RU2725632C1 - Pharmaceutical composition in form of capsules, having nootropic action - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.SUBSTANCE: invention refers to a pharmaceutical composition, namely a composition having nootropic action. Composition is capsule containing 6-[4-methoxy-3-(1H-pyrazol-1-ylmethyl)benzyl]-1,11 dimethyl-3,6,9-triazatricyclo[7.3.1.1]tetradecane-4,8 as an active substance, 12-trione - bicyclononane, and as auxiliary substances - sodium alginate, pregelatinized corn starch (PCS), sodium starch glycolate, silicon colloidal dioxide and calcium chloride, taken in certain amounts. Use of the above composition for treating and preventing memory disorders, dizziness, low concentration, emotional lability, dementia due to cerebral circulation (ischemic stroke), cerebral injuries, in Alzheimer disease in old age, to improve cognitive performance after suffering brain damage, for treating withdrawal and psychoorganic syndrome in chronic alcoholism and learning disabilities in children.EFFECT: above described nootropic composition has good solubility and stability.2 cl, 12 dwg, 15 tbl, 11 ex

Description

The invention relates to a pharmaceutical composition, namely to a capsule containing as an active ingredient the pharmaceutical substance 6- [4methoxy-3- (1H-pyrazol-1-ylmethyl) benzyl] -1,11dimethyl-3,6,9-triazatricyclo [7.3 .1.1] tetradecane-4,8,12-trion - bicyclononan, and as excipients - sodium alginate, pregelatinized corn starch (PAC), sodium starch glycolate, colloidal silicon dioxide and calcium chloride - for use as a nootropic agent in violation memory, dizziness, decreased attention span, emotional lability, dementia due to cerebrovascular accidents (ischemic stroke), brain injuries, Alzheimer's disease in old age, to improve cognitive performance after suffering brain damage, for the treatment of withdrawal symptoms and psycho-organic syndrome in chronic alcoholism and learning disabilities in children.

Nootropic drugs are a special group of neuropsychotropic drugs, the specific effect of which is characterized by the ability to improve memory and learning processes, intellectual and cognitive functions in healthy individuals and impaired in various diseases. As a synonym for nootropic drugs in the foreign literature, the term “cognition enhancers” is used. The mechanisms of action of nootropics are still the subject of discussion due to the heterogeneity of this group of drugs.

The concept of nootropic drugs was first formulated by the Belgian pharmacologists C. Giurgea and V. Skondia. In 1963, they synthesized piracetam (nootropil) - the first drug of this group, which was introduced to the world pharmaceutical market by the Belgian company UCB Pharma. In 1972, Cornelia Giurgea discovered that piracetam improves cognitive function and memory. After that, a new class was introduced into the classification of neuro- and psychotropic drugs - nootropics.

Piracetam affects the glutamatergic system, in particular, it is able to modulate the efficiency of glutamatergic transmission in the central nervous system mediated by ionotropic AMPA receptors. The chemical structure of piracetam is similar to the neurotransmitter gamma-aminobutyric acid (GABA, GABA). In relatively large doses and with repeated administration, piracetam can enhance glutamatergic inhibitory processes.

The role of glutamate as a neurotransmitter in the nervous system was established in the late 50s by a group of scientists led by David Curtis and Jeffrey. In their experiments on the isolated spinal cord of a toad, scientists first observed the effects of glutamate in vivo: at a concentration of 10 ^-2 -10 ^-4 M, the substance first excited neurons, and then, like the open and previously described GABA, showed inhibitory properties. Initially, glutamate and compounds close to it in structure were called “excitatory amino acids”. In the 70s, it was suggested that the excitatory amino acid receptors are divided into NMDA and non-NMDA. AMPA receptors - a subtype of ionotropic glutamate receptors capable of transmitting positively charged ions, was named AMPA in honor of the selective agonist - alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid.

Agonists promote the opening of ion channels coupled to the AMPA receptor. Depending on how deep the conformational changes the antagonists cause in the ligand-binding domain, the "pocket" S1 - S2, full or partial agonists are distinguished. The degree of desensitization of the AMPA receptor following these changes, respectively, can be complete or partial. Agonists are characterized by high therapeutic doses, a short interaction time with an agonist-binding site, and a quick transfer after interaction with the AMPA receptor biological target to a desensitized state.

Positive potentiation (or modulation) of AMPA receptors was found in the process of studying the mechanism of action of nootropics, when the ability of aniracetam to modulate the function of AMPA receptor was established, thereby enhancing glutamatergic neurotransmission. This fact gave impetus to the development of a new direction in neuropharmacology: the creation and study of the mechanism of action of substances that are positive modulators of AMPA receptors (RAM AMPA).

There are two mechanisms of action of individual AMPA RAMs. The difference is expressed in the fact that some PAM AMPA prevent receptor deactivation, while others block desensitization. Effective AMPA receptor potentiators have been found that can affect both deactivation and desensitization despite the fact that these processes are different. Both processes play a significant role in the work of the AMPA receptor, since its functional state is mediated by the nature of synaptic transmission and, as a result, stimulation of the processes of memory formation and improvement of cognitive functions.

The most important neurophysiological aspect of the action of positive AMPA RAMs is the so-called synaptic plasticity. One of its consequences is the effect of prolonged potentiation, which is considered as one of the main mechanisms of neuronal memory. In addition, the basis of the therapeutic potential of RAM AMPA is their ability, due to depolarization of the postsynaptic membrane, to significantly increase the expression of neurotrophic factors - NGF (nerve growth factor) and brain neurotrophic factor BDNF (brain-derived neurotrophic factor), which, in its First of all, it is associated with the improvement of cognitive functions and is a powerful mechanism for the restoration of nerve cells.

It is assumed that indications for the use of the drug, a positive modulator of AMPA receptors, but not an agonist or antagonist, will include acceleration and improvement of the quality of convalescence after brain disasters, since the acute phase and the phase of convalescence are provided by various pathogenetic mechanisms.

The progress of modern pharmacology and clinical medicine is largely determined by the discovery of new biologically active substances. This is especially relevant for neuro- and psychopharmacology, which is largely due to the steady increase in the importance of nervous and mental pathology as one of the leading causes of morbidity, disability and mortality, especially in developed countries. One of the most relevant and attracting increased attention in recent years groups of neuro- and psychopharmacological agents are the above-described nootropics.

The main fundamental feature of the action of these agents is the effect on the biochemical processes that underlie the implementation of intellectual-mnestic functions, i.e. regulation of cognitive processes, learning, memory - the basis of higher human nervous activity. A unique aspect of the action of nootropics is the fact that the scope of their application includes not only various forms of nervous and mental pathology, but also situations of prolonged psychoemotional stress. The leading place among the pharmacological effects of nootropics is also occupied by cognitive, cerebroprotective and stress-protective effects, which determines the appropriateness of their use under certain conditions in relatively healthy individuals. Another extremely important aspect of the action of nootropic drugs is the possibility of correcting with their help age-related disorders of metabolic processes in the brain during aging, which serve as the foundation for the development of various forms of age-related neuropsychic pathology. Representatives of none of the other groups of neuro- and psychopharmacological drugs do not possess such a comprehensive multilateral spectrum of pharmacological activity.

Currently, nine main structural classes of positive allosteric modulators (RAMs) of AMPA are distinguished: pyrromycin derivatives; derivatives of benzoylpiperidines / pyrrolidines; derivatives of benzothiadiazines; derivatives of piperonyl, non-dioxodanecarboxylic and quinoxaline acids; benzofurazan derivatives; derivatives of biarylsulfonamides; sulfonamide derivatives; derivatives of isothiourea; derivatives of 3,7-diazabicyclo [3.3.1] nonane (bispidine).

In the patent RU 2276992 C2 (priority of July 12, 2004), a composition having nootropic activity is described. The active substance contains a substance of plant origin, and the drug itself is similar in action to the Ginsana drug available on the market. However, the drug has a rather delayed effect and has significant contraindications and side effects.

Patent RU 2177783 C1 (Priority 02.02.2001) describes the composition of a nootropic agent in the form of a syrup. The disadvantage of this form is reduced storage stability, difficulties with dosing and low activity of the active substance compared to other forms when it comes to nootropic action.

There is a drug "Nootobril" in the form of a capsule, its composition is presented in patent document RU 2203660 C1. The disadvantage of this drug is its narrower range of prescriptions.

In addition, there are more than 100 preparations containing Piracetam on the market, however, their effectiveness does not vary much depending on the form and composition, and the main emphasis in the development of new compositions and forms is on the stability of the active substance. Despite the saturated market of Piracetam, the drug has a spectrum of side effects, among which headache and tremor of the limbs cause significant inconvenience.

