantine + melatonin and memantine on the death of neurons in areas CA1 and CA3 of the hippocampus and sensory-motor areas of the cerebral cortex of mice after injection of beta-amyloid. For this purpose we have used two-factor analysis of variance based on the matrix table 6.

Matrix for comparison of the influence of the combination of memantine+melatonin with memantine on the death of neurons caused by beta-amyloid in mice. The table shows the average values of density (N/mm2) microtechnic cells in areas CA1, CA3 of the hippocampus and sensory-motor areas of the cerebral cortex of mice ± standard error of the mean of a group of 9 animals.

Table 6
Factor (1): substancesFactor (2): dose (mg/kg)
510
CA1
Memantine + melatonin12,6±3,211,3±2,2
Memantine24,3±3,213,7±1,6
CA3
Memantine + melatonin20,6±1,516,8±2,6
Memantine26,1±1,918,4±3,4
Sensory-motor area of the cortex
Memantine + melatonin29,4±2,318,6±1,3
Memantine42,2±4,019,9±1,4

For area CA1 two-factor analysis of variance showed a significant effect of the factor "substance" (F1,32=to 7.77; p<0.01), and significant influence of the factor "dose" (F1,32=of 5.53; p<0.05) and no interaction between the factors "substance" and "dose" (F1,32=3,38; p=0.07)

For the region CA3 two-factor analysis of variance revealed no significant effect of the factor "substance" (f1,32=2.09; p=0,15), showed a significant influence of the factor "dose" (F1,325,46; p=0.03) and no significant interaction between the factors "substance" and "dose" (F1,32=0,63; p=0,4)

For sensory-motor areas of the cortex two-factor analysis of variance showed a significant effect of the factor "substance" (F1,32=13,35; p=0,0009), a significant influence of the factor "dose" (F1,32=44,17; p=0.0001) and a significant interaction between the factors "substance" and "dose" (F1,32=16,66; p=0,0003).

Thus, based on the results of the statistical analysis we can conclude that the combination of memantine + melatonin has more pronounced neuroprotective effect than separately administered memantine in neurodegeneration induced by beta-amyloid in the CA1 region of the hippocampus and sensory-motor areas of the cerebral cortex of mice.

In experiments on mice by using the model of neurodegeneration induced by beta-amyloid in the lateral ventricle of the brain, the following results were obtained.

1) Combination of substances memantine + melatonin with the inside within 8 days normalized motor activity of animals increased after injection of beta-amyloid. This effect was observed already at doses of combination memantine 5 mg/kg + melatonin 3 mg/kg and reached a maximum at doses of memantine 10 mg/kg + melatonin 6 mg/kg Memantine, administered separately, in contrast to the combinations had no effect on the hyperactivity of mice caused by beta-amyloid.

2) the Combination of substances memantine + melatonin with the inside within 8 days restored episodic memory in mice in the test of recognition of the new object, broken after injection of beta-amyloid. This effect was observed at doses of combination memantine 5 mg/kg + melatonin 3 mg/kg and memantine 10 mg/kg + melatonin 6 mg/kg On his antiamnesic action the combination of memantine + melatonin surpassed antiamnesic action separately input of memantine in appropriate doses of 5 and 10 mg/kg

3) Combination of substances memantine + melatonin when oral administration for 8 days showed a marked neuroprotective effect during neurodegeneration induced by beta-amyloid. This action is manifested in the decrease in the density pyknotic cells in all studied brain regions: CA1, CA3 of the hippocampus and sensory-motor areas of the cerebral cortex. The same neuroprotective effect was rendered and separately administered memantine at doses of 5 and 10 mg/kg, however, the neuroprotective effect of the combination was significantly higher in area CA1 and sensory-motor cortex compared with the effect of a separate input of memantine. In the hippocampus CA3 neuroprotective effect of the combination of memantine + melatonin did not differ from neuroprotective actions separately input of memantine.

