Cortexin rectal suppositories

Abstract

The invention relates to the field of pharmaceutics and medicine, namely, to a new dosage form of cortexin, rectal suppositories, capable of normalizing brain functions and characterized by efficacy and safety comparable to an injectable form. The invention also relates to a rectal cortexin composition in a suppository form, the composition comprising cortexin in a pharmacologically effective amount and a suppository base.

Description

[0001] CORTEXIN RECTAL SUPPOSITORIES

[0002] DESCRIPTION OF THE INVENTION

[0003] Field of invention

[0004] The invention relates to the field of pharmaceutics and medicine, namely, to a new dosage form of cortexin, i.e. rectal suppositories, capable of normalizing brain functions and characterized by efficacy and safety comparable to an injectable form. The invention also relates to a cortexin composition in the form of rectal suppositories, the composition comprising cortexin in a pharmacologically effective amount and a suppository base.

[0005] Background

[0006] Cortexin is a complex of low molecular weight water-soluble polypeptide fractions isolated from the cerebral cortex of cattle and pigs, with a molecular weight of up to 15,000 Da, an isoelectric point of 3.5-9.5 (the presence of absorption bands in the pl range of 3.5 to 9.5 upon isoelectric focusing in 2-8% polyacrylamide gel), and an absorption maximum in the UV spectrum at a wavelength of 275+6 nm (RU2275924, RU2104702). Cortexin is obtained from crushed frozen brain tissue of livestock by extraction with an acetic acid solution containing zinc chloride, separation of the precipitate, treatment of the supernatant with acetone, washing the resulting precipitate with acetone, drying, followed by purification, sterilization and lyophilization of the desired product.

[0007] Currently, a parenteral preparation of cortexin in two dosages is registered in the Russian Federation: "Cortexin®, a lyophilisate for the preparation of a solution for intramuscular administration, 5 mg" (LP-No.(000620)-(RG-RU)) and "Cortexin®, a lyophilisate for the preparation of a solution for intramuscular administration, 10 mg" (LP-No. (000636)-(RG-RU)); GEROPHARM LLC is the manufacturer and holder of marketing authorizations. Cortexin® is widely used in neurological diseases and pathological processes associated with impaired brain function. The protein fractions in Cortexin® are able to penetrate directly into the brain, providing a wide range of effects and indications for therapy. Cortexin® has a nootropic (affects the impaired cortical functions of the brain, improving activity, thinking, attention), neuro protective (protects brain cells from damage), anticonvulsant effect, stimulates recovery processes in the brain, reduces the harmful effects of substances toxic to the brain [1,2].

[0008] The mechanism of action of Cortexin® is associated with its metabolic activity: the drug regulates the ratio of inhibitory and excitatory amino acids, the level of serotonin and dopamine, has a GABA-ergic effect, has antioxidant activity, and is able to restore the bioelectric activity of the brain [2]. According to the approved Prescribing Information [1], Cortexin® is indicated for use:

[0009] - as part of comprehensive therapy in adult patients with impaired cerebral circulation, traumatic brain injuries and their consequences, encephalopathies of various origin, cognitive impairments (memory and thinking disorders), acute and chronic encephalitis and encephalomyelitis, epilepsy, asthenic conditions;

[0010] - as part of comprehensive therapy in children and adolescents with reduced learning ability; cognitive impairments (memory and thinking disorders); epilepsy; psychomotor and speech retardation; various forms of cerebral palsy.

[0011] While offering a large number of advantages, injectable drugs are known to have some drawbacks associated with the difficulty of self-administration, skin penetration, as well as pronounced pain during administration. These drawbacks are especially relevant when prescribing the drug in pediatric practice. Thus, there is a need for a set of studies aimed at finding and creating a new cortexin dosage form that is more convenient in terms of the method of use, but is not inferior to the previously registered injectable form in efficacy and safety.

[0012] The prior art discloses successful intranasal administration of a solution of cortexin lyophilisate in order to normalize brain functions [3,4]. Drug delivery through the nose has a number of advantages, including the rapid onset of the pharmacological effect, the possibility of bypassing the blood-brain barrier, reducing the likelihood of side effects, as well as a quick and non-invasive administration method. However, significant disadvantages of this route are relatively rapid washing out from the mucosal surface, poor drug penetration through the nasal mucosa, mucociliary clearance and the action of proteolytic enzymes [5].

[0013] Herewith, rectal forms of cortexin are not known from the prior art. The advantages of rectal forms include rapid onset of pharmacotherapeutic effect due to the rapid release of the active substance from the base and its very rapid entry into the blood due to high absorption capacity of the rectal mucosa. Rectal administration of drugs help achieving their high blood concentration, lengthening their circulation in the bloodstream and avoiding the barrier function of the liver.

[0014] The most common rectal dosage form is suppositories. Among the rectally administered drugs, there are no known means for normalizing brain functions.

[0015] The purpose of the present invention is to expand the range of drugs containing a complex of low molecular weight water-soluble polypeptide fractions isolated from the cerebral cortex of livestock, characterized by a molecular weight of up to 15,000 Da, an isoelectric point of 3.5-9.5 (the presence of absorption bands in the pl range of 3.5 to 9.5 upon isoelectric focusing in 2-8% polyacrylamide gel), and an absorption maximum in the UV spectrum at a wavelength of 275+6 nm) (cortexin), namely, to obtain a new dosage form - rectal suppositories, capable of normalizing brain functions, characterized by efficacy and safety comparable to injectable form.

[0016] SUMMARY

[0017] In one embodiment, the invention relates to a new rectal suppository dosage form capable of normalizing brain functions, comprising, in a pharmacologically effective amount, a complex of low molecular weight water-soluble polypeptide fractions isolated from the cerebral cortex of livestock, characterized by a molecular weight of up to 15,000 Da, the presence of absorption bands in the pl range of 3.5 to 9.5 upon isoelectric focusing in 2-8% polyacrylamide gel, and an absorption maximum in the UV spectrum at a wavelength of 275+6 nm. The aforementioned complex of polypeptide fractions is known in the art as "cortexin".

[0018] "Capability of normalizing brain functions" in the context of the present invention means the ability of cortexin to improve higher brain functions, learning and memory processes, concentration, increase tolerance to various stress factors, protect neurons from damage by various endogenous neurotoxic factors, reduce the toxic effects of psychotropic substances, stimulate recovery processes in the brain.

[0019] Due to the ability of protein fractions of cortexin to penetrate the brain, cortexin in suppository form can provide a wide range of effects and indications for therapy characteristic for Cortexin® in the form of lyophilisate, namely, it has a nootropic (affects the impaired cortical functions of the brain, improves activity, thinking, attention), neuroprotective (protects brain cells from damage), anticonvulsant effect, stimulates recovery processes in the brain, reduces the harmful effects of substances toxic to the brain [1,2], while exhibiting the property of normalizing brain functions.

[0020] A "pharmacologically effective amount" in the context of the present invention means an amount of cortexin capable of normalizing brain functions.

[0021] In one of the preferred embodiments, the invention relates to a rectal suppository dosage form capable of normalizing brain functions, comprising, in a pharmacologically effective amount, a complex of low molecular weight water-soluble polypeptide fractions isolated from the cerebral cortex of livestock, characterized by a molecular weight of up to 15,000 Da, the presence of absorption bands in the pl range of 3.5 to 9.5 upon isoelectric focusing in 2-8% polyacrylamide gel, and an absorption maximum in the UV spectrum at a wavelength of 275+6 nm (cortexin), in an amount of about 0.001 to about 1.000 g, preferably 0.004 - 0.413 g, with all intermediate values included in said ranges. The amount of cortexin is specified as the target dosage per 1 suppository, taking into account the quantitative content and weight loss when drying the lyophilisate intermediate or cortexin extract.

