RU2536270C1 - Combination for correction of neurologic and psychoemotional status in case of organic cns disorders - Google Patents

Abstract

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

Description

BACKGROUND OF THE INVENTION.

A derivative of adamantane, memantine, according to modern concepts, is a non-competitive antagonist of the subtype of glutamate receptors sensitive to P-methyl-D-aspartate (NMDA receptor).

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

Memantine was introduced into clinical practice in Europe in 2002 as a medicine for the treatment of Alzheimer's disease. Soon, memantine was also registered in the USA for the same indications. Numerous animal experiments have shown an improvement in cognitive function under the influence of memantine. First of all, this action of memantine was manifested in 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].

According to modern concepts, the effect of memantine on learning is associated with its neuroprotective effect, i.e. ability to protect nerve cells from the death of 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 effect of the neurotransmitter glutamate, which is released strongly 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).

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

Melatonin exerts its biological effect through melatonin receptors and is involved in the regulation of circadian rhythms. In addition to affecting biological rhythms, melatonin has a strong antioxidant effect, which, apparently, is not associated with its stimulating effect on melatonin receptors. The antioxidant effect of melatonin was clearly shown in cell-free models [Cagnoli C, Atabay CM, Kharlamova E, Manev N. Melatonin protects neurns from singlrt oxygen-induced apoptosis. J. Pineal Res., 1995, v. 18, 222-226]. Based on these experiments, it was suggested that melatonin may have a neuroprotective effect by suppressing oxidative stress. It has now been proven that in biological systems, oxidative stress causes cell death by direct damage to the DNA molecule, which leads to fragmentation and subsequent cell death by apoptosis [Cagnoli C, 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]. Oxidative stress plays a special pathogenetic role in nerve cell death in various pathologies, including postischemic reperfusion of the brain and traumatic brain damage [Yamamoto NA, 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 brain aging, epilepsy, and in patients with various neurodegenerative diseases, such as Alzheimer's, Parkinson's and Huntington’s diseases [Yamamoto NA, 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 brain neurons, melatonin reduces the toxic effect of many toxins, including quinolinic and kainic acid. In animal experiments, the protective effect of melatonin in seizures caused by cyanides and l-cysteine has been shown [Yamamoto HA, 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 in mice in which a learning disruption in the passive avoidance test was caused by the administration of the cholinoblocker scopolamine, melatonin at a dose of 20 mg / kg when given intravenously had a clear antiamnestic effect [Agrawal R, Tyagi E, Shukla R, Nath C. Effect of insulin and melatonin on acetylcholinesterase activity in the brain of amnestic mice. Behav. Brain. Res. 2008, v. 189, pp. 381-386]. With chronic administration, melatonin at a dose of 10 mg / kg eliminated memory impairment in mice caused by aging or chronic alcohol administration.

The share of the psycho-organic syndrome, which does not reach the degree of dementia, of vascular origin, accounts for 25% of diagnosed cases of mental pathology in people over 60 years old who have turned to a general clinic [Mikhailova NM, 1996]. In such patients, psycho-organic disorders with torpid phenomena, delayed psychomotor reactions, mild dysmnesthetic disorders, attention disorders can largely be determined and meet the criteria for “mild cognitive impairment” (ICD-10, heading F06.7 “Mild cognitive impairment”). Others have more pronounced personality changes, accompanied by passivity and a significant decrease in the circle of interests, with a persistent onset of complacency, or increased irritability with a tendency to psychopathic behavior (according to ICD-10, heading F07.0 "Organic personality disorder").

From the list of disorders included in the organic psychosyndrome, the proximity, if not identity, of these conditions with mildly expressed, at the initial stage of their development, symptoms of dementia follows.

There is evidence that in the acute and long-term effects of sports head injury (TBI), axon damage causes both regenerative and degenerative reactions in brain tissue and repeated tremors can initiate a long-term neurodegenerative process, causing the so-called boxing dementia or chronic traumatic encephalopathy (CTE). Chronic traumatic encephalopathy is in many ways similar to other neurodegenerative diseases, such as Alzheimer's disease, in particular with regard to the metabolism and aggregation of tau, amyloid β and TDP-43 (TAR-DNA binding protein) [Neuron. 2012 Dec 6; 76 (5): 886-99. doi: 10.1016 / j.neuron.2012.11.021. The neuropathology and neurobiology of traumatic brain injury. Blennow K, Hardy J, Zetterberg H.].

The use of NSAIDs, in particular indomethacin (Rogers J, et al., Clinical trial of indomethacin in Alzheimer’s disease Neurology, 1993 Aug; 43 (8): 1609-11 )

The role of cyclooxygenase-1 in β-amyloid-induced neuroinflammation was also studied, and attempts were made to use COX inhibitors (cyclooxygenases) as protectors for the development of Alzheimer's disease (A role for cyclooxygenase-1 in β-amyloid-induced neuroinflammation, Eduardo Candelario-Jalilario-Jalilario-Jalilario-Jalilario-Jalilario-Jalilario-Jalilario-Jalilario-Jalilario Vol 1, No. 4, pp 350-353).

