177 mature specific pathogen free (SPF) male SD rats, 7–10 weeks old were obtained from the Pushchino nursery of laboratory animals (Pushchino, Russia). All procedures and manipulations with animals were approved by the Committee for Control over Care and Use of Laboratory Animals of BIBCh RAS (IACUC) (protocol number 809/21 from 10.08.2021) and were carried out in accordance with the EU Directive 2010/63/EU. After receiving from the nursery, the animals underwent adaptation within 7 days. During this period, the animals were monitored for signs of deviation in health status.Animals without signs of health deviations were selected for the experiment (clinical examination was done). Animals were divided into groups using the principle of randomization, using body weight as a criterion, so that the average body weight of animals by the first day of administration did not differ statistically between groups.Each animal was assigned an individual number, according to which the animal was marked with a puncture of the auricle. The cell label indicated the group, animal number, label, study code, head full name, IACUC protocol number, group color code. During the study, the animals were kept under controlled environmental conditions in a barrier zone with a “clean” and “dirty” corridor system with controlled environmental conditions (temperature 20°C–24°C, relative humidity 30%–55%, 12-h light cycle). 08:00–20:00c“day”, 20:00–08:00—“night”, 10-fold change in air volume in the room per hour). SNIFF RI/M-H V1534-30 complete granular rodent food was autoclaved and fed ad libitum.

Polymorphs A, B, C, D, E, and F were prepared from latrepirdinedihydrochloride (JV “Organica”) as described in Patent WO 2009/111540. New polymorph E was prepared from latrepirdine free base (JV “Organica”) as described in Patent WO 2022/160066.

The preparations of six polymorphs of latrepirdine as a suspension in corn oil were orally administered to the animals for seven consecutive days, once daily at a dose of 10 mg/kg/5 mL. On the seventh day of administration, the animals were euthanized by anesthesia and subsequent exsanguination. According to the design of experiment (see Table 1), the animals were euthanized at different time points—15 min, 30 min, 60 min and 120 min after administration on the seventh day.

Individual administration doses of respective latrepirdine polymorph were prepared as follows: calculated amount of respective polymorph was weighed and mixed with corn oil in a volume that is equivalent to 5 mL/kg of an animal. Using an automatic pipette, the resulting suspension was transferred into a vial continuously stirred with magnetic stirrer and the suspension was dosed to animals as required. The preparation of doses for administration was carried out immediately before administration (no more than 3 h before administration) on the days of administration. Labeled doses ready for administration in vials were transferred to the animal holding area immediately before administration.

The preparations containing latrepirdine polymorphs were administered by gavage at a dose of 10 mg/kg/5 mL daily once a day for 7 days. The body weight of the animal was used to calculate the individual administration volume of 10 mg/kg for each latrepirdine polimorph. The administration of latrepirdine preparations was carried out according to the schedule of procedures at the same time of day (09:00–13:00). Vials with a suspension of preparations were kept on a magnetic stirrer when dosing.

On the day of necropsy, blood was taken from anesthetized animals from the inferior vena cava, placed in a test tube with sodium citrate 1:9 (1 part sodium citrate: nine parts blood). Blood was centrifuged at 21°C, 1600 g, 15 min, blood serum was collected (at least 600 µL), placed in marked Eppendorf tubes and frozen at −70°C and stored at −70°C until being analyzed. After blood sampling from the inferior vena cava, the brain was removed from the cranium, divided into two hemispheres in the sagittal direction, one of the hemispheres was frozen in liquid nitrogen and placed in labeled containers, which were stored in a freezer at −70°C until analyzed.

The concentration of latrepirdine in the liquid extracts of blood serum and brain tissue of rats was analyzed using liquid chromatography-mass spectrometer “LCMS-8050” equipped withg LC-20 Prominence chromatograph and SIL 20 Axrautosampler (Shimadzu, Japan) as described elsewhere (Nirogi et al., 2009). Polymorph Latrepirdine A substance, series No. 10121, was used as the analytical standard. Liquid extraction was used to extract the analyte. For quantitative determination, the HPLC-MS method (high performance liquid chromatography-mass spectrometry) was used with electrospray ionization and ion registration in the MRM mode (registration of multiple transitions), which allows achieving high sensitivity and selectivity of the technique. Quantitative determination of the substance content in blood serum and brain tissue was carried out by the method of an external standard or absolute calibration. Analyte identification is carried out by retention parameters and mass spectrometric data when comparing experimental data obtained for calibration and test solutions.