[0022] According to Example 10, in an experimental preclinical study of the neuroprotective effects of cortexin suppositories (GP20061) at the dosages of 1.6 mg / kg, 8 mg / kg and 16 mg / kg (1 / 10 therapeutic dose (TD), 1 / 2 TD and 1 TD) in models of mental and physical retardation in rat offspring caused by toxic damage during late pregnancy, the efficacy of all dosages studied was shown. The average weight of rats in the middle of the experiment (on the 12th day of life) was 40 g. The human equivalent dose (HED) for the minimum studied dosage of 1.6 mg / kg is: . „ , , Z0.04 ke (rat weight) \0'33> >_ , ,

[0023] 1 .6 mg a / kg a x — , ° \20 kg (child — wei ^g-h2t)- / = 0.20 mg a / kg a (tfor child /

[0024] . „ / i ( 0.04 kg (rat weight) \0 33,, . , ,

[0025] 1.6 mg a / kg a x — , , - ^-2- = 0.14 mg / kg (for adult \70 kg (adult weight) / '

[0026] Accordingly, provided that the child weight is about 20 kg, the content of cortexin in one suppository should be at least 4 mg. The upper limit of the cortexin content in the suppository is limited by the maximum load of a 4 g suppository equal to 413 mg. Example 9 of the present Specification “Toxicology Study” shows the pharmacological safety of higher dosages of cortexin. The safety of these dosages has also been proven in clinical trials.

[0027] In another preferred embodiment of the invention, the weight of the rectal suppository dosage form of cortexin capable of normalizing brain functions is 0.5 to 4 g. This range of the suppository weight is based on the permissible weight of a rectal suppository according to the SP (State Pharmacopeia): for an adult, it should be in the range of 1 to 4 g, for children - 0.5 to 1.5 g. Accordingly, the content of cortexin in a suppository for adults should be in the range of 0.004 g to 0.413 g, in a suppository for children - 0.004 g to 0.155 g.

[0028] In some of the preferred embodiments of the invention, the rectal suppository dosage form capable of normalizing brain functions comprises 4 mg, 10 mg, 20 mg, 40 mg, 80 mg, 155 mg or 413 mg of a complex of low molecular weight water-soluble polypeptide fractions isolated from the cerebral cortex of livestock, characterized by a molecular weight of up to 15,000 Da, the presence of absorption bands in the pl range of 3.5 to 9.5 upon isoelectric focusing in 2-8% polyacrylamide gel, and an absorption maximum in the UV spectrum at a wavelength of 275+6 nm (cortexin), and further comprises a suppository base in an amount sufficient to obtain a suppository weighing 0.5 - 4 g.

[0029] In yet another embodiment, the invention relates to a rectal composition comprising the above complex of low molecular weight water-soluble polypeptide fractions (cortexin) in a pharmacologically effective amount and a suppository base. In one preferred embodiment, the composition comprises 0.004 - 0.413 g of cortexin per one suppository. In another preferred embodiment of the invention, the weight of the compositions is between 0.5 and 4 g. In one embodiment, the present invention relates to the composition or the suppository in which the ratio of cortexin to the suppository base is 1:4000 to 1:4, preferably 1: 1000 to 1: 10, even more preferably 1:999 to 1:8.

[0030] In yet another embodiment, the invention relates to the use of the above dosage forms or compositions to normalize brain functions.

[0031] In a further embodiment, the invention relates to the use of a complex of low molecular weight water-soluble polypeptide fractions isolated from the cerebral cortex of livestock, characterized by a molecular weight of up to 15,000 Da, the presence of absorption bands in the pl range of 3.5 to 9.5 upon isoelectric focusing in 2-8% polyacrylamide gel, and an absorption maximum in the UV spectrum at a wavelength of 275+6 nm (cortexin), for the manufacture of a medicament in suppository form.

[0032] As the suppository base, the above dosage forms or compositions may comprise a base selected from a hydrophobic, hydrophilic, diphilic base or a mixture thereof, preferably a hydrophobic base, even more preferably a diphilic base.

[0033] In a preferred embodiment, the suppository base is selected from a hydrophobic, hydrophilic, diphilic base or a mixture thereof.

[0034] Any pharmaceutically acceptable suppository bases of lipophilic (hydrophobic, fatty), hydrophilic, diphilic (mixed or water-dispersible, including emulsion and absorption) type, as well as mixtures and combinations thereof, including combinations with any pharmaceutically acceptable excipients, can be used as the suppository base.

[0035] Water-soluble and water-insoluble (including mucoadhesive) polymers, solubilizers, surfactants, absorption enhancers, prolongators, solvents, emulsifiers, structure-forming agents, thickeners, heterogeneous system stabilizers, antioxidants, preservatives, fatty acid salts and other pharmaceutically acceptable salts, as well as combinations thereof, can be used as any pharmaceutically acceptable excipients, but this list is not limited to these classes of excipients.

[0036] In one embodiment, hydrophilic bases, preferably polyethylene oxides of different molecular weights (macrogols) and mixtures thereof, as well as other bases permitted for medical use, are used as the suppository base.

[0037] In one preferred embodiment, lipophilic bases are used as the suppository base.

[0038] In particular, solid fat or a mixture of solid fats is used as the suppository base. In one of the more preferred embodiments of the invention, a solid fat of a composition that has a narrow melting range and a small difference between the temperatures of melting and solidification, is selected. The narrow range of these characteristics confers the base the property of melting without the softening stage, i.e. having a steep melting profile and having a sufficiently high crystallization rate to form a stable polymorphic modification.

[0039] In a more preferred embodiment of the present invention, lauric type fatty bases are used, which are a mixture of mono-, di- and triglycerides of saturated fatty acids with carbon chain length of C10-C18. Lauric type fatty bases are less viscous in the molten state, but are more stable, processable and plastic. The viscosity in the molten state can affect both the sedimentation rate of the API during solidification and the release rate of the API from the base.

[0040] The so-called hydroxyl value is a characteristic of solid fats. According to the invention, the solid fats employed have a hydroxyl value from 1 to 50. In accordance a contemporary view of classification of suppository bases [6], bases with hydroxyl value of more than 5 mgKOH / g are classified as diphilic bases (they are also called miscellaneous bases / water dispersible bases), namely, absorption hydrophobic (self-emulsifying) bases, rather than traditional hydrophobic bases. In other words, chemically, these bases are triglycerides, but partial glycerides contained therein are surfactants, in particular water-in-oil emulsifiers, and promote partial hydrophilization of fat. The presence of glycerides contributes to better wettability of the mucous membrane, spreading of the molten mass over the intestinal wall, increased plasticity of the base, as well as stabilization of the suspension system.

[0041] In accordance with the principles of biopharmacy, suspension method is recommended for introducing solid active pharmaceutical ingredients (API) into a suppository base, since the release of active ingredients from a heterogeneous system occurs faster than from a homogeneous one, and the interaction between substances is minimized. However, when introducing powder APIs in the form of suspensions, it is necessary to take into account the changes in the structural and mechanical properties of suppository bases which can lead to structure strengthening, an increase in total deformation time and an increase in the melting temperature. In addition, to ensure rapid release, API particles must be fully wetted with the base, even though there is no dissolution process. Optimal technological and physicochemical properties (a combination of hydroxyl value and melting point) can facilitate selecting the type of base for the proposed API combination [6].