Since both neurodegenerative processes and brain ischemia play an important pathogenetic role in the hyperactivation of the glutaminergic system, researchers have suggested the possibility of using NMDA receptor antagonists to treat mild cognitive impairment.

The closest analogue may be indicated the use of memantine as a neuroprotective agent for amyloid intoxication (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 in monotherapy of moderate cognitive impairment (RBM) are negligible.

The objective of the present invention is the creation of an effective combination to eliminate disorders in patients with brain pathologies, for the prevention, correction and therapy of manifestations of amyloid intoxication.

This problem is solved by using a composition combined with Melatonin and Memantine for the prevention, correction and therapy of manifestations of amyloid intoxication.

This composition is prescribed to a mammal, including humans.

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

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

The form of the composition containing Melatonin and Memantine can be presented in the form of: tablets, including sublingual forms, capsules, dosage forms with a modified release, injection form, suppositories, powder for the preparation of a drink, drops, including drops in the nose, transdermal, buccal, aerosol forms.

Pharmaceutically acceptable excipients are selected to ensure the delivery of a therapeutically effective single dose amount of Memantine and Melatonin in a unit dosage form and to optimize the cost, ease and stability of the manufacturing process. A prerequisite for excipients is inertness, chemical and physical compatibility with Memantine and Melatonin. Excipients used in solid dosage forms, such as tablets and capsules, may further include colorants and pigments, taste masking agents, flavors, sweeteners and adsorbents.

Diluents help to increase tablet size with a small amount of active drug substance. Diluents include lactose, in forms - alpha-lactose or beta-lactose. Different types of lactose may consist of lactose monohydrate, alpha-lactose monohydrate, anhydrous alpha-lactose, anhydrous beta-lactose and agglomerated lactose. Other diluents may include sugars such as sucrose, invert sugar, dextrose, and dextrates. A more preferred diluent is lactose monohydrate. Another diluent may be microcrystalline cellulose, including micronized.

Diluents can include starch and starch derivatives. Starches include natural starches obtained from various cereals and / or other crops. Starches may also include pre-starch and sodium glycolate modified starch. Starches and starch derivatives also carry the property of disintegrants. Many diluents also act as disintegrants and binders, and these additional properties should be taken into account when manufacturing a pharmaceutical composition. Disintegrants are added to break down the tablets into particles of the active pharmaceutical component and auxiliary, to facilitate dissolution and increase the bioavailability of therapeutically active ingredients. Starch and starch derivatives, including the sodium salt of starch carboxymethyl ester such as starch modified with sodium glycolate, are disintegrants used. Gelatinized starch may be a preferred, but not exclusive, disintegrant. Another preferred disintegrant is sodium carboxymethyl cellulose.

Binders are used as auxiliary pharmaceutically acceptable substances for wet granulation to increase the concentration of therapeutically active substances and other auxiliary ingredients in the forming granules. A binder is added to improve the fluidity of the powder and to improve compression. Binders include cellulose derivatives such as microcrystalline cellulose, methyl cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose. Other binders are selected from substances such as povidone, polyvinylpyrrolidone, gelatin, natural gum, namely acacia, tragacanth, guar and pectin resin, starch paste, pregelatinized starch, polyethylene glycols and sodium alginate.

Anti-friction agents are lubricants and lubricants that are used in the manufacture of solid dosage forms in order to prevent the tablets from sticking to technological surfaces and to reduce sticking during the pressing stages. Such substances include stearic acid, salts of stearic acid, for example calcium stearate, magnesium stearate and sodium stearyl fumarate, talc, sodium benzoate, sodium acetate and sodium oleate.

Dosage forms for rectal administration may be solutions or suspensions, or they may be prepared in the form of suppositories containing the active substances in a mixture with a neutral fat base, or gelatin rectal capsules containing the active substances in a mixture with vegetable oil or liquid paraffin.

Solutions for parenteral administration by injection can be obtained in the form of an aqueous solution of a water-soluble pharmaceutically acceptable salt 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 buffering agents and can be conveniently prepared in ampoules with various dosage units.

The technical result of the invention: higher efficiency, including with moderate cognitive impairment.

The possibility of carrying out the invention can be demonstrated by the following examples.

Example 1 Study of the pharmacological activity of the claimed combination

The study was conducted in two directions

1) Study of the effect of the combination of memantine + melatonin in comparison with memantine on impaired motor activity and episodic memory caused by the introduction of the beta-amyloid peptide into the lateral ventricle of the mouse brain;

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

The study was conducted at the Institute of Pharmacology of the University of Tartu at the Biomedical Center of the University of Tartu, ul. Ravila 19, 51014 Tartu, Estonia.

Research methods

All experiments were carried out in accordance with the principles of working with laboratory animals (Principles of Animal Care, Directive 86/609 / EEC) according to the Helsinki Declaration. The purchase of animals and the conduct of experiments were made on the basis of an appropriate license from the Ethics Committee (Ministry of Agriculture of the Republic of Estonia). All animal caregivers and experimenters have personal licenses to conduct animal experiments. During the experiments, every effort was made to minimize the number of animals and their suffering.

