Evaluation of the transportation of kisspeptins through a blood-brain barrier after intranasal administration

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INTRODUCTION
Central nervous system (CNS) diseases are a leading cause of morbidity worldwide and are currently gaining increasing importance due to population growth, increasing life expectancy, and the COVID-19 pandemic [1]. These disorders, including neoplastic, neurodegenerative (Parkinson's and Alzheimer's diseases), and psychiatric disorders (depression, schizophrenia, and bipolar disorder), are difficult to treat pharmacologically due to the blood–brain barrier (BBB). The BBB is the microcirculatory system of the brain that tightly regulates the movement of ions, molecules, and cells between the blood plasma and the brain [2]. Although the BBB serves to protect the brain from toxins and pathogens, it also poses a significant barrier to the delivery of therapeutics, as 98% of small molecules and almost 100% of large molecules cannot penetrate the brain [3]. Thus, there remains a need to develop innovative strategies to deliver therapeutics to the brain for the treatment of CNS diseases [4]. Intranasal (IN) delivery is increasingly being considered as an alternative approach to deliver therapeutics to the CNS for the treatment of concomitant diseases. The INA route offers several advantages in drug delivery over traditional oral administration: the IN route bypasses major pharmacokinetic obstacles typically associated with oral drug delivery to the CNS, including gastrointestinal pH, enzymes, hepatic first-pass effect, renal filtration, and the BBB [5]; the nasal epithelium provides an optimal absorptive surface for drug delivery due to its high permeability, loose intercellular functional complex, and extensive vascularization [6]; the olfactory and trigeminal nerve pathways that innervate the nasal epithelium provide a direct route to the brain, resulting in increased therapeutic CNS bioavailability, decreased peripheral side effects, lower doses, and rapid onset of effect [5]; and from a patient care perspective, the IN pathway is noninvasive, easy to self-administer, and better suited for patients with movement disorders, nausea symptoms, gastrointestinal dysfunction, and/or salivary gland dysfunction (dry mouth). One promising and unexplored family in terms of intranasal delivery is the kiss1 gene-encoded peptide family, kisspeptins [7, 8]. One possible mechanism of action is that kisspeptin neurons interact with nitric oxide-producing neurons in the ventromedial hypothalamus, acting as a central regulatory node of the hypothalamic-pituitary reproductive axis [9]. Expression of kisspeptin has been identified in the limbic system, making it a participant not only in the reproductive function, but also a possible regulator of behavioral, emotional and cognitive reactions [10]. To date, there is not a single fundamental study assessing the possibility of using the intranasal route of administration for kisspeptins. In this regard, the aim of this work was to analyze kisspeptin-10 for its transport across the BBB when administered intranasally. The objectives were to evaluate the effect of different doses of kisspeptin-10 after intranasal and peripheral administration on the behavior of experimental animals. 

MATERIALS AND METHODS OF THE STUDY
Animals. The study involved 45 outbred mice weighing 20–30 g obtained from the Rappolovo nursery (Leningrad Region). The animals were kept in standard plastic cages in vivarium conditions with free access to water and food under inverted lighting from 8:00 to 20:00 at a temperature of 22 ± 2 °C.