[0042] In the manufacturing technology of suspension-type suppositories, there is no additional dissolution stage, water is excluded from the suppository, as the presence of water provides an increased risk of microbial contamination, and also accelerates physicochemical and hydrolytic processes in the system which may affect the stability of the finished dosage form.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Fig. 1 shows the overall study design. * Pathological pregnancy was simulated by administration of ethanol during the last week of pregnancy (3 administrations (at 48-h intervals) of 2 g / kg ethanol) description in the text);

[0045] ** hypoxic-ischemic brain injury - sequential modeling of ischemia (occlusion of the left common carotid artery) and brain hypoxia (placing animals in an atmosphere consisting of 92% nitrogen and 8% oxygen for 60 minutes);

[0046] *** animals were randomly assigned to experimental groups without sex-based division;

[0047] IM - intramuscular administration; PR - per rectum (rectal administration); TD - therapeutic dose.

[0048] Fig. 2 and 3 show body length and weight plotted against time (pathological pregnancy model). The data on Fig. 2 and 3 are presented as mean and a standard deviation. TD - therapeutic dose.

[0049] Fig. 4 shows neurological deficit assessment according to the mNSS scale (pathological pregnancy model).

[0050] Fig. 5 shows the animal latency to fall from a rotating rod of the RotaRod test (pathological pregnancy model).

[0051] Fig. 6 shows evaluation of horizontal locomotor activity (number of sectors crossed) in the Open Field test (pathological pregnancy model).

[0052] Fig. 7 shows evaluation of exploratory activity (number of burrow holes explored) in the Open Field test (pathological pregnancy model).

[0053] Fig. 8 shows evaluation of vertical motor activity (sum of unsupported and supported rearing events) in the Open Field test (pathological pregnancy model).

[0054] Figs. 9-14 show the time of detection (Fig. 9) and removal (Fig. 10) of a foreign object from the left paw palm, detection (Fig. 11) and removal (Fig. 12) of a foreign object from the right paw palm, the average time of detection (Fig. 13) and removal (Fig. 14) of a foreign object from the palms of the forepaws in the Adhesive Removal test (pathological pregnancy model).

[0055] The data on Figs. 4-14 are presented as median, interquartile range, maximum and minimum values; TD - therapeutic dose, *, **, *** - p < 0.05, p < 0.01, p < 0.001 when compared with the placebo group (Dunn's test).

[0056] Fig. 15 shows body length plotted against time (hypoxic-ischemic brain injury model).

[0057] Fig. 16 shows body weight plotted against time (hypoxic-ischemic brain injury model). The data on Figs. 15-16 are presented as mean and a standard deviation. TD - therapeutic dose.

[0058] Fig. 17 shows neurological deficit assessment: mNSS score (hypoxic-ischemic brain injury model). Fig. 18 shows an estimate of animal latency to fall from a rotating rod of the RotaRod test (hypoxic-ischemic brain injury model).

[0059] Fig. 19 shows evaluation of horizontal locomotor activity (number of sectors crossed) in the Open Field test (hypoxic- ischemic brain injury model).

[0060] Fig. 20 shows an evaluation of exploratory activity (number of burrow holes explored) in the Open Field test (hypoxic- ischemic brain injury model).

[0061] Fig. 21 shows the evaluation of vertical motor activity (sum of unsupported and supported rearing events) in the Open Field test (hypoxic-ischemic brain injury model).

[0062] Figs. 22-27 show the time of detection (Fig. 22) and removal (Fig. 23) of a foreign object from the left paw palm, detection (Fig. 24) and removal (Fig. 25) of a foreign object from the right paw palm, the average time of detection (Fig. 26) and removal (Fig. 27) of a foreign object from the palms of the forepaws in the Adhesive Removal test (hypoxic-ischemic brain injury model).

[0063] The data on Figs. 17-26 are presented as median, interquartile range, maximum and minimum values; TD - therapeutic dose, *, **, *** - p < 0.05, p < 0.01, p < 0.001 when compared with the placebo group (Dunn's test).

[0064] Fig. 28 shows drug blood level plotted against time at the time points of 0.5 - 2 - 6 hours, percentage of the injected dose per gram of tissue, %ID / g.

[0065] Fig. 29 shows drug brain level plotted against time at the time points of 0.5 - 2 - 6 hours, percentage of the injected dose per gram of tissue, %ID / g.

[0066] The data on Fig. 28 and Fig. 29 are presented as mean and a standard deviation. Cer. - Cerebrolysin, Cort. - Cortexin, IM - intramuscular administration, IV - intravenous administration, rect. - rectal administration.

[0067] EXAMPLES

[0068] The following exemplary specific embodiments illustrate the claimed invention, but do not limit it.

[0069] Examples 1 to 8. Preparation of rectal suppositories

[0070] For the production of cortexin suppositories, cortexin lyophilisate or extract is ground using a ball, rotary, disk or other types of mills; it is also possible to grind it during the preparation of the suppository mass using a rotary pulsating apparatus or a homogenizer.

[0071] The parameters “Particle size”, “Description” and “Microbiological purity” are monitored. All the active and auxiliary components necessary for the preparation of suppositories are weighed in the amounts specified in Table 1. The amount of the suppository base is adjusted depending on the dosage of the active substance in the suppositories. The suppository base is pre-melted at 55 to 65 °C. Then the molten base is cooled to 37.7 to 38.3 °C. The concentrate of cortexin lyophilisate or extract is prepared with a portion of the molten base, homogenized to obtain particles not greater than 100 pm, and then mixed with the remaining base to obtain a homogeneous mass at 37.0 to 45.0 °C.

[0072] The resulting suppository mass with a particle size of not greater than 100 pm is packaged in a suppository container (blister pack, metal or polymer molds) at 36.0-37.0 °C. Suppositories are cooled at up to 15 °C and stored at (5+3) °C.

[0073] Description of the obtained suppositories: torpedo-shaped suppositories, of white, yellowish-white or brownish- white color, or light yellow to yellow color, of uniform consistency. The presence of white deposit on suppository surface and the presence of an air rod or a funnel-shaped recess on the longitudinal section are allowed. A specific odor is allowed.

[0074] For the formulations developed, the values of the melting point, total deformation time, dissolution and disintegration time meet the requirements of the State Pharmacopoeia of the Russian Federation (SP XIV), namely the melting point does not exceed 37 °C, the total deformation time is less than 15 minutes, at least 75% (Q) should be released into the dissolution medium in 45 minutes, disintegration time is not more than 30 minutes.

[0075] The stability of the developed suppository formulations was studied by means of accelerated (at t of 30+2 °C, RH 65+5%) and long-term storage methods (at two temperature conditions, (25+2)°C / (60+5)% and 5 + 3 °C). The suppositories were stored in a polyethylene- laminated polyvinyl chloride film suppository container. This type of primary packaging is the most common for suppositories and meets the requirements of GPM.1.1.0025.18 "Packaging, labeling and transportation of medicinal products" for the packaging of suppositories. During storage, suppository samples were evaluated for the following indicators: "Description", “Identity", "Particle size", "Disintegration", “Mass uniformity", "Melting point", "Quantification", "High molecular weight impurities", "Uniformity of dosage units", "Microbiological purity", "Biological activity".