Despite a number of drugs with a nootropic effect that exist on the market, the use of nootropics in a wide range of relevant nosologies remains an urgent topic, as well as optimizing the production and increasing the availability of the drug in the treatment and prevention of memory impairment, dizziness, decreased concentration, emotional lability, and dementia due to cerebrovascular accidents (ischemic stroke), brain injuries in Alzheimer's disease in old age, to improve cognitive performance after suffering brain damage, for the treatment of withdrawal symptoms and psycho-organic syndrome in chronic alcoholism and learning disabilities in children. In addition, there is currently no dosage form for the derivative of 3,7-diazabicyclo [3.3.1] nonane on the market and the optimal composition is not presented.

Accordingly, it is an object of the present invention to provide, on the basis of the 3,7-diazabicyclo [3.3.1] nonane derivative, a domestic drug of nootropic effect, stable during storage, and having good solubility due to the qualitative selection of excipients and the quantitative content of the ingredients.

The technical result to which the claimed invention is directed is the creation of a pharmaceutical composition having a nootropic effect in capsules, characterized by good solubility and storage stability.

The technical result is achieved by developing a pharmaceutical composition including, in addition to the active substance, 6- [4methoxy-3- (1H-pyrazol-1-ylmethyl) benzyl] -1,11dimethyl-3,6,9-triazatricyclo [7.3.1.1] tetradecane- 4,8,12-trion-bicyclononan, excipients - sodium alginate, pregelatinized corn starch (PAC), sodium starch glycolate, colloidal silicon dioxide and calcium chloride, in the following ratio, wt. %

The closest analogue for comparing formulations according to the principle of action is Piracetam.

A pharmaceutical composition in the form of capsules containing, as an active ingredient, the pharmaceutical substance 6- [4methoxy-3- (1H-pyrazol-1-ylmethyl) benzyl] -1,11 dimethyl-3,6,9-triazatricyclo [7.3.1.1] tetradecane- 4,8,12-trion-bicyclononan (Z-109), belongs to the pharmacotherapeutic group of nootropic drugs, belongs to a new class of ampakines - derivatives of 3,7-diazabicyclo [3.3.1] nonane with a tricyclic skeleton, possessing the properties of RAM AMPA and provides prolonged activation of AMPA receptors and the production of neurotrophic factors. Accordingly, the most likely mechanism of action of the studied drug is similar to the mechanism of action of ampakines: allosteric binding to glutamate receptors such as AMPA (AMPK), as well as the expression of neurotrophic factors BDNF and NGF, triggering mechanisms responsible for the survival of existing neurons, as well as the growth and differentiation of new neurons and synapses.

The pharmaceutical substance of bicyclononan and a method for its preparation are disclosed in patent RU 2480470 C2, (priority from 04/06/2011), which describes tricyclic derivatives of 3,7-diazabicyclo [3.3.1] nonanes, potentially possessing pharmacological activity. This security document discloses the structure of a compound having a nootropic effect.

According to the task, the forms and compositions of the medicinal product were experimentally proposed and examined in order to obtain a quantitative and qualitative composition in such a form that would make it possible to obtain a stable preparation with good solubility, a pronounced specific effect with a minimum of adverse and allergic reactions.

Another objective of the invention is the expansion of the line of domestic drugs with nootropic effect over a wide range of purposes.

In the process of studying the compositional options, it was unexpectedly discovered that the substance of bicyclononan has low solubility, therefore, it was decided to choose hard capsules as the dosage form. The developed composition for capsules after a series of experiments made it possible to establish range quantitative characteristics for the selected excipients. As a result, the action of the active component of the pharmaceutical composition was enhanced by experimentally selected composition, in particular, its solubility was improved, and bioavailability was increased. An additional advantage of the developed medicinal product was the opportunity to achieve a relatively low cost of production on an industrial scale, as well as to show a high environmental level by implementing a virtually waste-free production of the finished dosage form.

In the process of manufacturing a pharmaceutical composition in the form of hard capsules, the pharmaceutical substance undergoes gentle processing, which relates to the biopharmaceutical benefits of this dosage form. It is extremely important that the technological stage does not have a pressing stage, which eliminates the negative impact on the release of the pharmaceutical substance from the dosage form. The main auxiliary substance in the formulation is sodium alginate as a filler and granule former, which is characterized not only by good technological characteristics, but also by its beneficial effect on the mucous tissue. The use of ethyl alcohol as a binder component allows to increase the solubility of the pharmaceutical substance in the aquatic environment, which positively affects the bioavailability of the drug. In addition, the technology for producing the finished dosage form of the pharmaceutical composition of the bicyclononan in the form of a capsule is distinguished by the simplicity of the instrumentation, low energy consumption of the process, the technology is practically waste-free, harmful compounds that pollute the environment are not used in the production process.

The results obtained in experimental studies indicate the promise of using the pharmaceutical composition of the bicyclononan in the form of capsules for the treatment of cognitive impairment and rehabilitation of patients undergoing acute brain hypoxia due to ischemia and other brain injuries. The developed composition in the studies showed high safety indicators.

The importance of the study of the composition is determined by the fact that up to 50% of problems with the technology and quality of the finished forms (granule flowability, tabletting process, disintegration and solubility of solid oral forms, etc.) depend on the components of the dosage form.

It is well known that compliance with pharmacopoeial requirements for active substances and excipients is not enough to ensure the stability of the processes and the desired pharmacokinetic properties of many dosage forms. The physicochemical and biological properties of the pharmaceutical substance that can affect the effect of the product and its manufacturability are determined and evaluated.

Thus, capsules have been developed as a dosage form. The composition of the auxiliary substances has a synergistic effect with the active substance, which is confirmed in the above examples of the invention. The claimed composition and form solve all the tasks.

Example 1. The choice of dosage form.

Currently, the most popular solid oral dosage forms are tablets and capsules. The technology of tablet pharmaceutical compositions is widely used by manufacturers. However, its features should be taken into account: multi-stage, a large number of technological excipients in the formulations, the impact on the pharmaceutical substance of stressful technological conditions such as moisture, high temperatures, pressing, which can lead to polymorphic transitions, as well as negatively affect the stability and effectiveness of the pharmaceutical substance.

Capsules have undeniable marketing, biopharmaceutical and technological advantages. In addition to their attractive appearance, the capsules mask the unpleasant taste and smell of the pharmaceutical substance, provide high accuracy of dosing, and most importantly, the pharmaceutical substance is not subjected to aggressive technological influence in the production of capsules, which positively affects the stability and effectiveness of the pharmaceutical composition. The biopharmaceutical capabilities of modifying the release of a substance from capsules allow it to be varied in a wide range from fast to phase and prolonged. Manufacturers also note the cost-effectiveness of the production of encapsulated pharmaceutical compositions compared to the production of tablets by reducing the number of process steps and types of production equipment needed.

Currently, the most common capsules are gelatin - a natural product of the hydrolysis of collagen containing raw materials, which is a waste of the food industry: meat and fish processing plants. Currently, it is widely used in the food industry [James Swarbrick. Encyclopedia of Pharmaceutical Technology Third Edition New York London edited by PharmaceuTech, Inc. v. 1 Brian E. Jones. - Capsules, Hard. - p. 406-418 and The two-piece gelatin capsule Handbook. - Qualicaps. - 2011. - 43 p.].

A gelatin molecule is a polypeptide, the molecule of which consists of amino acids, including glycine (about 30%), proline, hydroxyproline, alanine, glutamic and aspartic acids. Therefore, gelatin has undoubted advantages: it is biocompatible, easily digestible and does not give adverse reactions. Gelatin amino acids have an effect on the body: improve metabolism, increase mental performance and strengthen the heart muscle, have a beneficial effect on the tissues of the musculoskeletal system, gastrointestinal mucosa in case of erosion and peptic ulcer and are one of the main sources of energy of the central nervous system, muscles and the brain. Moreover, gelatin is used in medicine as a plasma replacement drug [Geloplasma balance, Gelofuzin, Gelatinol, https://www.medsovet.info/herb/5695].

It should be noted that gelatin, being a natural product, is easily absorbed by the body, quickly dissolves in the acidic environment of the stomach (which is facilitated by the presence of gastric pepsin protease) and releases the contents of the capsules, thereby facilitating the rapid entry of pharmaceutical substance into the body.