All experiments were performed on male mice of the C57BL / 6 line. Animals were purchased from Harlan (England). The average age of the animals at the time of arrival at the vivarium of the Biomedical Center of the University of Tartu was 5.5 (40 mice) and 6 weeks (20 mice). Upon arrival, animals were quarantined for 2 weeks at the vivarium of the Biomedical Center of the University of Tartu. After which the animals were transferred to the vivarium of the Institute of Pharmacology (room number 3028). The animals were kept in plastic cages (5 mice per cage) measuring 25 cm × 45 cm × 12 cm (W × D × H) without restriction in food and water with a 12-hour light cycle (the light turns on automatically at 8.00). Cells with animals were in special climatic containers (Scanbur, Denmark). The containers are equipped with HEPA filters for air purification and have 24-hour monitoring of humidity and air temperature. Relative humidity in containers is maintained within 50 ± 2%, temperature 22 ± 1 ° C. Access to animals is allowed only to personnel who have the appropriate license. Cell cleaning and provision of food and water are carried out once a day. Animal feed: granulated R70 (Lactamin, Stockholm, Sweden). Animals were kept in climatic containers for 1 week before the start of the experiments. Thus, the age of the animals at the beginning of the experiments was 8.5–9 weeks. The body weight of mice at the beginning of the experiments was 25.8 ± 0.30 g (n = 60). Before the start of the experiments, animals were divided into 6 groups (Table 1), 10 animals each, using a randomization program.

Test setup and administration of test substances

The layout of the experiments is presented in figure 1. Table 1 shows the scheme of administration of substances. The first administration of the test substances was carried out 24 hours after the injection of amyloid beta into the lateral ventricle of the mouse brain. Substances were administered within 8 days (once a day), at the same time from 9.00 to 11.00 hours. All solutions of the studied substances were prepared every day immediately before administration and after their preparation were encoded by the head and transmitted in encoded form to the experimenter. On the 6th and 7th day of the introduction of the test substances with animals, behavioral experiments were carried out (see figure 1).

Groups of animals (n = 10) and the scheme of administration of the studied substances.

Table 1 Group number
animals Doses of the studied substances The concentration and volume of amyloid beta introduced into the lateral ventricle The concentration and volume of the solutions of the studied substances administered orally one The control Sterile water (3 μl) Control solution 0.1 ml / 10 g body weight 2 Test substance control 48 pg / 3 μl Control solution 0.1 ml / 10 g body weight 3 Memantine 5 mg / kg 48 pg / 3 μl Memantine solution (0.5 mg / ml), 0.1 ml / 10 g body weight four Memantine 10 mg / kg 48 pg / 3 μl Memantine solution (1 mg / ml), 0.1 ml / 10 g body weight 5 Memantine 5 mg / kg + melatonin 3 mg / kg 48 pg / 3 μl A solution of memantine (0.5 mg / ml) and melatonin (0.3 mg / ml), 0.1 ml / 10 g body weight 6 Memantine 10 mg / kg + melatonin 6 mg / kg 48 pg / 3 μl A solution of memantine (1 mg / ml) + melatonin (0.6 mg / ml), 0.1 ml / 10 g body weight

Memantine was presented in the form of salt (memantine hydrochloride), molecular weight = 215.8, batch number 80611. There is a quality certificate. All doses of memantine were calculated on salt. Melatonin: molecular weight 232.278, batch number 20110915. There is a quality certificate.

Preparation of solutions of the investigated substances.

1) A solution of memantine hydrochloride (0.5 mg / ml) + melatonin (0.3 mg / ml)

5 mg of memantine HCl was dissolved in 9 ml of water. Separately, 3 mg of melatonin was weighed, placed in a mortar and 2 drops of Tween-80 emulsifier were added, triturated until a homogeneous mass was formed, adding 1 ml of water in portions. The resulting emulsion was mixed with a solution of memantine (9 ml). Before administration, the emulsion was vigorously shaken on a shaker. The resulting emulsion was administered in a volume of 0.1 ml per 10 g of mouse body weight, which corresponds to doses of memantine 5 mg / kg and melatonin 3 mg / kg.

2) A solution of memantine hydrochloride (1 mg / ml) + melatonin (0.6 mg / ml)

10 mg of memantine HCl was dissolved in 9 ml of water. Separately, 6 mg of melatonin was weighed, placed in a mortar and 2 drops of Tween-80 emulsifier were added, triturated until a homogeneous mass was formed, adding 1 ml of water in portions. The resulting emulsion was mixed with a solution of memantine (9 ml). Before administration, the emulsion was vigorously shaken on a shaker. The resulting emulsion was administered in a volume of 0.1 ml per 10 g of mouse body weight, which corresponds to doses of memantine 10 mg / kg and melatonin 6 mg / kg.