[0076] Based on the results of the study of the stability of suppositories, the temperature conditions of from 2 to 8 °C was recommended as the potential storage conditions of the drug, and the shelf life of 2 years was established.

[0077] Table 1. Formulations of suppositories according to Examples No. 1-8

[0078]

[0079] * The amount of cortexin is specified as the target dosage per 1 suppository, while the load of cortexin lyophilisate should take into account the quantitative content and weight loss when drying a specific batch of cortexin lyophilisate intermediate product used for the production of suppositories.

[0080] The solid fats Suppocire ("Gattefosse") used in Examples 1 to 6 are semisynthetic suppository bases isolated from coconut or palm kernel oil, whose fatty acid composition is characterized by a high content of lauric acid (C12) - up to 55%, and a minimum amount of unsaturated acids.

[0081] The bases with solid fats Suppocire NAI 25 A Pellets, Suppocire NA15 having the hydroxyl value of 20-30 mg KOH / g and 5-15 mg KOH / g, respectively (Examples 1, 3 and 4) and Suppocire AML (< 10 mg KOH / g, contains lecithin) (Example 2), belong to the diphilic bases [6].

[0082] The base which is a mixture of Suppocire NAI 5: Suppocire NAS 50 (1:4) (Example 5) is also diphilic. The base Suppocire NAS50 comprised in this mixture is characterized by a high hydroxyl value (40 - 50 mg KOH / g) due to the presence of a large number of monoglycerides which impart it hydrophilic properties.

[0083] The base Suppocire NA 0 (Example 6) essentially does not contain partial glycerides, and therefore belongs to hydrophobic bases.

[0084] Cocoa butter (Example 7) is a classic hydrophobic (lipophilic) base, a natural solid vegetable fat consisting of a mixture of triglycerides: tristearin, tripalmetin, triolein, trilaurin, triarachin. The base Macrogol 1500 (PEG-32) (“Jneos oxide”) (Example 8) is a hydrophilic base comprising polyethylene glycol 1500.

[0085] Example 9. Toxicology testing

[0086] Testing toxic properties, pharmacological safety with assessment of local tolerance of cortexin suppositories after single rectal administration to sexually mature rats

[0087] There was no intoxication pattern in male and female rats. Effects on body weight, organ weight coefficients, and individual animal behavior have not been established. No clinically significant changes were revealed in histopathological examination of organs (organ fragments) and tissues of laboratory animals. Based on the results of clinical examinations and observations, as well as pathological examination, the test drug after single rectal administration at a dose of 309 mg / kg does not have a local irritant effect. The test drug after single administration at a dose of 309 mg / kg (19TD) did not have a clinically significant negative effect on the cardiovascular and central nervous systems of male and female rats.

[0088] The average weight of the animals in the experiment was 170 grams. Based on this, the formula for calculating the human equivalent dose (HED) [7] will be as follows: nn / i / 0.17 kg (rat weight) \0 33, .n... . ..

[0089] 309 mg / kg a x I — ■ — — — — — — = 64 mg / kg (for child) \20 kg (child weight) / 6 6 Vnn / i / 0.17 kg (rat weight) \0'33n... .

[0090] 309 mg / kg x I — ■ — — — - — — = 42 mg / kg (for adult)

[0091] This method of calculating HED is described in a guidance published by the Food and Drug Administration (FDA), “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. Guidance for Industry”.

[0092] Comparative study of the toxic properties and local tolerance of cortexin suppositories after repeated 28-day rectal administration, and of Cortexin®, lyophilisate for the preparation of a solution for intramuscular administration, 10 mg (GEROPHARM LLC) after repeated 28-day intramuscular administration, with a period of delayed observation in sexually mature rats

[0093] The study was conducted on sexually mature rats of both sexes. The test articles were administered for 28 days; the period of delayed observation was 28 days. The test drug, cortexin rectal suppositories (doses of 16 mg / kg (1 TD) and 160 mg / kg (10 TD), and placebo were administered rectally using an insulin syringe without a needle, the reference drug Cortexin®, lyophilisate for the preparation of a solution for intramuscular administration (GEROPHARM LLC, Russia) (doses of 2 mg / kg (1 TD) and 20 mg / kg (10 TD)) was administered intramuscularly using syringes with a needle. The control substance (placebo) and the test drugs at maximum doses were administered in divided dose, twice a day, and at the minimum doses - once a day. To study toxic properties, the following monitored parameters were registered: intoxication pattern (clinical observation and clinical examinations), body weight, food and water consumption, individual behavior in the open field test, ECG, urinalysis, clinical blood test, hemostasis analysis, blood biochemistry, organ weight coefficients, pathological examination of internal organs, assessment of local tolerance (local irritant effect).

[0094] Based on the results of the comparative study of repeated dose toxicity, the test drug, cortexin rectal suppositories (doses of 16 mg / kg (1 TD) and 160 mg / kg (10 TD), and the reference drug, Cortexin®, lyophilisate for the preparation of a solution for intramuscular administration (GEROPHARM LLC, Russia) (doses of 2 mg / kg (1 TD) and 20 mg / kg (10 TD)), did not affect the body of laboratory animals, with the exception of changes in the administration site. The test drug, rectal suppositories at a dose of 10 TD, has a partially reversible moderate local irritant effect upon rectal administration, same as placebo. The reference drug Cortexin®, lyophilisate for the preparation of a solution for intramuscular administration, 10 mg (GEROPHARM LLC, Russia), when administered intramuscularly at a dose of 10 TD, has a reversible moderate local irritant effect.

[0095] Doses of the test drug ranging from 16 to 160 mg / kg were considered safe and did not affect the general condition of the animals. The average weight of the animals in the experiment was 250 grams. Based on this, the formula for calculating HED for a child will be as follows: . „ ( 0.25 (rat weight) \

[0096] 16 mgy / kgyx \20 , kg ( 'c ,0 33hild weight) / = 3.77 mga / kga

[0097] . - / i / 0.25 (rat weight) A0,33..

[0098] 160 mgy / kgyx \ —20 k —g— (ch —ild — weight) / = 37.7 mga / kga

[0099] The formula for calculating HED for an adult will be as follows:

[0100] 1. 6 „ 2.49 mga / kg a

[0101] .

[0102] 160 24.9 mga / kg a

[0103] Example 10. Experimental preclinical trial of the neuroprotective effects of cortexin suppositories (GP20061) (ZAO “Farm-Holding") in models of mental and physical retardation in offspring caused by toxic damage during late pregnancy or resulting from neonatal trauma in rats

[0104] Evaluation of drug efficacy on models of toxic damage or brain ischemia-hypoxia in rat offspring as a part of preclinical trials was carried out in accordance with the requirements of the Guidelines for preclinical trials of new drugs of the FSBI "Scientific Centre for Expert Evaluation of Medicinal Products" [8]. Materials and Methods

[0105] A study of the neuroprotective properties of cortexin was conducted in 2 models of mental and physical retardation in rats: CNS toxicity (oral administration of ethanol during the last week of pregnancy) or neonatal trauma (ischemia-hypoxia). The drug was administered for 20 days intramuscularly or rectally in suppository form. Treatment efficacy was evaluated using the mNSS scale, the Open Field, Rotarod and Adhesive Removal tests. Next, a histopathological examination of the brain was performed. In a separate experimental series, the concentration of test substances in the blood and brain was determined in mice.