The disadvantages of gelatin include the following:

- the polypeptide structure of gelatin determines its susceptibility to microbial contamination;

- the walls of the capsules are very sensitive to changes in moisture content. Both increasing and decreasing the moisture content of the capsules adversely affect the properties of the pharmaceutical composition;

- gelatin, being a raw material of animal origin, has restrictions on use for certain categories of consumers, such as vegetarians, as well as religious or ethnic groups that comply with dietary laws prohibiting the use of certain products of animal origin;

- gelatin can pose a threat to human health in connection with the appearance of serious animal diseases, such as spongiform encephalopathy, swine flu, etc.

The disadvantages inherent in gelatin capsules stimulated the search for alternative materials for the dosage form. An alternative material, in addition to the lack of the above disadvantages of gelatin, should have similar physicochemical properties for the possibility of using the developed technologies and the existing machine park without serious redesign. It must have proven safety and approval for pharmaceutical use. HPMC (Hypromellose) has become a successful alternative material for hard capsules, which are currently on the market [“The two-piece gelatin capsule Handbook. “Qualicaps.” - 2011. - 43 p.].

Hypromellose is obtained by the synthetic modification of cellulose and is considered safe for human consumption [Technical Reference File Hard Gelatin Capsules, 3rd edition. - CAPSUGEL. - 2010. - 55 p., And N.B. Demina, M.C. Demin Development of the technology for the production of capsule forms of drugs. Chapter in the monograph. Pharmaceutical development. Concept and practical recommendations. Moscow Publishing House Feather. - 2015. S. 196-236.]. It is practically insoluble in hot water, in acetone, in anhydrous ethyl alcohol and in chloroform, but it dissolves in cold water with the formation of a colloidal solution, and exhibits reversible temperature gelation.

Modification of the HPMC molecule by methoxy and hydroxy propoxy groups allows one to obtain products with variations of many properties, for example, gelation temperature, viscosity, elasticity, hydration.

This makes it possible to create products with a modified release and with increased resistance to storage conditions and mechanical processing.

HPMC is widely used in the pharmaceutical industry as coatings for tablets and bases for visco-plastic forms: gels and creams. HPMC is well compatible with known excipients and has gelatin-like properties. The advantage is that HPMC meets the dietary and cultural needs of all patients. Meets production requests - capsules can be manufactured and filled on existing equipment. It is characterized by proven safety information and is approved for pharmaceutical use. In addition, it provides improved characteristics of capsules, their strength, protection against moisture from microbial contamination, and high compatibility with products.

Hypromellose capsules are more stable against moisture than gelatine capsules. The moisture content of the capsules themselves is 6-7%, which is lower than the moisture content of gelatin capsules, which is 12-13%. This is extremely important for hygroscopic pharmaceutical substances and for the conditions of filling, transportation, storage of both empty capsules and dosage forms. However, since HPMC capsules appeared on the market of the Russian Federation recently, there are practically no publications on their properties and use when creating the drug.

Thus, since bicyclononan, as was established experimentally, is very slightly soluble in water, not tablets, but hard capsules were chosen as the dosage form. In the process of obtaining tablet pharmaceutical compositions, the substance is subjected to stressful technological influences (moisturizing, drying, pressing), which can negatively affect its already low solubility, and possibly subsequently bioavailability. As mentioned above, in the process of manufacturing a pharmaceutical composition in the form of hard capsules, the pharmaceutical substance undergoes gentle processing, which is attributed to the biopharmaceutical benefits of this dosage form. It is extremely important that the technological stage does not have a pressing stage, which excludes its negative effect on the release of the pharmaceutical substance from the dosage form.

Example 2. Research and selection of capsule filling form.

Traditionally, a pharmaceutical substance in the form of a powder or granules is placed in hard capsules. In the case of a small dosage of a pharmaceutical substance, in order to ensure the accuracy of dispensing by a capsule filling machine, excipients (diluents) are introduced into the formulations: mannitol, lactose, microcrystalline cellulose, corn starch, and other traditional excipients approved for pharmaceutical use.

To ensure the encapsulated mass is slipping relative to the working parts of the capsule filling machine, gliding substances are introduced into its composition in small amounts (often 1-3%): magnesium stearate, glyceryl monostearate, stearic acid, talc, aerosil, etc. [NB Demina, M.S. Demin Development of the technology for the production of capsule forms of drugs. Chapter in the monograph. Pharmaceutical development. Concept and practical recommendations. Moscow Publishing House Feather. - 2015. S. 196-236.].

The encapsulated mass is compressed during filling and, therefore, disintegrants are often present in the formulations: croscarmellose, corn starch, crospovidone, starch, alginic acid, sodium glyceryl starch, etc.

The choice of excipients is closely related to the technology of preparing the capsule mixture for filling; to select and justify the preparation technology, the technological properties of the pharmaceutical substance were first studied.

Since dosing into capsules is automatic, the powdered contents must first of all have good flowability so that the powder can move by gravity from the hopper of the machine to the dosing device. Flowability, in turn, depends on many characteristics of the powder: on the fractional composition, amorphousness and humidity of the substance. Since flowability is a complex characteristic, it is customary to determine it in two ways: by the rate of outflow from a standard funnel and by the angle of repose. The angle of repose is the angle between the generatrix of the cone of bulk material and a horizontal plane. The slope angle characterizes the effect of friction between particles and the effect of gravity on the powder mass, gives an estimate of the influence of particle size, shape and electrostatic interaction between particles during the precipitation of powder from the hopper of the capsule filling machine. The angle of repose depends on the shape, size and cohesive properties of the particles, it varies widely: from 25 ° to 30 ° for well-flowing materials, from 60 ° to 70 ° for poorly flowing materials.

Like other characteristics of bulk materials, bulk density is a complex indicator that depends on many others: particle size distribution, humidity, density of laying in the layer. It is important that this is not a constant value, it can change under the influence of vibration and even when stored in a warehouse. Therefore, the minimum bulk density of a freely sprinkled powder is distinguished from the maximum bulk density after compaction, for a powder sprinkled freely, but subjected to compaction by shaking. Bulk density is closely related to the volume of the capsule, the number of which is selected depending on the mass of the contents of the capsule.

Flowability and bulk density, being important characteristics of the powder mass in pharmaceutical technology, determine the rhythm and speed of the capsule filling machine, and therefore its performance. In addition to technological conditions, flowability also affects the homogeneity of the mass and the density of the encapsulated mass. This characteristic is complex and depends on many parameters: the shape and topography of the surface of the powder particles, fractional composition, humidity, and largely determines the bulk density of the powder mass.

According to the results of studies of the technological characteristics of the substance of bicyclononan, which are reflected in Table No. 1, data were obtained on the low technological properties of the substance. The flowability was only 1.5-1.7 g / s, while not all of the powder spontaneously spilled out of the funnel of the device, part of it hung. Corresponds to low friability and angle of repose, it is equal to 42-48 degrees. In addition, the powder is dusted and electrified. These characteristics can only be assessed as unsatisfactory. Based on the calculated values of the Carr and Hausner indices, the flowability of the substance is rated as “very poor”.

Table 1 - The results of determining the technological characteristics of the substance of bicyclononan obtained as a result of measurements of 6 samples.

Given the low dosage of the substance, which necessitates the introduction of a significant amount of filler, sliding and disintegrant into the composition of the dosage form, the powder mixture must be granulated before filling into capsules.

Example 3. The study of the granulation method.

The low technological characteristics of the pharmaceutical substance, its small dosage, small particle sizes, their amorphous nature, and volatility served as the basis for granulation of the encapsulated mixture. The goals that are achieved by granulation technology are to increase the flowability and ensure uniformity of the content of all components in the powder mixture.

The granulation methods currently used in production, depending on the consistency of the binder, are carried out with or without wetting of the powder mass. Dry granulation implies the use of significant compression or melting forces, which cannot be considered appropriate in the case of a pharmaceutical substance insoluble in the aquatic environment due to the possible loss of its pharmacological activity. Of the wet granulation methods, granulation with a high shear mixer-granulator is most acceptable. The formation of granules occurs when the powder mixture is moistened with a gluing solution, which is fed using a nozzle. The resulting agglomerates roll on the surface of the rotating bottom, made in the form of a plate. In the process, they build up layer by layer with a granular mixture and at the same time, the surface of the granules formed is polished. The obtained granules are durable, have a smooth surface. Side grinders are provided for leveling the fractional composition. In some cases, still wet granules are calibrated using an external nozzle. Z-shaped vanes of a disk rotor provide intensive mixing.