3) A solution of memantine hydrochloride 0.5 mg / ml

5 mg of memantine HCl was dissolved in 10 ml of water, to which 2 drops of Tween-80 emulsifier were added and mixed with vigorous shaking using a shaker. The resulting emulsion was administered 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) Solution of memantine hydrochloride 1 mg / ml

10 mg of memantine hydrochloride was dissolved in 10 ml of water, 2 drops of Tween-80 emulsifier was added and mixed with vigorous shaking using a shaker. The resulting emulsion was administered 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 of Tween-80 emulsifier and mixed with vigorous shaking using a shaker. The resulting emulsion was introduced in a volume of 0.1 ml per 10 g of mouse body weight.

Solutions 1-5 were introduced into the stomach using a metal probe for mice (FTSS-20S-38) from Salomon Scientific (USA).

6) A solution of the beta-amyloid peptide (48 picograms / 3 μl) The peptide fragment of the beta-amyloid corresponding to the amino acid sequence of human beta-amyloid (25-35) was obtained from Tocris (England) (Batch No: 6, molecular weight: 1060.27, there is a certificate of quality).

1 mg of the peptide substance was dissolved in 1 ml of sterile water and placed in a sterile incubator at 37 ° C for 96 hours to aggregate the peptide (oligomeric form). Immediately before use, the solution was diluted with sterile water to a peptide concentration of 16 nM and introduced into the left lateral ventricle of the mouse brain in the amount of 48 picograms in a volume of 3 microliters.

7) Control for amyloid beta

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

The introduction of the beta-amyloid peptide into the lateral ventricle of the mouse brain.

The introduction of amyloid beta into the lateral ventricle of the mouse brain was performed under general anesthesia. For anesthesia, a mixture of hypnorm (Hypnorm, VetaPharma; Lot P736 / 001; contains 0.315 mg of fentanyl and 10 mg of fluanisone in 1 ml), dormicum (Dormicum, Roche; Lot: F1038F71, 5 mg / ml) and water in a ratio of 1: 1 was used : 2. The mixture was administered ip in a volume of 0.1 ml per 10 g of body weight. 2-3 minutes after administration of the anesthetic, the animals developed a surgical stage of anesthesia, after which the animal was fixed in a David Kopf stereotactic apparatus (Leica Microsystems, Germany). An incision was made on the skull of the mouse, the bone surface was cleaned and bregma was found. The coordinates for introducing amyloid beta into the lateral ventricle were found using a Computer-assisted Stereotaxic system using the parameters of the mouse brain atlas (The Mouse Brain, in Stereotaxic Coordinates, KBJ Franklin and G. Paxinos, 2012). The coordinates for the introduction of amyloid beta with respect to Bregma were as follows: forward - 0.5 mm, laterally - 1 mm, ventrally - 2 mm.

A hole was drilled in the skull using boron and a needle was inserted into the left lateral ventricle of the brain, connected to a programmable micropump (Syringe pump SP101IZ, Gentaur, Germany), supplying a solution of amyloid beta. A solution of the substance was injected at a speed of 0.75 μl / min and after the end of the infusion, the needle remained in the brain for 1 min. The control group received an infusion of sterile water in a volume of 3 μl. After this, the wound was treated with an antiseptic solution, sutured and animals were placed on a heated table until the anesthesia was completely exited.

Surgical operation caused the death of four mice, one animal in groups intended for the introduction of the studied substances. Thus, the animal groups to which memantine or memantine + melatonin was to be administered consisted of 9 mice.

Determination of motor activity and episodic memory on the model of recognition of a new object in mice.

On the 6th day of the experiment, 1 hour after the administration of the test substances, the animals were taken into behavioral experiments to study cognitive functions.

To evaluate cognitive functions, a recognition test of a new object was used. This test is widely used to assess episodic memory in animals and its violation is characteristic of Alzheimer's patients. The test is based on the fact that a healthy animal examines a new object much more time than an old one.

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

1) The addictive phase.

During this phase, the mice were individually placed in a wooden box measuring 50 cm × 50 cm × 50 cm (length × width × height), located in an experimental room dimly lit by incandescent lamps, with constant light of 60 lux. The box floor was divided into 16 identical squares with a side length of 12.5 cm. The animal was in the box for 5 minutes and the experimenter recorded the number of crossed squares. This indicator, in the future, was used to assess the motor activity of mice. After 5 minutes, the animal was removed from the box and the floor of the box was rubbed with a 5% ethanol solution to eliminate odor.

2) Training phase.

2 hours after the end of the addictive phase, the training phase was conducted. For this, the animal was again placed in the center of the box, on the floor of which two identical objects were installed. The objects were two wooden cubes located in opposite corners of the box (Figure 2).

The animal was given the opportunity to examine objects for 5 minutes, and at the same time, the time during which the animal examined each of the objects was recorded. These data are necessary to assess the degree of motivation and research activity of animals. After each animal, the box floor was wiped with a 5% ethanol solution. At the end of the experiment, the animal was placed in a home cage.

3) Retention phase.

24 hours after the training phase, the test substances were administered to the mice (seventh administration) and 1 hour after the administration of the substances, the animals were again placed in the study box, in which one object was replaced with a new object of a different shape and color (Fig. 4).