[0106] Ethical principles

[0107] All experiments were performed in accordance with the legislation of the Russian Federation and the technical standards of the Eurasian Economic Union for good laboratory practice (GOST R 53434-2009, GOST R 51000.4-2011) and Directive 2010 / 63 / EU of the European Parliament and the Council of the European Union. The study protocol was reviewed and approved by the Regional Independent Ethics Committee (RNEC) of the Volgograd region, registration number: IRB 00005839 IORG 0004900 (OHRP), protocol No. 024 of January 21, 2022.

[0108] Characteristics of the test system

[0109] The experiment was performed on the offspring of Wistar rats (N=202) or on ICR (CD-I) mice (N=75) (FSUE Nursery for laboratory animals Stolbovaya, Moscow region). After arrival the animals were quarantined for 14 days in a separate section of the vivarium. Throughout the experiment, rats were kept in controlled environmental conditions (20-26 °C and 30-70% relative humidity), NH3 < 10 mg / m , CO2 < 0.15 vol%, with unlimited access to food and water. The light-dark cycle was 12 hours light and 12 hours dark. An air exchange mode was established, providing a change of about 15 room volumes per hour. All painful manipulations were performed under general anesthesia with a single intraperitoneal administration of 20 mg / kg zolazepam (Zolctil® 100, Valdepharm, France) + 8 mg / kg xylazine (Xyla, Interchemie, the Netherlands) or inhalation anesthesia. At the end of the experiment, the animals were euthanized in a CO2 chamber.

[0110] Study Design

[0111] The overall study design is shown in Figure 1.

[0112] The first part of the study consisted of two similar series which included the following steps: 1. Pairing of female rats with males for mating for 1 day (15 females for series 1 and 30 females for series 2).

[0113] 2. Pregnancy.

[0114] 3. Modeling complicated pregnancy (ethanol administration during the last week of pregnancy for series 1, and hypoxic-ischemic brain injury modeling in 5-day-old rat pups for series 2).

[0115] 4. Monitoring the animals during the development of pathology.

[0116] 5. Formation of groups.

[0117] 6. Treatment.

[0118] 7. Evaluation of treatment outcomes.

[0119] Modeling of developmental delay

[0120] Pathological pregnancy

[0121] In series 1, developmental delay in animals was induced by oral administration of 10% aqueous ethanol solution at a dose of 2 g / kg to females (during the last week of pregnancy). Ethanol was administered 3 times in total with 48-h intervals between administrations.

[0122] Modeling of hypoxic-ischemic brain injury

[0123] In series 2, hypoxic-ischemic brain injury was modeled in 5-day-old rat pups in 2 steps. First, irreversible occlusion of the left common carotid artery was performed under inhalation anesthesia. The duration of the surgery did not exceed 60 seconds. Next, hypoxia was modeled in rat pups by placing them for 60 minutes in an environment containing 8% oxygen and 92% nitrogen, after which they were returned to their home cage [9].

[0124] Tested objects

[0125] The tested complex of polypeptides isolated from the cerebral cortex of livestock (cortexin) was produced by ZAO “Farm-Holding" (Russia). In this study, cortexin was tested in 2 forms: rectal suppositories (155 mg) and lyophilisate for the preparation of a solution for intramuscular administration (10 mg).

[0126] Cortexin in solution form was administered at a therapeutic dose (TD) of 0.5 mg / kg (IM), and in suppository form at a TD of 16 mg / kg (1 TD, study series 1 and 2), 8 mg / kg (1 / 2 TD, study series 2) or 1.6 mg / kg (1 / 10 TD, study series 2). The drug in suppository form was administered into the rectum of immature rats in a molten form (t = 37 °C) at weight- appropriate doses. For this purpose, a dispenser with a plastic probe was used. A melted base substance was used as the placebo. Placebo was administered in an equivalent volume.

[0127] Evaluation of treatment efficacy In study series 1 and 2, the efficacy of the therapy was determined by assessing the neuropsychological parameters of animals (mNSS score, motor and exploratory activity in the Open Field test, sensorimotor functions in the Adhesive Removal test, coordination disorders in the Rotarod test), as well as by analyzing pathomorphological changes in the brain in histopathological examination. mNSS Scale

[0128] Neurological deficit in animals was assessed according to the mNSS scale (Modified Neurological Severity Scores)

[0010] . This scale included tests to identify locomotor activity disorders (muscle status and abnormal movements), sensory functions (visual, tactile and proprioceptive), reflexes and motor coordination. Evaluated parameters included: rat’s motor activity when suspended by the tail; rat’s walking pattern on a surface; motor coordination when walking on a beam; strength of reflexes (auricular reflex and corneal reflex). The maximum score was 14, the minimum score was 0.

[0129] Open Field test

[0130] In this study, the "Open Field" apparatus (Open Science, Russia) was used, which is white, circular and has a floor divided into sectors with holes. During a 3-minute test session in the Open field test, the number of sectors crossed was recorded, which was interpreted as an indicator of motor activity

[0011] . The number of events of exploring the burrow holes, as well as the number of supported or unsupported rearing events were also registered. These indicators in the Open Field test were interpreted as indicators of motor and exploratory activity natural to rodents when exploring a new territory.

[0131] Adhesive Removal test

[0132] Fine motor skills and sensitivity of the palmar surface of each of the animal's forepaws were assessed using the Adhesive Removal test

[0012] . During the test, square pieces of a fabricbased adhesive tape (5 mm2) were placed on the volar surface of the forepaws. Then, after returning to a cage (similar to the home cage), the test time taken for the animal to notice the sticker (interpreted as an indicator of sensory function), as well as the test time taken for the rat to remove the sticker (interpreted as an indicator of fine motor skills) were recorded over a 3- minute period.

[0133] Rotarod test

[0134] In the Rotarod test, the latency to fall from a rotating (25 rpm) rod of the apparatus (Neurobotics EEC, Russia) was recorded over a 3-minute period. This time, compared to the result of a group of animals without pathology, was interpreted as the severity of coordination disorders

[0013] . Morphometric analysis

[0135] Brain tissue damage was evaluated with standard methods of morphometric studies. The degree of neuronal damage was assessed according to the following procedure. The neurons were divided into three groups: normal, unchanged neurons (NN); slightly modified neurons (SN) with preservation of the nucleus, but with structural or tinctorial disorders of the components of the cytoplasm (swelling, hyperchromatosis, chromatolysis, central tinctorial acidophilia); roughly altered neurons (RN) - pronounced wrinkling, “severe change”, homogenizing change in neurons, shadow cells. Morphometry was performed by staining the sections according to the Nissl method. The relative numerical density of unchanged neurons and neurons with mild and pronounced changes was determined. Microphotography of histological preparations was carried out with an Olympus digital camera (Japan) using a MICROS microscope (Austria).

[0136] Drug distribution study

[0137] All tested drugs were iodinated using Nal25I according to a standard protocol with chloramine T

[0014] . To purify the drug from free radioactive label, gel filtration was performed on PD MiniTrap G-10 (Cytiva). The radiochemical purity of the prepared drug was controlled using thin-layer chromatography (TLC). Samples of the mixture before addition, after incubation with chloramine T, and after gel filtration were applied to a Silufol TLC plate, using 96% ethanol as the liquid phase. TLC radiography was performed on an Amersham Typhoon 5 laser scanner (GE, Cytiva).

[0138] In series 3, the content of cortexin in different forms was assessed in blood and brain after a single administration. Cerebrolysin® was used as the reference drug.