Compared with the traditional punching granulation scheme, the use of a granulator-mixer reduces energy costs, production areas, and product cost. Using a scientifically based selection of excipients as a result of applying this technology, it is possible to minimize the amount of a humidifier, as a result of which it is possible to exclude the granulate drying stage from the technological process, which is accompanied by adverse effects of temperature on the active pharmaceutical ingredients and cumbersome instrumentation of the processes of air preparation and air purification, as well as a decrease in the duration and energy intensity of the process of mixing the moistened mass.

Despite the many developed and applied granulation technologies, new methods are currently being proposed with the goal of minimizing the disadvantages of the known methods. One of these technologies has become moisture activated granulation (VAG).

VAG is designed as an alternative to wet granulation, in which a significant amount of granulating liquid is used, and the granulated material is not only negatively affected by moisture, but also goes through the stage of thermal drying. On the one hand, this can be detrimental to labile biologically active substances and, on the other, it makes the process more expensive, since it requires significant energy consumption and the use of several types of special equipment.

The main role for conducting VAG is played by the correct choice of excipients, which should promote the formation of agglomerates when tiny drops of a humidifier get into a powdery mixture. As the latter, it is necessary to use liquids with a low viscosity, which will contribute to fine atomization. From a technological point of view, a good moisturizer is ethyl alcohol, which has a low viscosity, as well as water, which, with a reasonably selected formulation, can be used to granulate even hydrophobic PSs [N. B. Demina Development of technology for the production of tablets Chapter in the monograph Pharmaceutical development. Concept and practical recommendations. Moscow, Publishing House Perot. - 2015.S. 83-134.].

Previously, at the Department of Pharmaceutical Technology, Federal State Autonomous Educational Institution of Higher Education First MGMU named after THEM. Sechenova of the Ministry of Health of Russia (Sechenovskiy University) formulations were developed for conducting VAG of dry plant extracts and crystalline substances [M.V. Hojava, NB Demina, S.A. Skatkov, V.A. Kemenova Technological aspects of moisture-activated granulation. Pharmacy. - 2013. - No. 4, - p. 34-36.]. Based on previous studies on other objects, in this study, we studied as fillers: beckoning, lactose monohydrate, microcrystalline cellulose, corn starch; as disintegrants - crospovidone, croscarmellose sodium; colloidal silicon dioxide is used as a release agent.

Example 4. The study of the composition and selection of excipients.

As a result of a multivariate experiment, a formulation of the bicyclononan dosage form was developed. Three model compositions of capsule masses were prepared using different excipients, which included bicyclononan and excipient in various combinations. During the experiment, the compositions were evaluated by the following indicators: flowability, angle of repose, bulk density before compaction and after compaction, uniformity of dosage. The results are shown in table No. 2. The flowability of sample No. 1 compared with the substance increased from 1.7 to 5.93 g / s, the angle of repose was 35 degrees, bulk density before compaction increased from 0.15 to 0.40 g / ml, bulk density after compaction - from 0.24 to 0.55 g / ml, which indicates the advisability of using capsule composition No. 1 for the pharmaceutical composition. For the first time, sodium alginates were used for the BAG of bicyclononan in the present study, which showed the best results not only by the above criteria, but also to achieve the maximum solubility level of the substance.

The optimal composition indicating the qualitative and quantitative composition for capsules of any shape is presented in the following form, wt. %:

Bicyclononan - 6-8%,

Sodium Alginate - 60-62%,

Pregelatinized Corn Starch (PAC) - 15-17%,

Sodium starch glycolate - 3-5%,

Silicon dioxide colloidal - 3-5%,

Calcium chloride - 3-5%.

Selected excipients provide the necessary volume and compactness of the contents of the capsule.

Example 5. Preparation of a pharmaceutical substance for filling into capsules. Granulation.

With VAG, a humidifier is introduced into a dry mixture of ingredients (pharmaceutical substance, excipients, dry binders) with stirring by fine spraying to form agglomerates - granules. The resulting moistened granules with continuous stirring are dusted with silicon dioxide, which sorb and redistribute moisture in the resulting product, "dries" it. For uniform redistribution of moisture, the granules are simply left for some time in a closed container in a normal room, the drying operation is no longer necessary, which is an undoubted advantage when working with labile substances in conditions of high humidity and temperature.

Comparison of the process conditions of wet and moisture-activated granulation is presented in table No. 3.

The resulting granules have a close fractional composition, high flowability and compressibility. Their moisture content corresponds to the moisture content of granules obtained by standard technology of wet granulation. If necessary, at the final stage, disintegrants and moving ones are added.

Description of granulation technology. Excipients: sodium alginate, pregelatinized starch and sodium starch glycolate were mixed, the substance of bicyclononan was added and again thoroughly mixed, a humidifier was added from the nozzle to form agglomerates, and then calcium chloride was added to stimulate granulation, and it was dusted with pre-sieved 0.3 mm sieve with openings. After unloading from the granulator, the product was left to redistribute moisture in a closed bin at room temperature for 10-12 hours.

Humidifier selection. For VAG, it is advisable to use low viscosity humidifiers. The simplest, cheapest, most environmentally friendly and technologically safe granulating agent is purified water. Its main drawback is hydrolysis processes occurring during wetting. During the experiment it was found that the formation of granules when the powder mixture is moistened with purified water, in which the substance is insoluble, proceeds only due to pregelatinized corn starch and partially sodium alginate. The resulting product is represented by particles of irregular shape, among which there were both granules of excipients and free particles of a pharmaceutical substance.

When using 95% ethanol as a granulating liquid, in which the substance is readily soluble, the granules have a more rounded shape and a smooth surface, which favorably affects the flowability of the product; there are no particles of the substance in it. In this case, not only excipients, but also a pharmaceutical substance take part in the formation of granules. As a result of dissolving the pharmaceutical substance in a humidifier (95% ethanol), when applied to a mixture of the powdered components of excipients, the product is more uniform. The technique used will ensure uniform dosage, which is extremely important in the manufacture of dosage forms with a low dosage of the substance. The amount of moisturizing liquid used in the granulation process was precisely determined during the experiment for each composition and did not exceed 5% of the total mass of the powder mixture.

The results of determining the technological characteristics of the granulate obtained as a result of measurements of 6 samples are shown in table No. 4.

The obtained results indicate good technological properties of the granulate: flowability compared to the substance (results in Table No. 1) increased from 1.7 to 5.9 g / s, bulk density before compaction increased from 0.15 to 0.44 g / ml , bulk density after compaction is from 0.24 to 0.51 g / ml, which indicates the advisability of using capsule No. 3 for the dosage form. Based on the calculated values of the Carr and Hausner indices, the flowability of the substance is rated as “good”. Thus, the quality indicators of the granulate allow us to conclude that the developed technology of granulation and the suitability of the obtained product for further research are rational.

According to the results of the study, it was possible to develop the optimal composition and dosage form in the form of capsules, which have such advantages as: high bioavailability, production technology sparing for the pharmaceutical substance, high patient compliance, etc. The developed dosage form meets modern requirements for pharmacopoeial quality indicators, including in terms of release kinetics: according to the results obtained, over 45 minutes, 77.6 ± 2.5% of the substance passes into the dissolution medium with a pH of 4.5, which corresponds to the pharmacopoeial norm. It should be taken into account the important point that the substance itself is very slightly soluble in water, an increase in its solubility is achieved as a result of the proposed technology for the preparation of the dosage form and the developed optimal composition.

Example 6. A study of the pharmacokinetics of a drug in a single and multiple administration.

With a single administration, the study was conducted as open prospective, with the inclusion of animals in various groups to study pharmacokinetics and bioavailability. According to the results of the selection of animals, a sequential set of four groups of rats was made. A total of 270 male rats were used (Table 5).

18-20 hours before the study, the animals were deprived of food with open access to water. The drug was dissolved in DMSO, then in a 2% starch gel, and was administered orally with an atraumatic probe in a volume of 2 ml. The drug was dissolved in DMSO, then in 0.9% saline, administered intravenously in a volume of 1 ml.

Before blood sampling, rats were placed in a CO ₂ chamber until breathing was completely stopped. Blood was taken from the jugular vein 1 ml with EDTA at the following time points: 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 hours. There were 5 animals per time point. Also, at a dose of 25 mg / kg in animals, organs were taken (brain, thymus, heart, lungs, liver, kidneys, spleen) to study the distribution of the drug at the following time points: 0, 0.25, 0.5, 0.75, 1, 1.5, 2 , 3, 4, 6, 8, 12, 24, 36, 48 hours. Plasma was obtained from blood in the usual way: centrifugation at 3500 rpm at 4 ° C and storage at minus 20 ° C in 1 ml aliquots.