The animal was again given the opportunity to explore the old and new objects for five minutes and the time of investigation of each object was recorded by the experimenter.

The research preference coefficient of a new object was presented as the ratio of the time of research of a new object in relation to the total time of research of the old and new objects according to the formula (Tnov × 100) / (Tst + Tnov), where Tst and Tnov are the time of investigation of the old and new objects.

Preparation of histological preparations and determination of cell death.

24 hours after the end of the behavioral tests, the animals were tested for the last time and, 1 hour after the introduction, the mice were injected with an anesthetic solution of chloral hydrate (350 mg / kg, ip). Within 5 minutes, the animals developed deep anesthesia. Upon reaching anesthesia, the animals were fixed, the chest was opened and a perfusion needle was inserted into the left ventricle of the heart. At the same time, the right atrium was opened and transcardial perfusion of the circulatory system was performed, first with physiological saline (0.9% NaCl), then 4%) with 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, the brain was removed and placed for 24 hours in a 4% solution of paraformaldehyde (postfixation).

Sagittal sections of the brain (40 μm thick) passing through the dorsal hippocampus were prepared using a vibrating microtome (Leica, Germany). Slices were placed on plates (one slice per well) filled with 0.1 M phosphate buffer and stored at 4 ° C (shelf life 3-4 days). For each animal, 4 sections were selected passing through the dorsal hippocampus. For this, all slices from each animal were distributed into a series of 6 slices each. For each animal, the selection of slices was carried out based on the Cavalieri principle: first, a slice was randomly selected from the first series and then a slice with the same serial number was taken from each subsequent series.

Staining was performed on free-floating sections in a 24-seater plate. 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 min. After washing the sections in phosphate buffer, 0.25% Triton x-100 solution (Sigma, USA) was added to them and the sections were incubated for 1 hour. After that, the sections were washed again and a hematoxylin-eosin solution was added to them for 2 minutes, after which the sections were washed with running water and immersed for 5 seconds in an alcoholic solution of hydrochloric acid (1% HCl in 70% ethanol) and washed again with water. Stained sections were placed on glass slides, filled with Vectashild medium (Vector Laboratories, USA) and covered with coverslips.

Analysis of cell death was carried out using a BX51 microscope (Olympus, Japan). The microscope is equipped with a DFC495 video camera (Leica, Germany). The microscope is controlled by NewCAST software from Visiopharm (Denmark). First, at low magnification, using the UPlan APO 4 × / 0.16 lens and using the Newcast program, a super image was constructed on which the structures of interest to us were determined (Fig. 3) and the program recorded the coordinates of the structures in which the number of pyknotic cells should be determined.

Then, using an LCAch 20 × 0.40 PhC ∞ / 1 lens, pycnotic cells were analyzed and counted in the structures CA1 and CA3 of the hippocampus and sensory-motor region of the cortex. Pycnotic cells were defined as cells containing condensed hyperchromic nuclei, nuclear fragments, and condensed cytoplasm. Counting was carried out in each square, as shown in FIG. 7, 8 and 9. The square area was set by the NewCast program and, depending on the size of the structure and cell density, it ranged from 0.1-0.5 mm ² . For each animal, the number of pycnotic cells and the area on which they were determined on four selected sections were summed up and the average density of pycnotic cells per 1 mm ² section was calculated and calculated. The obtained average densities of pycnotic cells in the CA1, CA3 regions of the hippocampus and the sensory-motor region of the cortex for each animal were subsequently used for statistical analysis.

Statistical analysis

For each group of mice, the mean values (M) ± the standard error of the mean (m) were calculated. Further, the data was analyzed using Student's t-test (control and beta-amyloid), one-way analysis of variance, followed by the retrospective Bonferroni test (action of the studied substances, dose effect). In a comparative assessment of the action of combinations of substances, two-way analysis of variance was used. Differences between groups were considered significant at p <0.05. Statistical data processing was performed using statistical software GraphPad PRISM-5 (USA).

Research results and discussion of the results.

Justification of the choice of doses of memantine and melatonin for research.

In clinical practice, a therapeutic daily dose of memantine of 20 mg / day provides a plasma concentration level of 0.5-1 μmol / liter of plasma (12). In rodents (mice), corresponding memantine concentrations (1 μmol / liter of plasma) were observed after administration of memantine at a dose of 30 mg / kg / day (9). Since the aim of the study was to analyze whether melatonin can enhance the action of memantine, in the study, memantine was used in doses lower than those that give the maximum therapeutic plasma concentrations. In this study, doses of memantine 5 and 10 mg / kg were used, and, accordingly, the concentrations of memantine in these doses should be lower than the maximum therapeutic (1 μmol / liter of plasma).

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

Thus, the studied doses of the combination of memantine + melatonin were 5 + 3 mg / kg and 10 + 6 mg / kg. Separately, taken for comparison, memantine was studied at doses of 5 and 10 mg / kg.

Characterization of the animal population used in the experiments.