Animal testing. Animal behavior was studied using the open field, elevated plus maze, and sexual motivation tests.
Elevated plus maze test. This test is used to identify anxiety behavior in experimental animals. Outbred mice were placed in the center of the experimental chamber — a plus maze consisting of 4 arms 30 cm long and 6 cm wide, connected at a right angle. Two arms have 30 cm high walls on both sides, while the other two are open and illuminated with diffused artificial light. The maze is located on a stand 40 cm above the floor. The following parameters were systematically recorded visually for 5 minutes: time spent in the illuminated arms; number of exits from the dark arms to the illuminated ones; time spent by mice on the central platform; number of hangings from the open arms.
Open field test.
This test was used to study the free motor activity of animals. The design was a round platform 80 cm in diameter, limited in circumference by opaque walls 30 cm high. 16 passages (burrows) were evenly distributed over the entire area of ​​the open field. The diameter of an individual burrow was 3 cm each. They were designed to reveal an element of exploratory activity in rats (burrow reflex). The illumination of the open field was 100 lux. The duration of one experiment was 5 minutes. Based on the characterological behavioral atlas for rodents, a series of motor acts and postures were extracted, the totality of which characterizes the holistic behavior in the "open field". To determine the orienting reaction, the mouse was placed in an open field, the floor of which was divided into sectors. The number of rearings on the hind legs (the vertical component of the orienting reaction), the number of crossed squares (the horizontal component), the number of sniffs (the exploratory component), grooming, freezing and bolus secretions, as well as the number of peeks into holes in the floor (burrowing behavior, reflecting exploratory activity) for 5 minutes of observation were counted.
Test "Sexual motivation". To assess sexual motivation, a setup was used consisting of 4 experimental chambers (15 × 30 cm 2 ), each of which was adjacent to a stimulating cage separated by a permeable wall [11]. A perforated partition allowed male rats to explore (smell) a potential partner (an estrus female) in the chamber, but prevented tactile interaction or copulation. The day before testing motivational behavior, all experimental animals were acclimated to the conditions of the setup for 30 minutes. Behavior was recorded as a video in a dark room under red light for 10 minutes. Between exposures, the setup and partition were cleaned with 3% hydrogen peroxide to remove odor. To measure sexual motivation, the number of attempts to reach the female, the time spent near the partition, and the latency to the onset of the response to the female were recorded for each animal. Pharmacological agents. Kisspeptin-10 (Sigma, USA) was used in the study, which was diluted in distilled water for 3 doses: 1. 0.1 μg in 20 μl 2. 1 μg in 20 μl and 3. 10 μg in 20 μl and administered intranasally. For intraperitoneal administration, kisspeptin-10 was used in doses of 1 μg per 0.2 ml, 10 μg per 0.2 ml and 100 μg per 0.2 ml. A 0.9% sodium chloride solution served as a control. Rats were administered kisspeptin-10 intranasally and intraperitoneally 10–15 min before placing them in the behavioral assessment setups. Statistical analysis methods. The statistical significance of differences was assessed using the GraphPad Prism 8.3.4 software package using one-way analysis of variance. One-way analysis of variance ANOVA was used to compare the control and experimental groups. Of the nonparametric criteria, the Kruskal–Wallis test was used to compare the groups. Differences were considered statistically significant at p < 0.05. The obtained data were presented using descriptive statistics such as the arithmetic mean and the error of the mean.
RESULTS OF THE STUDY
Test "Sexual motivation"
When studying sexual motivation, the time spent near the partition and the latent time before the onset of the reaction to the female were recorded for each animal (Fig. 1-2). The first group of experimental animals was not exposed to any influence - the intact group. In the second, third and fourth groups, the animals received kisspeptin-10 intranasally in doses of 0.1, 1 and 10 μg (in 20 μl, 10 μl in each nostril), respectively. In the fifth, sixth and seventh groups, the experimental animals received kisspeptin-10 in doses of 1, 10, 100 μg intraperitoneally in a volume of 0.2 ml, respectively. In the eighth group, the animals received physiological solution intranasally 20 μl (10 μl in each nostril) (false intranasal group), the ninth group of animals "false intraperitoneal group" received physiological solution intraperitoneally 0.2 ml. The control latent time before the onset of the male's reaction to the female was 9.1 ± 1.0 sec. Intranasal administration of kisspeptin-10 significantly reduced the latent time. With the introduction of 1 μg of kisspeptin, the latent time was significantly different from the intact group of mice (p ≤ 0.05) and was 4.0 ± 0.4 sec, and with the introduction of 10 μg, the latent time decreased to 3.4 ± 0.4, compared with the intact group (p ≤ 0.01). Intraperitoneal administration of kisspeptin-10 only at the maximum dosage significantly reduced the latent time (p≤0.05) compared to the intact group and was 4.1±0.5 sec. Other dosages of intraperitoneal administration of kisspeptin-10 (1 and 10 μg) did not significantly change the control latent time. Intranasal and intraperitoneal administration of physiological solutions did not show significant changes compared to the intact group, which indicates that the level anxiety in animals did not increase significantly and the manipulations were performed correctly. Thus, intranasally administered kisspeptin-10 at a dosage of 1 μg caused a decrease in the time to the onset of the reaction, as with intraperitoneal administration, but at a dosage 10 times higher than the intranasal one.
Table 1. The behavior of animals in the test “Sexual motivation” according to the indicator after intranasal and intra-Brushyes of the physiological solution and kisspeptin-10;