[0139] The study was conducted on female outbred ICR (CD-I) mice. At the start of the experiment, all mice were clinically healthy and examined by a veterinarian. The mice were housed in groups of 5 per cage with a 12-h light-dark cycle at a temperature of 22 °C and were fed with a standard diet. Before the start of the study, the animals were food-restricted. The study was conducted in accordance with the rules for working with laboratory animals and bioethical standards.

[0140] Experimental groups were randomly formed: mice receiving Cerebrolysin® (Cerebrolysin®, 2.5 mL / kg, IM, n = 15); mice receiving cortexin (Cortexin®, 16 mg / kg, IV, n = 15); mice receiving rectal suppositories with cortexin in two doses (8 mg / kg and 16 mg / kg, rectally, n = 15). In the study groups, animals were withdrawn from the experiment 5 mice at a time at checkpoints of 30 minutes, 2 and 6 hours after drug administration. Blood and brain samples were taken from each mouse. Weight and accumulated dose were measured for each sample. Dose accumulation was measured using a TRI-CARB 5110 TR liquid scintillation alpha- beta radiometer (PerkinElmer) with gamma vials

[0015] .

[0141] Data analysis

[0142] Statistical analysis of the study results was performed using the following software: Microsoft Office Excel 2013 (Microsoft, USA), Prism 6 (GraphPad Software Inc., USA). The Shapiro-Wilk test was used to check for the normality of distribution. Intergroup differences were analyzed using parametric or non-parametric methods depending on the type of distribution. Depending on the nature of the data, the following methods of statistical analysis were used: one-way analysis of variance (One-Way ANOVA) or Kruskal-Wallis one-way analysis of variance by ranks (kw), followed by post hoc tests (Student's t-test with Bonferroni correction or Dunn's test). For normally distributed data, results are described as mean ± standard deviation (mean+o). For non-normally distributed data, results are described as medians and interquartile ranges (median [Q1;Q3]). Differences were considered significant at p < 0.05.

[0143] Study results and discussion

[0144] Neurological deficit assessment

[0145] Pathological pregnancy model (ethanol administration during the late week of pregnancy)

[0146] In experimental series 1, all animals showed consistent increases in body length and weight throughout the experiment; there were no statistically significant intergroup differences (Figures 2 and 3).

[0147] Animals in the placebo group exhibited signs of mild to moderate neurological deficits, with a median mNSS score in this group of 7 (2-9) points, whereas in the other groups (in the intact group, in the groups treated with cortexin suppositories at the three tested dosages (1 / 10 TD, * TD and TD) and in the IM cortexin treatment group (TD)), the median score was 0 points (or 1 point in the group receiving suppositories at 1 / 2 TD) (Fig. 4). Statistically significant differences from the placebo group were observed in both the intact group and the treatment groups (p < 0.001). No statistically significant differences were found between the intact group and the treatment groups (p > 0.05), which confirms the efficacy of therapy with cortexin suppositories at all tested dosages and IM cortexin injections. No statistically significant differences were found between the groups treated with cortexin suppositories at the three tested dosages and IM cortexin injections (p > 0.05), therefore, the effect of cortexin suppositories at all tested dosages is comparable to the effect of cortexin IM injections.

[0148] In the Rotarod test, the placebo group showed a tendency towards a decrease in the latency to fall from a rotating rod compared to intact animals (Figure 5). All treatment groups showed a tendency towards an increase in the latency to fall from a rotating rod; herewith the group receiving cortexin suppositories at a dose of 1 / 10 TD showed statistically significant differences at p = 0.0177.

[0149] The experimental pathology was accompanied by a statistically significant reduction in horizontal locomotor activity of the animals in the Open Field test: the median number of sectors crossed by rats in the placebo group was 14 (9-24), while the intact rats had a statistically significantly (p < 0.01) higher value of 40 (29-49). In animals treated with cortexin both in IM injection form and in suppository form at the three tested dosages, increase of this value to 30-40 was noted, and the increase was statistically significant (p < 0.001-0.01; Figure 6) in the groups that received suppositories.

[0150] Exploratory activity (number of burrow holes explored) was also significantly reduced in the placebo group compared to the intact group (2 [1-3] vs. 6 [4-9], p < 0.001). Animals treated with cortexin both in IM injection form and in suppository form at the three tested dosages showed comparable improvement of this value (up to 4-5), resulting in statistically significant differences from the placebo group (p < 0.01-0.001; Figure 7). No statistically significant differences were found between the groups treated with cortexin suppositories at the three tested dosages and IM cortexin injections (p > 0.05), therefore, the effect of cortexin suppositories at all tested dosages is comparable to the effect of cortexin IM injections.

[0151] Vertical motor activity (supported and unsupported rearing) in animals from the placebo group was minimal, and the median sum of rearing events was 1 (0-3) vs. 15 (12-17) in the intact group. In rats from other groups (intact group, group treated with cortexin suppositories at the three tested dosages, and the IM cortexin treatment group) the number of rears (Figure 8) was statistically significantly higher than in animals from the placebo group (p < 0.001), but lower than in the intact group. No statistically significant differences were found between the groups treated with cortexin suppositories at the three tested dosages and IM cortexin injections (p > 0.05), therefore, the effect of cortexin suppositories at all tested dosages is comparable to the effect of cortexin IM injections.

[0152] In animals with experimental pathology given a placebo, significant deterioration was observed in the Adhesive Removal test (Figures 9-14): the median foreign object detection time on the left and right paws was 70 (39-180) and 64 (40-180) seconds, respectively (vs. 7 [4-22] and 4 [2-17] seconds in the intact group; p < 0.001) (Figures 9 and 11); the median average (between the left and right paws) detection time was 83 (38-180) seconds (vs. 5.5 [3-23] seconds in the intact group; p < 0.001).

[0153] The median removal time from the left and right paws was 120 (78-180) and 180 (101— 180) seconds, respectively (vs. 10 [6-93] and 11 [8-34] seconds in the intact group; p < 0.001) (Fig. 10 and Fig. 12); the average (between the left and right paws) removal time was 129.5 (97- 180) seconds (vs. 10.5 [7.5-100] seconds in the intact group; p < 0.001). Rats treated with cortexin both in IM injection form and in suppository form at the three tested dosages showed an improvement in the results of the Adhesive Removal test. Statistically significant differences from the placebo group in detection and removal times were noted in the groups treated with cortexin in solution form and in suppository form at 1 / 10 and i of the therapeutic dose (p < 0.05).

[0154] Hypoxic-ischemic brain injury model

[0155] As in the previous series, all animals showed consistent increases in body length and weight throughout the experiment; there were no statistically significant intergroup differences (Fig. 15 and Fig. 16). As in the previous series of experiments, animals in the placebo group (untreated group with pathology) exhibited signs of moderate neurological deficits, with a median mNSS score of 6 (2-7.25) points, whereas in the other groups (the intact group (untreated group without pathology), cortexin suppository treatment group and IM cortexin treatment group), the median score was 0 points, which was statistically significantly lower than in the placebo group (p < 0.001, Fig. 17), which confirms the efficacy of therapy with cortexin both in the IM injection and suppository form. No statistically significant differences were found between the cortexin suppository treatment group and IM cortexin treatment group (p > 0.05) which supports that the effect of both forms is comparable. No statistically significant differences were found between the intact group and the treated groups (p > 0.05), i.e. the treatment with cortexin both in the IM injection form and in suppository form led to recovery.

[0156] In this experiment series, the Rotarod test showed a significant decrease in the latency to fall from a rotating rod in the placebo group compared to intact animals (20 (10-28.25) vs. 52.5 (35.5-70.75), (p < 0.01) (Fig. 18). All treatment groups showed a tendency towards an increase in the latency to fall from a rotating rod, but the differences were not statistically significant (p > 0.05).