During the entire experiment, including the numbers and its biological part, no differences were observed in the behavior, appearance, and manifestations of physiological functions in experimental rats before and after they received the drug.

When determining the test drug in rat blood plasma, standard techniques known from the prior art to a general specialist were used. Solutions were prepared: 0.1% solution of TFU in acetonitrile, 0.1% solution of TFU in water, 50% solution of acetonitrile. A stock solution of CO was prepared. Calibration range for bicyclononan: 0.1-100 mcg / ml. The test solution was prepared. Plasma samples were placed in a 10-ml volumetric flask, 9 ml of the solution was added and mixed until the components were completely dissolved, the volume was adjusted to the mark with solution 3. 100 μl of the resulting solution was placed in a 10 ml flask and the solution 3 was added to the mark. 100 μl of the resulting solution was placed in a 0.5 ml microtube, centrifuged for 2 min at 14000 rpm to precipitate insoluble components. The supernatant was taken for analysis.

A blank solution was prepared: 130 mg of sodium alginate, 35 mg of pregelatinized corn starch, 10 mg of sodium starch glycolate, 10 mg of colloidal silicon dioxide and 10 mg of calcium chloride were placed in a 10 ml glass, 9 ml of solution 3 was added and mixed until the components were dissolved maximally. . The resulting solution was transferred to a flask with a capacity of 10 ml, the volume of the solution was brought to the mark with solution 3, and mixed. 100 μl of the resulting solution was placed in a flask with a capacity of 10 ml and brought solution 3 to the mark. 100 μl of the resulting solution was placed in a 0.5 ml microtube, centrifuged for 2 min at 14000 rpm to precipitate insoluble components. The supernatant was taken for analysis.

A model solution was prepared. 15 mg of a standard sample of bicyclononan, 130 mg of sodium alginate, 35 mg of pregelatinized corn starch, 10 mg of sodium starch glycolate, 10 mg of colloidal silicon dioxide and 10 mg of calcium chloride were placed in a 10 ml glass, 9 ml of solution 3 was added and mixed until maximum dissolution components. The resulting solution was transferred to a flask with a capacity of 10 ml, the volume of the solution was brought to the mark with solution 3, and mixed. 100 μl of the resulting solution was placed in a flask with a capacity of 10 ml and brought solution 3 to the mark. 100 μl of the resulting solution was placed in a 0.5 ml microtube, centrifuged for 2 min at 14000 rpm to precipitate insoluble components. The supernatant was taken for analysis.

Figure 1 presents the experimental data for determining the concentration of the test drug in rat plasma samples at different times after a single oral administration at doses of 2.5 mg / kg, 13 mg / kg and 25 mg / kg. Individual values of the concentration of the drug in plasma after a single oral administration are presented in Tables No. 6-8.

Values of pharmacokinetic parameters were obtained from experimental data in an automatic mode using the program of the PK Solver Excel module (Table No. 9).

After a single oral administration of the drug to rats in three increasing doses of 2.5 mg / kg, 13 mg / kg and 25 mg / kg, the linear pharmacokinetics of the drug was studied. Based on the obtained data, a hypothesis of linear pharmacokinetics of the drug was put forward. To test this hypothesis, the statistical significance of the deviation from zero of the free member of the linear regression AUC (0-t) was evaluated. The calculation is shown in Figure 2. The results showed that the free term is slightly different from zero and the hypothesis should be considered true.

Individual values of the concentration of the drug in plasma after a single intravenous administration are presented in Table No. 10. Figure 3 presents the experimental data for determining the concentration of the test drug in rat plasma samples at different times after a single intravenous dose of 13 mg / kg. Values of pharmacokinetic parameters were obtained from experimental data in an automatic mode using the program of the PK Solver Excel module (Table 11).

The relative bioavailability of the drug after oral administration was 42.3% relative to the intravenous administration of the drug. An analysis of the concentrations and distribution of the drug in the organs of animals was also carried out. The main pharmacokinetic parameters of the drug in rat organs after a single oral administration at a dose of 25 mg / kg are presented in Table 12.

The analysis showed that the greatest retention time (MRT) of the drug was in the following organs: thymus (6.09 h), heart (3.81 h) and brain (3.76 h). The half-life (T1 / 2) of the drug was the largest for the following organs: thymus (5.20 h), brain (4.17 h) and spleen (2.24 h).

The half-life (T1 / 2) of the drug in doses of 2.5 mg / kg, 13 mg / kg and 25 mg / kg after oral administration was 4.49 hours, 4.31 hours and 4.23 hours, respectively. The time to reach the maximum concentration of Tmax was 1 h for all doses when administered orally. The average retention time (MRT) of the drug at doses of 2.5 mg / kg, 13 mg / kg and 25 mg / kg after oral administration was 5.12 hours, 4.79 hours and 4.61 hours, respectively. The apparent total clearance (Cl / F_obs) of the drug in doses of 2.5 mg / kg, 13 mg / kg and 25 mg / kg when administered orally was 0.07 (mg / kg) / (μg / ml) / h, 0 08 (mg / kg) / (μg / ml) / h; 0.09 (mg / kg) / (μg / ml) / h, respectively.

With repeated administration, the study was conducted as open prospective, with the inclusion of animals in various groups to study pharmacokinetics and bioavailability. According to the results of the selection of animals, a sequential set of one group of rabbits was produced. A total of 6 male rabbits were used.

18-20 hours before the study, the animals were deprived of food with open access to water. The drug was dissolved in DMSO, then in a 2% starch gel, and was administered orally with an atraumatic probe in a volume of 5 ml at a dose of 13 mg / kg. Animals received the drug every 24 hours for 6 days (144 hours).

Blood was taken from the central ear vein 1 ml EDTA at the following time points: 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 25, 28, 48, 49, 52, 72, 73, 76, 96, 97, 100, 120, 120.25, 120.5, 120.75, 121, 121.5, 122, 123, 124, 126, 128, 132, 144 hours.

At one time point there were 6 animals. Plasma was obtained from blood in the usual way: centrifugation at 3500 rpm at 4 ° C and storage at minus 20 ° C in 1 ml aliquots.

When determining the investigational medicinal product in the blood plasma of rabbits, standard techniques known from the prior art to the average specialist and similar to the previously described principle were used.

Figure 4 presents the experimental data for determining the concentration of the test drug in rabbit plasma samples at different times after repeated oral administration at a dose of 13 mg / kg.

The individual values of the concentration of the drug in plasma after repeated oral administration are presented in table No. 13. Values of pharmacokinetic parameters were obtained from experimental data in an automatic mode using the program of the PK Solver Excel module (table No. 14).

A comparison of the pharmacokinetics of the studied medicinal product after repeated administration to rabbits at a dose of 13 mg / kg with a dosing interval of 24 hours did not show an acceleration in the elimination of the drug from the body after repeated administration. The total visible clearance did not change, the average retention time (MRT) decreased by only 1.06%. The maximum concentration created in plasma immediately after the next injection was 40.92 μg / ml and did not significantly differ from the maximum concentration in the first 24 hours of the experiment. It can be concluded that repeated administration of a drug at a dose of 13 mg / kg does not affect its basic pharmacokinetic parameters and there is no cumulation of the drug in the body.

Example 7. Acute and chronic toxicity.

In experiments on laboratory animals, the acute toxicity of a drug based on bicyclononane derivatives was studied. In acute experiments in rats and mice (males and females), the test drug was administered orally at doses of 600, 1300 and 2000 mg / kg and intraperitoneally at doses of 600, 1300 and 2000 mg / kg.

In all acute experiments, the studied drug did not cause a deterioration in the condition of the animals. Spontaneous motor activity of animals was similar to the control group. Death was not observed at all doses in both animal species. In this regard, the calculation of lethal dose indicators was not possible (LD50 more than 2000 mg / kg). The acute toxicity experiment did not allow the determination of lethal doses for the study drug.

There was no lag in the increase in body weight of animals (mice and rats) that received the study drug from that in animals of the control group.

During the study, no deviations in hematological and biochemical parameters were recorded in all the studied groups in relation to the control. Thus, experiments in mice and rats indicate the absence of a toxic effect on the blood formation and biochemistry of the studied drug. Microscopic examination of the organs of mice and rats in an acute experiment in all groups of animals showed no deviations from the clinical norm at the injection site of the studied medicinal product.

The mucous membranes and viscera maintained integrity. Analysis of necropsy did not reveal pathological changes in internal organs. The research results showed that all injected doses of the studied medicinal product do not cause side effects in animals.