In this study, to analyze the homogeneity of the animal population, body mass indices were used (Table 2). As can be seen from table 2, the groups of animals did not differ in body weight (analysis of variance: F5.59 = 0.93; p = 0.47; the differences are not statistically significant). This indicates that, according to this indicator, a homogeneous group of animals was used in the experiments.

The influence of the studied substances on motor activity and the level of research motivation in mice after the introduction of amyloid beta into the lateral ventricle of the brain.

Data on the effect of the studied substances on the motor activity in the addictive phase and the level of research motivation in the training phase are shown in table 2. As can be seen from table 2, 6 days after the introduction of amyloid beta into the lateral ventricle of the brain, the animals showed increased motor activity compared to with a control group of mice. Repeated administration of memantine at doses of 5 and 10 mg / kg did not affect the increased locomotor activity. The introduction of a combination of substances memantine + melatonin in doses of 5 + 3 mg / kg and 10 + 6 mg / kg reduced motor activity to a control level. Univariate analysis of variance showed a significant effect of the combination of substances (F2.27 = 16.97; p <0.0001), and the Bonferroni test showed highly reliable action 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 the introduction of amyloid beta and the test substances, no statistically significant differences were detected (Table 2).

Numerous studies indicate that in patients with Alheimer's disease, circadian rhythms are disturbed, and during the night phase they have psychomotor activation. Since mice have reverse rhythms, and their sleep phase coincides with daytime, the calming effect of the memantine + melatonin combination on hyperactivity of mice in the daytime indicates the restoration of normal circadian rhythms, which may have therapeutic value in patients with Alzheimer's disease.

Body weight, locomotor activity (number of sectors crossed) in the addictive phase and the time of study of objects (level of motivation) in the training phase in mice after administration of beta amyloid and test substances. The mean values ± standard errors of the mean value (M ± m) for groups of 9-10 animals are presented. ## p <0.0001 compared to control; ** p <0.01 compared with amyloid beta.

table 2 Test substances Number of animals Animal body weight, g (M ± m) Physical activity (M ± m) Time (s) of object research (M ± m) The control 10 25.0 ± 0.9 100.7 ± 6.2 7.0 ± 1.1 Amyloid beta 10 26.1 ± 0.8 156.0 ± 8.7 ## 8.1 ± 0.9 Memantine 5 mg / kg 9 24.2 ± 0.9 146.8 ± 11.6 ## 8.5 ± 1.0 Memantine 10 mg / kg 9 26.2 ± 0.7 142.2 ± 13.4 ## 7.6 ± 1.2 Memantine 5 mg / kg + Melatonin 3 mg / kg 9 26.0 ± 0.7 107.0 ± 7.0 ** 6.1 ± 0.6 Memantine 10 mg / kg + Melatonin 6 mg / kg 9 26.4 ± 0.3 100.0 ± 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 a new object.

The results of a study of the effect of the test substances on impaired episodic memory in mice caused by intracerebral administration of beta-amyloid are presented in Table 3 and Figure 4.

Animals of the control group showed a pronounced preference in the study of a new object: the preference index in the control group was 87.8 ± 2.3%. 7 days after the introduction of beta-amyloid into the lateral ventricle of the brain in mice, a decrease in the preference index of the new object to 49.7 ± 14% was observed. Statistical analysis showed a highly reliable (p <0.0001, Student t-test) difference compared with the control group. A decrease in the preference index indicates memory impairment in mice after administration of beta-amyloid. Memantine and memantine in combination with melatonin increased the preference index, which indicates an improvement in memory impaired by the introduction of beta-amyloid. For a statistical analysis of the action of the investigated substances, one-way analysis of variance was used, where a group with the introduction of amyloid beta was used as a control. An analysis of the action of memantine revealed an improvement in memory (F2.27 = 34.23; p <0.0001), and this action of memantine was manifested in both doses of 5 mg / kg (p <0.01) and 10 mg / kg (p <0 , 01). Studying the effect of the combination of memantine + melatonin revealed a more significant improving effect on memory: F2.27 = 71.90; p <0.0001, and the Bonferroni test revealed a highly reliable effect of both combinations of 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 a new object. Presents the average values of the preference index of the new object ± standard errors of the mean (M ± m) for groups of 9-10 animals.

Table 3 Test substances Number of animals New Object Preference Index Confidence level The control 10 87.8 ± 2.3 Amyloid beta 10 49.7 ± 1.4 p <0.0001 cf. with control Memantine 5 mg / kg 9 67.4 ± 2.7 p <0.01 cf. with amyloid beta Memantine 10 mg / kg 9 71.0 ± 1.8 p <0.01 cf. with amyloid beta Memantine 5 mg / kg + Melatonin 3 mg / kg 9 77.4 ± 1.7 p <0.0001 cf. with amyloid beta Memantine 10 mg / kg + Melatonin 6 mg / kg 9 80.2 ± 2.8 p <0.0001 cf. with amyloid beta