To 300 μL of blood serum was added 50 μL of 0.1 M NaOH solution, stirred, then 1.2 mL of methyl tert-butyl ether: hexane (80:20) was added. Thoroughly mixed on an orbital shaker for 10 min, then the sample was centrifuged at 13,000 rpm for 5 min. The top layer was taken and evaporated to dryness. To the dry residue was added 0.300 mL of a 30% aqueous solution of acetonitrile, thoroughly mixed. The solution was transferred to an autosampler vial.

A sample of brain tissue (300 mg) was homogenized using an ultrasonic homogenizer. To the homogenate was added 50 μL of 0.1 M NaOH solution, stirred. Then 1.2 mL of methyl tert-butyl ether was added, thoroughly mixed on an orbital shaker for 15 min. The sample was centrifuged at 13,000 rpm for 5 min. The top layer was taken and evaporated to dryness. To the dry residue was added 0.3 mL of a 30% aqueous solution of acetonitrile, thoroughly mixed. The solution was transferred to an autosampler vial.Chromatography conditions are shown in Table 2.

A standard solution of a standard sample (RS, latrepirdine hydrochloride) was prepared as follows: a standard sample of 5 mg was placed in a 2 mL microtube, dissolved in 1 mL of a 30% aqueous solution of acetonitrile with 0.1% formic acid (30 ACN +0.1FA), thoroughly mixed (hereinafter using a shaker for 1 min). 200 μL of standard solution was added to a microtube, 800 µL of 30 ACN + 0.1FA was added to obtain a 1 mg/mL solution. By successive dilution, solutions were obtained with a concentration of 500; 100; fifty; 10 and 5 μg/mL. To establish the calibration characteristics, samples for calibration were prepared by adding the appropriate working solutions (WS) of latrepirdine to intact rat blood blood serum to obtain concentrations of latrepirdine 1, 5, 15, 30, 60, and 75 ng/mL and the introduction of the appropriate working solutions (WS) of latrepirdine in the homogenizate of intact rat brain tissue to obtain concentrations of 3, 5, 15, 30, 60, and 100 ng per 300 mg of brain tissue. The prepared solutions were labeled and used to construct calibration curves. Chromatograms were registered and processed using the LC Solution software (Shimadzu, Japan) for quantitative processing of the analytical results.

Analytical data for concentrations of latrepirdine in blood serum and in the brain tissue were processed using the Statistica 7.1 program. Descriptive statistic approach was applied: for the concentrations of latrepirdine in blood serum and in the brain tissue, the mean value and standard deviation were calculated, which are presented in the tables below. Two-way analysis of variance factorial ANOVA (ANOVA 2) was used to establish statistically significant differences associated with the factors “time” and “drug”. To determine statistically significant differences in concentration between administered polymorphs at different time points, one-way ANOVA (ANOVA 1) and Kruskal–Wallis ANOVA analysis were used. Differences were determined at the 5% significance level (p ≤ 0.05).

The study of nootropic activity was carried out on 64 laboratory Wistar rats of both sexes (32males and 32 females), 6–8 weeks old, weighing 200–250 g. The animals were obtained from the Rappolovo nursery of the National Research Center Kurchatov Institute, Russia. All procedures and manipulations with animals were approved by the Committee for Control over Care and Use of Laboratory Animals of Federal Research Center of Kazan Scientific Center, Russian Academy of Sciences, Russia (IACUC) (protocol number two from 02.07.2022) and were carried out in accordance with the EU Directive 2010/63/EU. Laboratory animals were kept in cages for at least 3 days prior to the start of the research. During this period, the animals were monitored daily for clinical condition by visual inspection. Clinically healthy animals were transferred to the experiment. During the period of adaptation and experiment, the animals were kept in standard transparent polycarbonate cages in groups of four individuals, on the bedding; cages are covered with steel lattice covers with aft recess. The animals were given drinking water ad libitum. The animals were kept under controlled environmental conditions (temperature from 19°C to 25°C and relative humidity from 30% to 70%) and light mode (day/night) 12/12 h.