1. Intact – Intact group of animals without exposure,
2. Sat. r. IP – intraperitoneal administration of saline solution
3. Sat. r. IN – intranasal administration of saline
4. Kisspeptin IN 0.1 – intranasal administration of kisspeptin-10 at a dosage of 0.1 μg
5. Kisspeptin IN 1 – intranasal administration of kisspeptin-10 at a dosage of 1 μg
6. Kisspeptin IN 10 – intranasal administration of kisspeptin-10 at a dosage of 10 μg
7. Kisspeptin IP 1 – intraperitoneal administration of kisspeptin-10 at a dosage of 1 μg
8. Kisspeptin IP 10 – intraperitoneal administration of kisspeptin-10 at a dosage of 10 μg
9. Kisspeptin IP 100 – intraperitoneal administration of kisspeptin-10 at a dosage of 100 μg
Note: the study results are presented as M±m;
* – p≤0.05, ** – p≤0.01- difference between the group with the introduction of kisspeptin-10 and the intact group;
## – p≤0.01, difference between intranasally administered kisspeptin and intraperitoneally administered kisspeptin of the same dosage
1. Intact - an intact group of animals without influence,
2. Phys.r. WB - intraperitoneal administration of a physiological solution
3. Phys.r. IN - intranasal administration of physiological solution
4. Kisspeptin IN 0.1-intranasal administration of Kisspeptin-10 in a dosage of 0.1 μg
5. Kisspeptin IN 1-intranasal administration of Kisspeptin-10 in a dosage of 1 μg
6. Kisspeptin IN 10-intranasal administration of Kisspeptin-10 in a dosage of 10 μg
7. Kisspeptin IP 1- intraperitoneal administration of Kisspeptin-10 in a dosage of 1 μg
8. Kisspeptin IP 10- intraperitoneal administration of Kisspeptin-10 in a dosage of 10 μg
9. Kisspeptin IP 100- intraperitoneal administration of Kisspeptin-10 in a dosage of 100 μg
Note: The results of the study are presented in the form of M ± M;
*- p≤0.05, **- p≤0.01- The difference between the group with the introduction of Kisspeptin-10 and the intact group;
## - p≤0.01, the difference between intranasally introduced by Kisspeptin and intra -abdominal kisspeptin of one dosage