[0157] The experimental pathology was accompanied by a significant reduction in horizontal locomotor activity of the animals in the Open Field test: the median number of sectors crossed by rats in the placebo group was 21.5 (13-31), while in the intact rats, the value was statistically significantly (p < 0.001) 2.5 times higher, at 56 (36-68) (Fig. 19). Animals treated with cortexin showed an increase in this value (compared to the placebo group): 34.5 (27.75-44) (p < 0.05) and 45.5 (27.75-50), p < 0.001 for the test drug in suppository and solution forms, respectively. No statistically significant differences were found between the cortexin suppository treatment group and IM cortexin treatment group (p > 0.05), therefore, the effect of cortexin in suppository form is comparable to the effect of cortexin upon IM administration. Exploratory activity (number of burrow holes explored) was also significantly reduced in the placebo group compared to the intact group (1 [0-2] vs. 9 [7-10] (p < 0.001) (Fig. 20). Animals treated with cortexin both in IM injection and suppository form showed comparable improvement of this value (up to 5), resulting in statistically significant differences from the placebo group (p < 0.001). No statistically significant differences were found between the two treatment groups (cortexin suppositories and cortexin IM injections) (p > 0.05) which supports that the effect of tested forms of cortexin is comparable.

[0158] Vertical motor activity (supported and unsupported rears) in animals from the placebo group was minimal, and the median sum of rears was 0.5 (0-2) vs. 15.5 (11-17.75) in the intact group (Fig. 21). In rats from other treated groups the number of rears was statistically significantly higher than in animals from the placebo group (p < 0.001). These data support the efficacy of treatment with the two tested forms of cortexin. No statistically significant differences were found between the cortexin suppository treatment group and IM cortexin treatment group (p > 0.05), therefore, the effect of cortexin in suppository form is comparable to the effect of cortexin upon IM administration.

[0159] In animals with experimental pathology given a placebo, significant deterioration was observed in the Adhesive Removal test (Figures 22-27): the median foreign object detection time on the left and right paw was 61 (37.5-180) and 50 (29.5-180) seconds, respectively (vs. 9.5 [4- 15.5] and 6 [2.5-13.25] seconds in the intact group; p < 0.001), the median average detection time was 59.5 (43.38-180) seconds (vs. 7.75 [3-12.88] seconds in the intact group; p < 0.001), the median removal time from the left and right paw was 117.5 (54-180) and 180 (56.75-180) seconds, respectively (vs. 10.5 [6.25-21.5] and 13.5 [9.25-25.75] seconds in the intact group; p < 0.001), the average removal time was 124 (58.75-180) seconds (vs. 12 [8.5-23.63] seconds in the intact group; p < 0.001). In both experimental groups treated, there was a significant reduction in the adhesive tape detection and removal time in the Adhesive Removal test (p < 0.01-0.05). In the groups of cortexin in suppository form and cortexin in IM injection form, the median average detection time was 25.5 (15.38-33.13) and 22 (9.375-57.25) seconds, respectively, and the median average removal time was 33 (21.5-47.75) and 36.5 (24.13-96.38) seconds, respectively, which was significantly lower compared to the placebo group (p < 0.01-0.05). No statistically significant differences were found between the two treatment groups (cortexin suppositories and cortexin IM injections) (p > 0.05) which supports that the effect of both tested forms of cortexin is comparable. Conclusions

[0160] Cortexin suppositories and cortexin solution for intramuscular administration had a comparable effect in pathological pregnancy model (toxic effects of ethanol in late pregnancy), as well as in neonatal ischemia-hypoxia model, reducing the severity of signs of neurological deficit hypoactivity in the Open Field test and the severity of fine motor skills impairment.

[0161] The similarities in the magnitude of the neuroprotective effect upon the rectal and parenteral route of administration of cortexin allow considering the tested dosage form (suppositories) of neuroactive peptides as promising in terms of treatment of mental and physical retardation.

[0162] Histopathological examination results

[0163] In this series of experiments studying the effects of various pathogenic factors on the developing brain, extremely heterogeneous pathomorphological changes affecting various cerebral structures were noted. This heterogeneity can be attributed to the fundamentally different nature of the experimental impacts (ischemia-hypoxia and fetal alcohol syndrome) and, apparently, the varying degrees of adaptive and neuroplastic processes in the growing brains of the rat pups under these experimental conditions. Therefore, to unify and ensure the continuity of the obtained results, we primarily investigated the structural characteristics of different functional areas of the neocortex, where pathomorphological changes were consistently observed under all types of experimental impacts. The results of the study demonstrate that the most frequent forms of pathomorphological changes in nerve cells were: 1) chromato lysis of varying degrees, often accompanied by cytoplasmic vacuolization; 2) hyperchromatosis, where in its extreme form, nerve cells appeared as shrunken, dark homogeneous formations with poorly defined nuclei and nucleoli.

[0164] Naturally, the effect of pharmacological correction varied significantly depending on the nature of the pathogenic impact and the doses of the drugs used. Nonetheless, it is worth noting that the test drug cortexin, administered in various forms (rectal suppositories and lyophilisate for the preparation of a solution for intramuscular injection) and dosages, demonstrated some efficacy in reducing the severity of neurodegenerative processes.

[0165] When analyzing the neuroprotective activity of the therapy conducted in cases of fetal alcohol syndrome, we noted approximately equal efficacy of the therapy with injectable and rectal administration of cortexin at therapeutic doses (0.5 and 16 mg / kg, respectively). Our data indirectly indicate that the neuroprotective effect of rectally administered cortexin is dosedependent. The therapy with suppository forms of cortexin at doses of 1 / 2 TD or 1 / 10 TD was characterized by a lesser protective effect. A similar trend was observed upon pharmacological correction of combined brain damage in rat pups induced by general hypoxia with ligation of the left carotid artery. Injection of cortexin had a pronounced neuroprotective effect, which, compared to the placebo group, manifested in a lower number of damaged neurons in most functional regions of the cortex. Rectal administration of cortexin also significantly prevented the development of neurodegenerative changes, especially in the auditory and entorhinal cortex.

[0166] Summarizing the obtained morphological data, it can be concluded that under the influence of various negative factors on the developing brain of experimental animals, the use of cortexin, both injectable and rectal, exhibited a pronounced, largely consistent neuroprotective effect compared to placebo.

[0167] Example 11. Determination of the concentration of test substances in the blood and brain after intravenous, intramuscular and rectal administration

[0168] For cortexin formulations, regardless of the route of administration and dose, the distribution profile of the drug in brain tissues can be considered similar, depending primarily on cortexin concentration in the blood. It can also be noted that the distribution profile of cortexin, regardless of the dose and administration regimen, is close to the distribution profile of the control substance Cerebrolysin® containing peptides that effectively cross the BBB; cortexin concentrations in organs and tissues depend on its blood level. The maximum concentration of cortexin after intravenous and intramuscular administration was observed at 30 min postadministration, which aligns well with previously obtained data on the distribution of peptide drugs

[0016] . Blood concentration of the drug after rectal administration is characterized by a lower relative decline over time, with the maximum concentration also occurring at 30 min postadministration. The analysis of the distribution of drugs in organs and bedding showed that the primary route of excretion for the tested drugs is through urine within the first 2 hours postadministration. The trend of accumulation and the coincidence of the blood concentration profile of cortexin after rectal administration at doses of 8 mg / kg and 16 mg / kg suggest the presence of a linear dependence of drug absorption in the rectum on the concentration / amount of the administered drug in suppository form. Thus, by varying the administered dose of the drug when administered rectally in a suppository form, the required concentration of the drug in the blood and, consequently, in the brain can be achieved (Fig. 28 and Fig. 29).