During the study of chronic toxicity of the studied medicinal product, there was no influence on the dynamics of the gain in mass of laboratory animals compared to the control. An analysis of the data showed that chronic administration of the investigational medicinal product does not reduce motor and cognitive activity compared with animals in the control group at a dose of 25 mg / kg. However, at a dose of 2.5 mg / kg and 13 mg / kg, motor and cognitive activity in animals was significantly increased, compared with the control.

Urinalysis data from rats and rabbits indicate the absence of pathological changes in the kidneys after 30 days of drug administration. In the urine of animals of the experimental group (25 mg / kg — rats and 10 mg / kg — rabbits), erythrocytes and protein are not detected, which indicates the integrity of the filtration apparatus of renal nephrons. The analysis of the presented data did not reveal the toxic effect of the studied drug on the blood system of rats and rabbits in a chronic experiment. Fluctuations of all quantities were within the physiological norm.

A biochemical study of the blood serum of rats and rabbits of the experimental groups that received the study drug did not reveal a statistically significant change in enzyme activity compared with the control group. All values of biochemical parameters varied within the physiological norm for these animal species.

Assessment of the level and nature of pathological changes in the internal organs (systems of internal organs) of experimental animals (rats, rabbits) did not reveal pathological changes.

Histological analysis of the injection site and adjacent tissues of the studied medicinal product did not reveal pathological changes. The epithelium lining the esophageal, cardiac, fundal and pyloric parts of the stomach is a normal structure. The submucosa is formed by loose connective tissue with a significant content of elastic fibers. The small intestine has 3 layers: the mucosa and submucosa, muscle and serous membrane. The mucous membrane of the small intestine forms villi and crypts, in its composition goblet cells and Packet cells, in the stroma lymphoid infiltration with an admixture of eosinophils, the submucosa is well vascularized. In the large intestine in the mucosa, crypts covered with goblet cells, in the stroma, more pronounced lymphoid infiltrates, up to the formation of lymphoid follicles. Thus, the test drug does not have a local irritant effect.

A pharmaceutical composition based on bicyclononan derivatives does not affect the functions of internal organs and vital systems of the body; does not have the effect of delayed body reactions and toxic side effects that may occur with the clinical use of high doses of the drug.

The results of studies on rats show that a pharmaceutical composition based on bicyclononan derivatives with repeated oral administration does not have reproductive toxicity: it does not affect the reproductive (generative) function of females and males and does not have embryo- and fetotoxic effects recorded in the antenatal and postnatal periods development

Thus, it can be concluded that the drug does not have an immunotoxic effect, does not worsen the proliferative activity of antibody-producing cells, phagocytosis, macrophage activation - the main components of an active immune response. A study of the immunological safety of the investigational medicinal product suggests that, with respect to immunological safety, the investigational medicinal product is safe in the range of doses tested.

As a result of studying the allergenic effect of a drug based on bicyclononan derivatives: conjunctival tests in guinea pigs are negative; test of general and active anaphylaxis - negative; skin applications are negative. Therefore, this study showed that the test drug lacks allergenic properties in the range of doses tested.

Example 8. The study of the specific activity of the drug.

The study used five experimental groups of animals, ten males and ten females e ”in each group. The experiments were conducted during the chronic administration of the test drug and the reference drug (Piracetam, 200 mg / kg). The drug was dissolved in DMSO, then in a 2% starch gel and was administered in a volume of 2 ml to rats. Animals were randomly assigned to groups using body weight as a criterion, so that the individual weight of the animals did not differ by more than 10% from the average weight of animals of the same sex.

The effect of the drug on learning in the methodology of the conditioned reflex of passive avoidance (passive avoidance reaction)

The basic model for assessing the effect of substances on the formation and reproduction of a commemorative trace is normal is the development of a conditioned passive avoidance reflex (passive avoidance reaction). In this test, we used an installation consisting of two compartments: darkened and illuminated, connected by a door. During training, the animal was placed in a bright compartment (tail to the door). As soon as the animal passed into the dark compartment, the door was closed, and in the compartment along the mesh metal floor, an electric current of 0.5 mA and a duration of 3 seconds was applied to the legs. After the door opened. Thus, the animal was trained not to enter the dark compartment. When playing the test, the latent period was measured - the period when the animal passively avoided the dark compartment. During the day, animals were trained. A control test was performed 24 hours after the betrothal.

Study of the effect of the drug on the input and initial processing of information

To obtain data on the effect of the drug on the input process and the initial processing of information, the drug and the comparison drug were introduced before the training procedure, and the trained was tested after 24 hours. The animal was in the installation for 180 seconds. Piracetam at a dose of 200 mg / kg was used as a comparison drug.

As a result of statistical analysis, a significant difference from the control was observed, both in the comparison drug group and in the studied drug groups at all doses (p <0.05). The maximum effect was observed in the study drug group at a dose of 13 mg / kg (animals did not leave the light compartment for 180 seconds), as well as in the comparison drug group. It is worth noting that males and females showed similar results in this test.

A study of the effect of a drug on the process of extracting information.

To obtain data on the effect of the drug on the process of extracting information, the drug and the comparison drug were administered immediately before the reflex was reproduced (24 hours after training, a test was performed for 25 hours). Piracetam at a dose of 200 mg / kg was used as a comparison drug.

As a result of statistical analysis, a significant difference from the control was observed, both in the comparison drug group and in the studied drug groups at all doses (p <0.05). The maximum effect was observed in the study drug group at a dose of 13 mg / kg (animals did not leave the light compartment for 180 seconds), as well as in the comparison drug group. It is worth noting that males and females showed similar results in this test.

The effect of the drug on learning in the method of conditioned reflex with positive reinforcement

The conditioned food reflex is produced in animals, usually in a T-shaped labyrinth. Rats with food deprivation for 48 h were placed in the starting compartment of the T-shaped labyrinth, in one of the arms of which a feeder with food was placed. 30 s after landing, the start compartment door was opened. The sound signal served as a conditioned stimulus. The running time of the animal from the starting compartment to the feeder was recorded.

During the first day, animals were trained. After, tests were carried out for 4 days. Animals received the test drug 1 hour before the test. Piracetam at a dose of 200 mg / kg was used as a comparison drug.

As a result of statistical analysis, a significant difference from the control was observed, both in the comparison drug group and in the groups of the studied drug at all doses (p <0.05) at all time intervals. On the 4th day of the test, the maximum effect was observed in the group of the studied medicinal product at a dose of 13 mg / kg, as well as in the comparison drug group. No significant differences between females and males were found.

Influence on the strategy of behavior in the research radial maze.

This test was carried out in 3 stages.

• 1 stage (Addiction), 4 days:

Four sleeves of the labyrinth were discovered, in each lay a bait. The experimental animal was placed in the central compartment. The stage ended when the rat found all 4 baits or after 15 minutes.

• Stage 2 (Training), 10 days.

Eight sleeves of the labyrinth were discovered, in each lay a bait. The experimental animal was placed in the central compartment. The stage ended when the rat found all 8 lures; or after 15 minutes.

• 3 stage (Checking working memory), 10 days.

This stage consisted of two parts. In the first part 4 sleeves of the maze were discovered, in each lay a bait. The experimental animal was placed in the central compartment. The first part of stage 3 ended when the rat found all 4 baits. After the animal was removed from the maze for 5 minutes. In the second part, all 8 arms of the labyrinth were opened, the bait lay in 4 arms, which were closed in the first part of 3 stages. The stage ended when the rat found all 4 baits or after 15 minutes. Each repeated entry into the sleeve, where the bait was located in 1 part of 3 stages, was counted as an error. The results were calculated using the following formula:

where A is the number of errors, B is the total number of entries into the arms.

All groups of animals received the test drug and the comparison drug for 14 days, during stages 1 and 2, orally. The animals received the test drug 1 hour before the test in step 3. Piracetam at a dose of 200 mg / kg was used as a comparison drug.

As a result of statistical analysis, a significant difference from the control was observed, both in the comparison drug group and in the groups of the studied drug at all doses (p <0.05) at all time intervals. In the groups of the studied medicinal product and the comparison drug, positive dynamics are visible, animals by 10 days of testing were significantly less mistaken in comparison with the control and in comparison with the initial time points of stage 3. The maximum effect (animals were the least mistaken) was observed in the study drug group at a dose of 13 mg / kg, as well as in the comparison drug group. No significant differences between females and males were found.

Recognition of new objects in the process of research behavior.