The administration of scopolamine, an antagonist of muscarinic acetylcholine receptors, causes a temporary blockade of receptors, impaired cognitive functions and cerebral metabolism, which is considered to be a reliable model of AD (Buccafusco, 2009). Scopolamine aqueous solution was prepared by dissolving weighed sample in water for injection. A solution of scopolamine was injected intraperitoneally at a dose of 1.5 mg/kg/5 mL once 30 min before testing of the passive avoidancereflex (only on the first day - the day of training).

Individual administration doses of respective latrepirdine polymorph were prepared as follows: calculated amount of respective polymorph was weighed and mixed with corn oil in a volume that is equivalent to 5 mL/kg of an animal. The mixture was triturated with aid of porcelain mortar and pestle until a homogeneous suspension is obtained. The resulted suspension was sonicated for 10 min at ambient temperature the prepared doses were administered within 3 h on the days of experiment to avoid polymorph transitions. Suspensions of the polymorphs were administered orally (per os—po) using by gavageat a dose of 10 mg/kg/5 mL daily once a day for 9 days.

The administration of scopolamine, an antagonist of muscarinic acetylcholine receptors, causes a temporary blockade of these receptors that impaired cognitive functions (Buccafusco, 2009).

For 8 days, animals received oraly latrepirdine polymorphs according to group affiliation (Table 3). Polymorph preparations of latrepirdine at a dose of 10 mg/kg were administered to experimental animal groups (groups 3–8) once a day for 7 days. The groups 1 and 2 were administered once a day with an equivalent amount of corn oil.

To study cognitive-enhancing nootropic effect of the polymorphs the passive avoidance (PA) test was performed as described by Burwell et al. (Burwell et al., 2004). The PA apparatus (Neurobotics, Moscow, Russia) consisting of two compartments (light and dark chambers) separated by sliding guillotine door was used. PA consisted of an acquisition trial on the first (training) day and a retention trial 24 h later. On the seventh day of corn oil or the test polymorph administration, the animals were placed for 5 min in the PA unit to familiarize themselves with the space in order to avoid the stress caused by the new environment. The next day (eighth day) the training phase of PA test was started. 60 min before the start of training, corn oil or the test polymorph was administered, as per the group design. The experimental groups (2–8) were injected intraperitoneally with scopolamine at a dose of 1.5 mg/kg 30 min before training. During the training phase of PA test each rat was placed in the lighted chamber. After 10 s, the door was opened, and latency to enter the dark chamber was recorded. An animal was considered to have entered the dark chamber when all fours paws were beyond the guillotine door. After entry, the door was closed. After 2 s had elapsed, a foot shock stimulus was delivered. The shock level and duration were set at 1 mA for 2 s. The rat was immediately removed back to its home cage. Twenty-4 hours later (ninth day), a retention test consisted of placing the rat back into the lighted chamber, waiting 10 s, opening the door, and the latent period (LP) of the first entry into the dark chamber was recorded. If a rat did not enter the chamber in 180 s, the trial was terminated, and the rat was removed to the home cage.

The most effective polymorph was determined.

Statistical processing of the results of a comparative study of the cognitive-enhancing activity of polymorphs was performed using the Statistics 7.1 software. Pairwise comparison using the Mann-Whitney U-test was used to identify differences between each polymorph with control and with scopolamine, as well as differences between polymorphs. The significance level was determined at p ≤ 0.05.

Mean LP values are presented in Table 10.

Analysis of the presented data allows us to say that only one of the latrepirdine forms studied, namely, polymorph E, has a statistically significant increase in LP at a dose of 10 mg/kg compared with the group of animals treated with scopolamine, but not receiving latrepirdine. It is important to note that LP values of the control group of animals (group 1) did not differ from the group that received polymorph E.Also, when polymorphs and scopolamine were compared in pairs, only polymorph E differed from scopolamine (Table 10).

Pairwise comparison of polymorphs with each other revealed that polymorph E statistically differs from all other polymorphs, exceptpolymorph C, in the direction of increasing latency into the dark chamber. The remaining polymorphs do not differ from each other (Table 11).

Thus, it can be concluded that polymorph E is most effective in comparison with other polymorphs when testing long-term memory in the PA test.