[0169] References

[0170] 1. Package Insert - Patient Information Sheet, Cortexin®, lyophilisate for the preparation of a solution for intramuscular administration, 5 and 10 mg [https: / / grls.rosminzdrav.ru / ]. Accessed on: 01.06.24. 2. Lebedev A.A. et al. Psychopharmacology & Biological Narcology. 2006, 6 (3): 1275-1283.

[0171] 3. Buynov L. G., Sorokina L. A., Shabanov P. D. Correction of statokinetic resistance in cerebrovascular pathology with cortexin. Cerebrovascular pathology - new opportunities for low-dose neuroprotection. Collection of scientific articles. St. Petersburg, 2014, 140- 147.

[0172] 4. Patent RU2469730. Publication date 20.12.2012.

[0173] 5. Porfiryeva N. N., Semina I. I., Moustafine R. I., Khutoryanskiy V. V. Intranasal administration as a route to deliver drugs to the brain (Review). Drug Development & Registration. 2021, 10 (4): 117-127.

[0174] 6. Orlova T.V. Biopharmaceutical justification of the optimal composition, technology and methods of study of suppositories as illustrated by the pharmacological group of nonsteroidal anti-inflammatory drugs. Thesis for the degree of Doctor of Pharmaceutical Sciences in specialty 14.04.01. Kursk, 2013. 388 p.

[0175] 7. E. V. Shekunova et al. Dose selection in preclinical studies: cross-species dose conversion. The Bulletin of the Scientific Centre for Expert Evaluation of Medicinal Products, 10 (2020): 1: 19-28.

[0176] 8. Guidelines for Preclinical Trials of Medicinal Products. Edited by Mironov A.N., 2012.

[0177] 9. Rice J. E., Vannucci R. C., Brierley J. B. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Annals of Neurology. 1981, 9(2): 131-141.

[0178] 10. Morkovin E.I., Kurkin D.V., Tyurenkov I.N. The assessment of the psychoneurological impairments in rodents: Basic methods. I.P. Pavlov Journal of Higher Nervous Activity. 2018. 68: 1: 3-15.

[0179] 11. Prut L., Belzung C. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol. 2003; 463(1-3): 3-33. PMID: 12600700. doi: https: / / doi.org / 10.1016 / s0014-2999(03)01272-x

[0180] 12. Bouet V., Boulouard M., Toutain J., Divoux D., Bemaudin M., Schumann-Bard P., Freret T. The adhesive removal test: a sensitive method to assess sensorimotor deficits in mice. Nature Protocols. 2009; 4(10): 1560-1564.

[0181] 13. Monville C, Torres EM, Dunnett SB. Comparison of incremental and accelerating protocols of the rotarod test for the assessment of motor deficits in the 6-OHDA model. J. Neuroscience Methods. 2006; 158(2):219-223. PMID: 16837051. doi: https: / / doi.Org / 10.1016 / j.jneumeth.2006.06.001

[0182] 14. Hunter W. M, Greenwood F. C. Preparation of iodine- 131 labelled human growth hormone of high specific activity. Nature. 1962; 194:495-496. PMID: 14450081. doi: https: / / doi.org / 10.1038 / 194495a0 Meunier J. C. A low cost gamma-vial for counting 1251 with a liquid scintillation counter. Clin Chim Acta. 1976; 66(1): 141-144. PMID: 1261036. doi: https: / / doi.org / 10.1016 / 0009- 8981(76)90382-x

[0183] Perret P., Ahmadi M., Riou L., Bacot S., Pecher J., Poillot C., et al. Biodistribution, Stability, and Blood Distribution of the Cell Penetrating Peptide Maurocalcine in Mice. Int J Mol Sci. 2015; 16(l l):27730-27740. PMID: 26610471. doi: https: / / doi.org / 10.3390 / ijmsl61126054

Claims

CLAIMS1. A rectal suppository dosage form capable of normalizing brain functions, comprising, in a pharmacologically effective amount, a complex of low molecular weight water- soluble polypeptide fractions isolated from the cerebral cortex of livestock, characterized by a molecular weight of up to 15,000 Da, the presence of absorption bands in the pl range of 3.5 to 9.5 upon isoelectric focusing in 2-8% polyacrylamide gel, and an absorption maximum in the UV spectrum at a wavelength of 275+6 nm.

2. The dosage form according to claim 1 comprising 0.004 to 0.413 g of the complex of low molecular weight water-soluble polypeptide fractions.

3. The dosage form according to claim 1 having a weight of 0.5 to 4 g.

4. The dosage form according to claim 2 comprising 4 mg, 10 mg, 20 mg, 40 mg, 80 mg, 155 mg or 413 mg of the complex of low molecular weight water-soluble polypeptide fractions, and further comprising a suppository base in an amount sufficient to obtain a suppository of 0.5 to 4 g.

5. The rectal suppository dosage form according to claim 4, wherein it comprises a base selected from a hydrophobic, hydrophilic, diphilic base or a mixture thereof as the suppository base.

6. The rectal suppository dosage form according to claim 5, wherein it comprises, as the hydrophobic base, cocoa butter or a solid fat, the solid fat being a mixture of mono., di- and triglycerides of natural saturated fatty acids with a carbon chain length of Cio-Cis, substantially free of partial glycerides.

7. The rectal dosage form according to claim 5, wherein it comprises, as the diphilic base, a solid fat that is a mixture of mono-, di-, and triglycerides of natural saturated fatty acids with a carbon chain length of Cio-Cis, comprising a residual amount of partial glycerides and having a hydroxyl value greater than 5 mgKOH / g.

8. The rectal dosage form according to claim 5, wherein the diphilic base comprises an emulsifier.

9. The rectal dosage form according to claim 5, wherein it comprises a mixture of high and low hydroxyl value solid fats as the diphilic base.

10. The rectal dosage form according to claim 5, wherein it comprises polyethylene oxide or a mixture of polyethylene oxides of different molecular weights as the hydrophilic base.

11. A rectal composition comprising, in a pharmacologically effective amount, a complex of low molecular weight water-soluble polypeptide fractions isolated from the cerebral cortex of livestock and characterized by a molecular weight of up to 15,000 Da, the presence of absorption bands in the pl range of 3.5 to 9.5 upon isoelectric focusing in 2-8% polyacrylamide gel, and an absorption maximum in the UV spectrum at a wavelength of 275+6 nm, and a suppository base.

12. The composition according to claim 11, comprising 0.004 to 0.413 g of the complex of low molecular weight water-soluble polypeptide fractions per one suppository.

13. The composition according to claim 11, having a weight of 0.5 to 4 g.

14. Use of the dosage form according to claims 1 to 10 or the composition according to claims 11 to 13 for normalizing brain functions.

15. Use of a complex of low molecular weight water-soluble polypeptide fractions isolated from the cerebral cortex of livestock and characterized by a molecular weight of up to 15,000 Da, the presence of absorption bands in the pl range of 3.5 to 9.5 upon isoelectric focusing in 2-8% polyacrylamide gel, and an absorption maximum in the UV spectrum at a wavelength of 275+6 nm, for the manufacture of a medicament in a suppository form.

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