The next task of the experiment in the study of sexual behavior in male rats was to determine the number of attempts by the male to reach the female at the septum. Each experimental group received the same drugs as in the previous study. The number of attempts by the male to reach the female at the septum in the intact group was 13.5±1.0. Intranasal administration of kisspeptin-10 significantly (p≤0.05 and p≤0.01, respectively) increased the number of attempts by the male to reach the female at the septum in doses of 1 and 10 μg compared to the intact group and amounted to 22.2±2.0 and 22.8±1.8 sec. Intraperitoneal administration of kisspeptin-10 at a dosage of 100 μg did not significantly increase the number of attempts by the male to reach the female at the septum compared to the intact group and amounted to 15.7±2.1 sec. When comparing groups of mice after intranasal and intraperitoneal administration of kisspeptin, a reliable increase in the number of approaches to the female was found almost twice as compared to the intraperitoneal group at the same dosage of 10 μg. Thus, intranasal administration of kisspeptin-10 at dosages of 1 and 10 μg increased by 1.5 times (22.2±2.0 sec. and 22.8±1.8) the number of attempts by the male to reach the female at the septum compared to the intact group (13.5±1.0 sec.). Intraperitoneal administration of kisspeptin-10 in doses of 1 and 10 μg did not affect the number of attempts to reach the female by the male at the partition compared to the intact group. Intranasal and intraperitoneal administration of physiological solution did not show reliable changes compared to the intact group and amounted to 12.5 ± 1.2 and 14.0 ± 1.2, respectively, which indicates that the level of anxiety in animals did not increase significantly and the administration manipulations were performed correctly. Intranasal kisspeptin-10 significantly increased the number of attempts to reach the female by the male at the partition in two doses of 1 and 10 μg, while intraperitoneal administration of even 100 μg did not cause reliable changes in this indicator. Kisspeptin-10 significantly increases the preference of male rats for females after intranasal administration. Intranasal administration of kisspeptin-10 affected the sexual behavior of male rats by decreasing the latency to the onset of a response to a female and increasing the number of attempts to reach a female at concentrations 10 times lower than those required for intraperitoneal administration. A dose of 100 μg for intraperitoneal administration was insufficiently effective in changing the behavior of mice in the Sexual Motivation test.
Elevated Plus Maze Test.
With intranasal administration of kisspeptin-10, an increase in the number of runs was observed in all three doses compared to the intact group of animals, but a reliable increase was in the groups after the administration of kisspeptin-10 1 μg and 10 μg, indicating a decrease in anxiety and an increase in exploratory activity in the group of rats after intranasal administration of kisspeptin-10 (Fig. 1). After intranasal administration of kisspeptin-10 in the amount of 1 μg, the number of runs increased from 2.2±0.3 in the intact group to 5.7±1.0. After intranasal administration of kisspeptin-10 in the amount of 10 μg, the number of runs significantly increased to 6.1±0.9. A dose-dependent effect was observed after intranasal administration of kisspeptin-10. There were no differences in the number of runs in the group of intranasal administration of saline (2.5±0.4) and intraperitoneal administration of physiological solution (2.4±0.3) and the intact group (2.2±0.3) were not detected. Intraperitoneal administration of kisspeptin at doses of 1 and 10 μg did not show reliable changes compared to the intact group and was 2.0±0.3. With an increase in the dosage to 100 μg intraperitoneally, a reliable increase in runs to 5.1±1.1 was observed compared to the intact group.
The next assessed parameter was the time spent in the open arms.
With intranasal administration of kisspeptin-10 in two dosages, an increase in the time in the open arms was observed compared to the intact group of animals, while intraperitoneal administration caused an effect only at one dosage. (Fig. 2). A significant increase in the indicator was observed in the groups after the introduction of 1 μg kisspeptin, which amounted to 32.8±5.4 s compared to the intact group of 15.1±2.1 s. When 10 μg kisspeptin was administered intranasally, the time spent in the open arms significantly increased to 35.8±6.0 compared to the intact group of 15.1±2.1 s. With intraperitoneal administration of kisspeptin, significant changes were observed only at the maximum dosage, showing an increase in the time in the open arms to 32.5±2.5 compared to the intact group of 15.1±2.1 s. The remaining groups - intranasal and intraperitoneal administration of saline, intraperitoneal administration of 1 and 10 μg kisspeptin, as well as intranasal administration of kisspeptin-10 at a dose of 1 μg did not show reliable changes compared to the intact group.
When assessing the indicator of the number of hanging from the sleeve, it was found that intranasal administration of 1 and 10 μg kisspeptin-10 caused a reliable increase due to a possible decrease in the stress level and an increase in exploratory and motor activities. The indicator after intranasal administration of the average dose increased to 4.1 ± 0.3 (1 μg), and after the maximum dose (10 μg) - to 4.4 ± 0.5, compared to the intact group 1.7 ± 0.2. Kisspeptin-10, administered intraperitoneally in the amount of 100 μg, also caused a reliable change in behavior, increasing the number of hangings to 4.4 ± 0.5 compared to the intact group.
It can be assumed that the dosage of 1 μg with intranasal administration and 1 and 10 μg with intraperitoneal administration are ineffective, since they did not cause changes in behavior. The dosage of 10 μg with intranasal administration of the pharmacological agent caused reliable changes in all the parameters assessed, as well as after intraperitoneal administration of 100 μg. Thus, for a reliable change in the behavior of mice in the elevated plus maze test, kisspeptin-10 was required 10 times less than with intraperitoneal administration.
For the other assessed parameters - time in closed arms and the number of grooming acts, no reliable differences were found between the groups 