The next step in the statistical analysis was to compare whether the combination of memantine + melatonin is different from the action of memantine in terms of anti-amnestic effect. For comparison, two-way analysis of variance was used, where one factor was “substance” and another factor was “dose” (Table 4). The analysis showed a strong influence of the factor “substance” (F1.32 = 17.72; p = 0.0002) and the absence of influence of the factor “dose” (F1.32 = 1.837; p = 0.18), as well as the absence of interaction “substance” "×" dose "(F1.32 = 0.04; p = 0.8). From the analysis, it follows that the anti-amnestic effect of the memantine + melatonin combination significantly exceeds the anti-amnestic effect of memantine on the model of amnesia in mice induced by the administration of beta-amyloid in the test for the recognition of a new object. In addition, the improving effect on memory impairment caused by beta-amyloid was already evident in doses of the combination of memantine 5 mg / kg + melatonin 3 mg / kg.

A matrix for comparing the antiamnestic effect of the combination of memantine + melatonin with the effect of memantine on memory impairment in mice caused by beta-amyloid in the test for recognition of a new object. The table shows the mean values of the preference index of the new object ± standard errors of the mean value per group of 9 animals.

Table 4 Factor (1): substances Factor (2): dose mg / kg 5 10 Memantine + Melatonin 77.4 ± 1.7 80.2 ± 2.8 Memantine 67.4 ± 2.7 71.0 ± 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 the CA1, CA3 hippocampus and the sensorimotor region of the cortex after administration of beta-amyloid are presented in Table 5. As can be seen from the table, single pycnotic cells were detected in the brains of mice of the control group in all the studied structures. which, apparently, is associated with mechanical damage to brain tissue with the introduction of a control solution into the lateral ventricle. The introduction of amyloid beta caused a significant death of neurons in all the studied brain structures: CA1, CA3 of the hippocampus and the region of the sensory-motor cortex. Statistical analysis showed a highly reliable (p <0.0001; Student's t-test) action of beta-amyloid, compared with the control in all studied structures (Table 5). The introduction of memantine reduced cell death, and a one-way analysis of variance revealed a significant effect of the substance: the analysis of variance for the CA1 region was (F2.27 = 9.42; p <0.001). Subsequent analysis using the Bonferroni test showed a significant effect of a dose of memantine of 10 mg / kg (p <0.01) and did not reveal a significant effect of memantine at a dose of 5 mg / kg. The effect of the combination melatonin + memantine on neuronal death caused by the administration of amyloid beta in the CA1 region is shown in FIG. 5. The results of the statistical analysis are shown in Table 5. The combination of memantine + melatonin significantly reduced the density of pycnotic cells in the CA1 region of the hippocampus (F2.27 = 14.13; p <0.0001), and this effect was manifested after the introduction of combinations of memantine 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 the CA3 region, and one-way analysis of variance revealed a significant effect of the substance: the analysis of variance for the CA3 region was (F2.27 = 20.84; p <0.0001). Subsequent analysis using the Bonferroni test showed a significant effect of memantine doses of 5 mg / kg (p <0.001) and 10 mg / kg (p <0.0001) (Table 5). The effect 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 presented in Fig.6.

The results of the statistical analysis are shown in Table 5. The combination of memantine + melatonin significantly reduced the density of pycnotic cells in the CA3 region of the hippocampus (F2.27 = 34.45; p <0.0001), and this effect was manifested after the introduction of combinations of 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 of pycnotic cells in the sensory-motor cortex (F2.27 = 48.08; p <0.0001), with both doses of 5 mg / kg and 10 mg / kg having a significant effect (Table 5). The combination of memantine + melatonin also significantly reduced the density of pycnotic cells in the sensory-motor region of the cortex (F2.27 = 34.45; p <0.0001), and this effect was manifested after doses of the combination of memantine 5 mg / kg + melatonin 3 mg / kg (p <0.001) and memantine 10 mg / kg + melatonin 6 mg / kg (p <0.001) (Fig. 7 9, Table 5).

The effect of the test substance on the density of pycnotic cells in the brain of mice after the introduction of amyloid beta into the lateral ventricle of the brain. Average density values (N / mm2) ± standard error of the mean value (M ± m) per group of 9-10 mice are shown. ## p <0.001 (Student's t-test) compared with the control, ** p <0.01; *** p <0.0001 compared with the group receiving beta-amyloid.

Table 5 Brain structure The control Amyloid beta Amyloid Beta + Memantine 5 mg / kg Amyloid Beta + Memantine 10 mg / kg Amyloid Beta + Memantine 5 mg / kg and Melatonin 3 mg / kg Amyloid Beta + Memantine 10 mg / kg
and melatoni n 6 mg / kg CA1 hippocampus 8.6 ± 2.1 35.7 ± 4.9 ^## 24.3 ± 3.2 13.7 ± 1.6 ** 12.6 ± 3.2 *** 11.3 ± 2.2 *** CA3 Hippocampus 13.7 ± 3.1 43.9 ± 3.1 ^## 26.1 ± 1.9 ** 18.4 ± 3.4 *** 20.6 ± 1.5 *** 16.8 ± 2.6 *** Sensory motor region of the cerebral cortex 7.8 ± 1.7 70.0 ± 4.5 ^## 42.2 ± 4.0 *** 19.9 ± 1.4 *** 29.4 ± 2.3 *** 18.6 ± 1.2 ***

The next step in the statistical analysis was to compare the effect of the combination of memantine + melatonin and memantine on neuronal death in the CA1 and CA3 regions of the hippocampus and in the sensory-motor region of the cerebral cortex of mice after administration of amyloid beta. For these purposes, we used two-way analysis of variance based on matrix table 6.