Thus, the results of the study indicate that the combination of memantine + melatonin is more effective than memantine to eliminate functional (increased Moto the ICA, disorders of episodic memory) and morphological (neurodegeneration) disorders in mice, caused by the introduction of beta-amyloid in the lateral ventricle. The proposed combination of memantine + melatonin is more effective in patients with pathologies of the brain, caused by increased production of beta-amyloid.

Example 2 Dosage forms

Composition of tablets: Memantine and Melatonin

Substance mg

Memantine 100

Melatonin 5

Lactose 5,25

Microcristalina cellulose 1,25

Starch 2,25

Povidone 2

Crosscarmellose 2,25

Calcium stearate 2

The capsules

Memantine 100

Melatonin 5

Lactose 5,25

1. Pharmaceutical composition for correction and treatment of the manifestations of amyloid toxicity in patients with pathologies of the brain, characterized in that it contains Melatonin 3-10 mg Memantine 5 are 300 mg.

2. The pharmaceutical composition under item 1, where the composition is represented in the form: tablets, including sublingual form, capsules, dosage forms with modified release, injectable forms, suppositories, powder for preparation of the drink, drops, including nose drops, transdermal, transbukkalno, aerosol form.

3. The use of a composition according to any one of paragraphs.1 and 2 for the correction and treatment of the manifestations of amyloid toxicity in patients with pathologies of the brain.

4. Application under item 3, g is e manifestation of amyloid toxicity is observed in cases of cognitive impairment in vascular lesions and post-traumatic conditions.



 

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60 cl, 3 tbl, 65 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: pharmaceutical composition contains 11-deoxy-prostaglandine compound and fatty acid ester prepared of fatty acid having C6-C24 carbon atoms, and monobasic C1-C6 alcohol. The 11-deoxy-prostaglandine compound may be stabilised by mixing it with the above fatty acid ester. Preferentially, the 11-deoxy-prostaglandine compound represents the type E1 11-deoxy-13,14-dihydro-15-keto-16,16-difluoro-prostaglandine compound; the above fatty acid ester is preferentially isopropyl palmitate. What is also described is a preparation in the form of a soft gelatin capsule that comprises a soft gelatin capsule shell containing polyol and/or sugar alcohol as a softening agent, and a pharmaceutical composition containing the 11-deoxy-prostaglandine compound and the fatty acid ester (as an excipient), wherein the above composition is encapsulated into the gelatin capsule shell.

EFFECT: invention provides stabilising the 11-deoxy-prostaglandine compound.

31 cl, 4 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to medicine. A pharmaceutical formulation for the treating diseases associated with endothelial dysfunction contains an active ingredient presented by a methyl pyridine derivative - 1.0-6.0 wt %; purine - 10.0-80.0 wt % and additive agents - the rest. The active substance is presented by compounds of a group: 3 -(N,N-dimethyl carbamoyloxy)-2-ethyl-6-methylpyridinium succinate, 3-methylpyridinium succinate, 2-ethyl-6-methyl-3-hydroxypyridinium hydrochloride, 6-trichloromethyl-2-chloropyridine (nitrapyrin), 2-ethyl-6-methyl-3-hydroxypyridine succinate. Purine is presented by inosine, adenosine, hypoxanthine. The pharmaceutical formulation may be presented in the form of injections, lyophilisate, solid capsules, tablets and suppositories.

EFFECT: formulation according to the invention provides creating the stable drug dosage form which considerably exceeds the existing analogues in pharmacodynamics activity on the endothelial dysfunction and toxicological properties.

4 cl, 4 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: composition representing a solid oral dosage form containing 4-Aminopyridine (INN fampridine, USAN dalfampridine), which is an organic compound having chemical formula C5H4N-NH2 as an agent, as well as a substance prolonging release.

EFFECT: improving the composition.

18 cl, 1 dwg, 3 tbl, 8 ex

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