The open field test was carried out in 2 stages. At stage 1, 2 identical objects were placed on the open field site. The animal was placed in the center of the arena for 3 minutes. At stage 2 (after 24 hours) one of the objects was replaced, the animal was placed in the arena for 3 minutes. The recognition index was calculated as the ratio of time spent near an old subject to the time that an animal spent near a new subject. All animal groups received the test drug and the reference drug for 7 days, orally. On day 8, the animals received a pharmaceutical composition 1 hour before the start of stage 1. On day 9, animals received a pharmaceutical composition 1 hour before the start of stage 2.

As a result of statistical analysis, a significant difference from the control was observed only in the groups of the studied medicinal product at all doses (p <0.05). The highest index was observed in the groups of the studied medicinal product at doses of 2.5 and 13 mg / kg. No significant differences between females and males were found.

The cruciform labyrinth test was carried out in 2 stages. At stage 1, two identical objects were placed in two arms of a cross-shaped labyrinth opposite each other. The animal was placed in the center of the maze for 3 minutes. At stage 2 (after 24 hours) one of the objects was replaced, the animal was placed in a maze for 3 minutes. The recognition index was calculated as the ratio of time spent near an old subject to the time that an animal spent near a new subject. Animals received a drug and a comparison drug (Piracetam, 200 mg / kg) 1 hour before the start of stage 1. On the 9th day, the animals received a pharmaceutical composition and Piracetam 1 hour before the start of stage 2.

As a result of statistical analysis, a significant difference from the control was observed both in the groups of the studied medicinal product at all doses, and in the comparison drug group (p <0.05). The highest recognition index was observed in the groups of the studied medicinal product at doses of 2.5 and 13 mg / kg. No significant differences between females and males were found.

Morris Water Maze.

The Morris water labyrinth was a circular pool (150 cm in diameter) filled with opaque water, into which a small platform (10 cm in diameter) was immersed, not visible to the animal (rat).

The test protocol consisted of five training sessions, five attempts each. Sessions were held at the same time after 24 hours. Animals were placed from the edge of the pool. The maximum duration of the first session was 90 s. In the event that the animal could not independently find the platform, the experimenter helped the animal. After the platform was discovered, the animal was on it for 30 s. The next 4 sessions in duration were 60 s. If the platform was not found, the animal was taken out of the pool and was outside the boundaries of the maze for 30 s. The location of the platform for the entire duration of the test did not change and was in the opposite sector from the start of the animal sector at the border of the tigmotaxis zone. The time during which the animal found the platform and climbed onto it was recorded.

Before each training session, the animals received the studied pharmaceutical composition and the comparison drug (Piracetam, 200 mg / kg) 1 hour before the test.

As a result of statistical analysis, a significant difference from the control was observed in the groups of the studied medicinal product at 5 sessions at a dose of 2.5 mg / kg and at 4 and 5 sessions at a dose of 13 mg / kg. No significant differences between females and males were found.

The study of antihypoxic properties of the drug.

Occlusion of the middle cerebral artery (OSMA) was performed in anesthetized (“Zoletil 100”, intramuscularly) rats. A hole with a diameter of about two millimeters was made at the site of the suture of the zygomatic bone with the frontal, in order to see the intersection of the middle cerebral artery with the inferior cerebral vein. Under the microscope, a special metal hook was brought under the left middle cerebral artery. Using the coagulator, the middle cerebral artery was occluded proximal to the site of its bifurcation on the frontal and parietal branches. In the field of view of the microscope, a cessation of blood flow through the middle cerebral artery above the site of occlusion was observed. All surgical operations were performed under sterile conditions.

The test drug and the reference drug Piracetam (200 mg / kg) were administered to rats 4 hours after surgery and then every 24 hours for 21 days. 2 hours before surgery, animals were trained in the conditioned reaction of passive avoidance (passive avoidance reaction). The reflex was reproduced on days 1 and 21 after the operation. Neurological status on the severity scale of neurological deficit (NSS) also served as a criterion for the effectiveness of the drug. This test evaluates the animal’s ability to perform 10 different tasks (table 15), which evaluate basic reflexes, as well as the ability to move and maintain balance. One point is assigned for failure to complete the task, so a normal healthy animal should have 0 points for all points on the scale. Neurological status was determined in animals at 1, 7, 14 and 21 days after surgery.

Interpretation of results:

From 7 to 10 points - severe damage to the central nervous system;

From 4 to 6 points - moderate damage to the central nervous system;

From 1 to 3 points - slight damage to the central nervous system;

Figure 5 shows the neurological status of animals in dynamics, in which reversible occlusion of the middle cerebral artery was simulated.

As a result of statistical analysis, a significant difference from the control was observed, both in the comparison drug group and in the studied drug groups at all doses (p <0.05) by 21 days. The maximum effect (lowest neurological status) was observed in the study drug group at a dose of 2.5 mg / kg. No significant differences between females and males were found.

Figure 6 presents the results of the passive avoidance reaction test at different time points (1 and 21 days). In all groups, positive dynamics is visible (by 21 days, latent time in all groups is increased). The maximum effect (the greatest latent time) was observed in the group of the studied medicinal product at a dose of 2.5 mg / kg. No significant differences between females and males were found.

Example 9. Photoinduced thrombosis.

To reproduce one-sided photochemical damage to the cerebral cortex of the rat, the animal’s head, which was under anesthesia (flying, intramuscularly), was fixed in the head holder of the SR-5R stereotactic device (Narishige, Japan). A unilateral focal ischemic focus in the prefrontal (fields Fr1 and Fr2) cortex of rat brain using the atlas G. Paxinos and C. Watson was created by photochemical thrombosis.

Photochemical thrombosis is achieved using a TTG001-100 laser with a luminosity of 100 mlV / cm ² . A 532 nm light beam was brought to the skull surface at a distance of 0.5 cm. Immediately prior to irradiation, anesthetized animals were injected intravenously (into the jugular vein) with a 3% solution of the Bengal Pink dye, at a dose of 40 mg / kg. The exposure time was 15 minutes. As a result of the interaction of the fluorescent dye with a light beam, free oxygen is released, which damages the vascular endothelium, which leads to adhesion and aggregation of platelets, the formation of blood clots and impaired local blood flow. False operation was carried out according to the same scheme, with the exception of the intravenous administration of the Bengal pink dye. All surgical operations were performed under sterile conditions.

The test drug and the reference drug Piracetam (200 mg / kg) were administered to rats 4 hours after surgery and then every 24 hours for 21 days. 2 hours before surgery, animals were trained in the conditioned reaction of passive avoidance (passive avoidance reaction). The reflex was reproduced on days 1 and 21 after the operation. Neurological status on the severity scale of neurological deficit (NSS) also served as a criterion for the effectiveness of the drug. Neurological status was determined in animals at 1, 7, 14 and 21 days after surgery.

Figure 7 shows the neurological status of the animals in dynamics, in which the focal ischemic focus was simulated by photochemical thrombosis.

As a result of statistical analysis, a significant difference from the control was observed, both in the comparison drug group and in the studied drug groups at all doses (p <0.05) by 21 days. The maximum effect (lowest neurological status) was observed in the study drug group at a dose of 2.5 mg / kg. No significant differences between females and males were found.

Figure 8 shows the results of the passive avoidance reaction test at different time points (days 1 and 21). In all groups, positive dynamics is visible (by 21 days, latent time in all groups is increased). The maximum effect (the greatest latent time) was observed in the group of the studied medicinal product at a dose of 2.5 mg / kg. No significant differences between females and males were found.

Example 10. Global brain ischemia.

Global cerebral ischemia was performed by complete ligation of both carotid arteries. It is known that after ligation of two carotid arteries in rats, the blood pressure in the willis circle of the brain drops to 40 mm Hg. Art. Developing ischemia is accompanied by mass adhesion of leukocytes to the walls of the cerebral venules and the smallest veins. Two hours after ligation, the blood pressure in the vessels of the willis circle drops to 16-20 mm RT. Art. The number of acts of adhesion increases dramatically. ”In these vessels, leukocyte conglomerates are formed, which cause complete vascular occlusion and death of animals. To simulate global brain ischemia, the following was done:

• Conducted anesthesia (“flying 100”, intramuscularly)

• Made a longitudinal incision in the neck (2-3 cm)

• Identified the left and right common carotid arteries

• Conducted electrical coagulation of the branches of the common carotid artery

• Ligated the left and right common carotid arteries.

The test drug and the reference drug Piracetam were administered to rats 4 hours after surgery and then every 24 hours for 21 days. 2 hours before surgery, animals were trained in the conditioned reaction of passive avoidance (passive avoidance reaction). The reflex was reproduced on days 1 and 21 after the operation. Neurological status on the severity scale of neurological deficit (NSS) also served as a criterion for the effectiveness of the drug. Neurological status was determined in animals at 1, 7, 14 and 21 days after surgery.