A matrix for comparing the effects of the combination of memantine + melatonin with memantine on neuronal death caused by beta-amyloid in mice. The table shows the average density (N / mm2) of pycnotic cells in the CA1, CA3 areas of the hippocampus and the sensory-motor region of the cerebral cortex of mice ± standard errors of the mean value for a group of 9 animals.

Table 6 Factor (1): substances Factor (2): dose (mg / kg) 5 10 CA1 Memantine + Melatonin 12.6 ± 3.2 11.3 ± 2.2 Memantine 24.3 ± 3.2 13.7 ± 1.6 CA3 Memantine + Melatonin 20.6 ± 1.5 16.8 ± 2.6 Memantine 26.1 ± 1.9 18.4 ± 3.4 Sensory-motor area of the cortex Memantine + Melatonin 29.4 ± 2.3 18.6 ± 1.3 Memantine 42.2 ± 4.0 19.9 ± 1.4

For the CA1 region, two-way analysis of variance revealed a significant effect of the factor “substance” (F1.32 = 7.77; p <0.01), a significant effect of the factor “dose” (F1.32 = 5.53; p <0.05) and the lack of interaction between the factors “substance” and “dose” (F1.32 = 3.38; p = 0.07)

For the CA3 region, two-way analysis of variance did not reveal a significant effect of the “substance” factor (f1.32 = 2.09; p = 0.15), showed a significant effect of the “dose” factor (F1.32 = 5.46; p = 0.03) and the lack of reliable interaction between the factors “substance” and “dose” (F1.32 = 0.63; p = 0.4)

For the sensory-motor region of the cortex, two-way analysis of variance revealed a significant effect of the factor “substance” (F1.32 = 13.35; p = 0.0009), a significant effect of the factor “dose” (F1.32 = 44.17; p = 0 , 0001) and a reliable interaction between the factors “substance” and “dose” (F1.32 = 16.66; p = 0.0003).

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

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

1) The combination of substances memantine + melatonin when administered orally for 8 days normalized the motor activity of animals, increased after administration of beta-amyloid. This effect was already apparent at doses of the combination of 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 alone, unlike combinations, did not affect the hyperactivity of mice caused by beta-amyloid.

2) The combination of memantine + melatonin when administered orally for 8 days restored episodic memory in mice in a new object recognition test, impaired after administration of amyloid beta. This effect was manifested in doses of the combination of memantine 5 mg / kg + melatonin 3 mg / kg and memantine 10 mg / kg + melatonin 6 mg / kg. The combination of memantine + melatonin was superior in its anti-amnestic effect to the anti-amnestic effect of separately administered memantine in appropriate doses of 5 and 10 mg / kg.

3) The combination of memantine + melatonin when administered orally for 8 days showed a pronounced neuroprotective effect in neurodegeneration caused by the introduction of beta-amyloid. This action was manifested in a decrease in the density of pycnotic cells in all studied areas of the brain: CA1, CA3 of the hippocampus and sensory-motor region of the cerebral cortex. Separately administered memantine at doses of 5 and 10 mg / kg had the same neuroprotective effect, however, the neuroprotective effect of the combination was significantly higher in the CA1 region and sensory-motor cortex compared to the effect of separately administered memantine. In the CA3 hippocampus, the neuroprotective effect of the memantine + melatonin combination did not differ from the neuroprotective effect of separately administered memantine.

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

Example 2 Dosage Forms

Composition of tablets: Memantine and Melatonin

Mg substance

Memantine 100

Melatonin 5

Lactose 5.25

Microcrystalline cellulose 1.25

Starch 2.25

Povidone 2

Croscarmellose 2.25

Calcium Stearate 2

Capsule composition

Memantine 100

Melatonin 5

Lactose 5.25

Claims (4)

1. A pharmaceutical composition for the correction and treatment of manifestations of amyloid intoxication in patients with brain pathologies, characterized in that it contains Melatonin 3-10 mg and Memantine 5-300 mg. 2. The pharmaceutical composition according to claim 1, where the composition is presented in the form of: tablets, including sublingual forms, capsules, dosage forms with modified release, injection form, suppository, powder for preparing a drink, drops, including drops in the nose, transdermal, buccal, aerosol form. 3. The use of the composition according to any one of claims 1 and 2 for the correction and treatment of manifestations of amyloid intoxication in patients with brain pathologies. 4. The use according to claim 3, where the manifestation of amyloid intoxication is observed in cases of cognitive decline in vascular lesions and post-traumatic conditions.

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