Figure 9 shows the neurological status of animals in dynamics in which brain ischemia was modeled by bilateral occlusion of the carotid arteries.

As a result of statistical analysis, a significant difference from the control was observed, both in the comparison drug group and in the studied drug groups at all doses (p <0.05) by 21 days. The maximum effect (lowest neurological status) was observed in the study drug group at a dose of 2.5 mg / kg. No significant differences between females and males were found.

Figure 10 shows the results of the passive avoidance reaction test at different time points (days 1 and 21). Positive dynamics is visible only in the groups of the studied medicinal product (latency is increased by 21 days). The maximum effect (the greatest latent time) was observed in the group of the studied medicinal product at a dose of 2.5 mg / kg. No significant differences between females and males were found.

Modeling global brain ischemia by bilateral occlusion / reperfusion of rat carotid arteries.

To create a reperfusion model of ischemic damage, clips were applied to both common carotid arteries for 1 h, after which the blood flow through the common carotid arteries was restored, achieving reperfusion.

The test drug and the reference drug Piracetam were administered to rats 4 hours after surgery and then every 24 hours for 21 days. 2 hours before surgery, animals were trained in the conditioned reaction of passive avoidance (passive avoidance reaction). The reflex was reproduced on days 1 and 21 after the operation. Neurological status on the scale of the severity of neurological deficit (NSS) also served as a criterion for the effectiveness of the drug. Neurological status was determined in animals at 1, 7, 14 and 21 days after surgery.

Figure 11 shows the neurological status of animals in dynamics, in which brain ischemia was modeled by bilateral occlusion / reperfusion of the carotid arteries.

As a result of statistical analysis, a significant difference from the control was observed, both in the comparison drug group and in the studied drug groups at all doses (p <0.05) by 21 days. The maximum effect (lowest neurological status) was observed in the study drug group at a dose of 2.5 mg / kg. No significant differences between females and males were found.

Figure 12 shows the results of the passive avoidance reaction test at different time points (days 1 and 21). Positive dynamics is visible only in the groups of the studied medicinal product and the comparison drug (by 21 days the latent time is increased). The maximum effect (the greatest latent time) was observed in the group of the studied medicinal product at a dose of 2.5 mg / kg. No significant differences between females and males were found:

The study of the effect of the drug on the input process and the initial processing of information on the passive avoidance reaction model showed a positive effect for the studied drug in all doses (2.5 mg / kg, 13 mg / kg and 25 mg / kg). The maximum effect was observed in the group of the studied medicinal product at a dose of 13 mg / kg. Similar results were obtained when studying the effect of a drug on the process of extracting information, on learning in the method of conditioned reflex with positive reinforcement, and on the behavior strategy in the research radial maze.

Recognition of new objects in the process of research behavior in an open field, cruciform labyrinth and Morris water labyrinth showed that the studied drug improved object recognition and increased the efficiency of the research behavior of rats. The best results were also obtained with the use of drugs at a dose of 13 mg / kg. The less effective use of drugs in high doses is probably associated with the activation of glutamatergic inhibitory processes.

In a model of unilateral photochemical damage to the cerebral cortex of a rat, drugs eliminated neurological deficit, while the maximum effect was also observed when using the drug at a dose of 2.5 mg / kg

The study of drugs on the model of global cerebral ischemia reproduced by bilateral occlusion of rat carotid arteries revealed its ability to contribute to the restoration of the brain after damage. Similar data were obtained using drugs for global cerebral ischemia reproduced by bilateral occlusion / reperfusion of rat carotid arteries. The greatest effect was observed in the groups of the studied medicinal product at a dose of 2.5 mg / kg.

Example 11. Impact on the reproductive system.

The mutagenic effect of the drug was studied by accounting for chromosomal aberrations in the bone marrow cells of 1/57 C57B mice with a single dose of 5 and 50 mg / kg and repeated oral administration at a dose of 5 mg / kg. The positive control group was injected once intraperitoneally with cyclophosphamide at a dose of 20 mg / kg. As a result of the study, in the groups receiving the test drug at doses of 5 and 50 mg / kg, there was no significant increase in the number of cells with chromosomal aberrations relative to the negative control groups, the number of cells with chromosomal aberrations in animals did not exceed 2%. The qualitative characteristics of cell damage after exposure to the analyte also did not change: the bulk of all rearrangements were chromatid-type aberrations (terminal deletions), cells with multiple aberrations were not detected. With single and multiple oral administration of the drug, no mutagenic effect on somatic cells was detected. Drugs in the Ames test at concentrations of 0.1; 1; ten; one hundred; 1000 μg per cup does not have a mutagenic effect on indicator strains of Salmonella typhimurium.

The results of studies on rats show that the drug, when administered orally repeatedly, does not have reproductive toxicity: it does not affect the reproductive (generative) function of females and males and does not have embryo-fetotoxic effects recorded in the antenatal and postnatal periods of development.

The results of the research can be fixed by the following theses.

From the results of a study of the specific activity of the pharmaceutical composition of bicyclononan, it follows that the studied medicinal product showed properties confirming the presence of nootropic properties. The pharmaceutical composition positively influenced cognitive activity in various models, and, judging by the results of use in oxygen-deficient conditions, it also increased levels of trophic factors, such as cerebral neurotrophic factor, in case of brain damage. This confirms the mechanism of action proposed on the basis of the chemical structure, and is consistent with preliminary studies. Particularly noteworthy is the dose-response effect typical of AMPA receptor modulators.

The study of specific types of toxicity (immunotoxicity, mutagenicity, reproductive toxicity and allergenicity) showed the absence of allergenic properties in the tested medicinal product in the range of doses tested: conjunctival tests in guinea pigs are negative; test of general and active anaphylaxis - negative; skin applications are negative. The drug does not have an immunotoxic effect, does not impair the proliferative activity of antibody-producing cells, phagocytosis, macrophage activation - the main components of an active immune response.

The drug at doses of 5 and 50 mg / kg does not induce dominant lethal mutations in mature, late and early spermatozoa of F1 mice (CBAxC57BL6). Mutagenic effect, in the form of increased embryonic mortality, was not detected.

A study of the mechanism of action of the drug. The effect of the 3,7-diazabicyclo [3.3.1] nonane (PS) derivative on desensitization and deactivation of receptors was investigated. It was shown that in the absence of a drug, desensitization occurred in two exponential phases, with a dominant component of 1.1 ± 0.05 ms and a minor component of 4.0 ± 0.3 ms. With the addition of a pharmaceutical substance with increasing concentration, the slower exponential components of desensitization dominated; at first, the 7 ms component began to dominate, and at high concentrations of the derivative of 3,7-diazabicyclo [3.3.1] nonane, an additional component of 30 ms appeared. Current deactivation after rapid removal of glutamate in the absence of drugs was described by single-exponential decay from 500 to 600 μs. In the presence of 0.1 mM of the 3,7-diazabicyclo [3.3.1] nonane derivative, the deactivation time constant was doubled. Over the entire range of drug concentrations, the decontamination time increased from 0.55 ± 0.19 ms in the control to 1.95 ± 0.52 ms at 5 mM of the drug. During stimulation, the relationship between deactivation and drug concentration is described by a steady decrease in the channel closure rate constant. The magnitude of the effect of the drug on desensitization was greater and occurred at a higher concentration of the drug than the decontamination effect.

Bicyclononan has little effect on kainate AMPA receptors. The pharmaceutical composition leads to a slight increase in the amplitude of the stationary state, but does not affect the peak or the temporary regime of desensitization or deactivation.

As a result of the work done, the effectiveness and safety of a drug based on bicyclononan derivatives for the rehabilitation of patients after brain damage were evaluated.

Those skilled in the art will appreciate that numerous changes and / or modifications can be made to the above described embodiments without departing from the broad general scope of the present invention. Thus, the presented embodiments should be considered illustrative in all aspects, but not limiting.

Claims (3)

1. The pharmaceutical composition in the form of a capsule for use as a nootropic agent in the following ratio of components, wt. %:

2. The use of the pharmaceutical composition according to claim 1 for the treatment and prevention of memory impairment, dizziness, decreased attention span, emotional lability, dementia due to cerebrovascular accidents (ischemic stroke), brain injuries in Alzheimer's disease in old age, to improve cognitive performance after suffering brain damage, for the treatment of withdrawal symptoms and psycho-organic syndrome in chronic alcoholism and learning disabilities in children.

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