Figure 1 - the number of dashes between the sleeves in the dough "Raised Cross Labyrinth"

1. Intact - an intact group of animals without influence,
2. Phys.r. IP - intraperitoneal administration of a physiological solution
3. Phys.r. IN - intranasal administration of physiological solution
4. Kisspeptin IN 0.1-intranasal administration of Kisspeptin-10 in a dosage of 0.1 μg
5. Kisspeptin IN 1-intranasal administration of Kisspeptin-10 in a dosage of 1 μg
6. Kisspeptin IN 10-intranasal administration of Kisspeptin-10 in a dosage of 10 μg
7. Kisspeptin IP 1- intraperitoneal administration of Kisspeptin-10 in a dosage of 1 μg
8. Kisspeptin IP 10- intraperitoneal administration of Kisspeptin-10 in a dosage of 10 μg
9. Kisspeptin IP 100- intraperitoneal administration of Kisspeptin-10 in a dosage of 100 μg

  * - p≤0,05, ** – p≤0,01 - differences between intact and experimental animals

Figure 2 - Results of the test "Raised Cross Labyrinth"

I- Evaluated parameter: time spent in open sleeves

II- Assessed parameter: The amount of might from the sleeve

1. Intact - an intact group of animals without influence,
2. Phys.r. IP - intraperitoneal administration of a physiological solution
3. Phys.r. IN - intranasal administration of physiological solution
4. Kisspeptin IN 0.1-intranasal administration of Kisspeptin-10 in a dosage of 0.1 μg
5. Kisspeptin IN 1-intranasal administration of Kisspeptin-10 in a dosage of 1 μg
6. Kisspeptin IN 10-intranasal administration of Kisspeptin-10 in a dosage of 10 μg
7. Kisspeptin IP 1- intraperitoneal administration of Kisspeptin-10 in a dosage of 1 μg
8. Kisspeptin IP 10- intraperitoneal administration of Kisspeptin-10 in a dosage of 10 μg
9. Kisspeptin IP 100- intraperitoneal administration of Kisspeptin-10 in a dosage of 100 μg

* - p≤0.05, ** - p≤0.01, differences between intact and experimental animals

Open Field Test
It is known from literature that kisspeptin-10 neurons are localized in the posterior dorsal medial amygdala of the brain, which in turn is responsible for emotional behavior and motivation for sexual behavior, and is also functionally associated with fear and anxiety. Therefore, a study was conducted on the effect of intranasal kisspeptin-10 on emotional-exploratory and motor behavior in male rats. The results of the study of the behavior of male rats in the Open Field test are presented in Fig. 3 and 4. The group of animals that were administered 1 mg of kisspeptin-10 intranasally showed a slight increase in horizontal motor activity (an increase in the number of crossed squares) compared to the intact group, an increase in exploratory activity: the number of sniffs (p≤0.05), the number of explored holes (p≤0.05) and crossed squares (p≤0.05) significantly increased compared to the intact group.
It was found that after intranasal administration of 10 μg of kisspeptin-10, animals showed a significant increase in exploratory activity (an increase in the number of explored holes (p≤0.01), sniffs (p≤0.01), vertical stands (p≤0.05), crossed squares (p≤0.01)) compared to the intact group. In the groups of animals with intranasal and intraperitoneal administration of physiological solution, no reliable differences were found with the intact group in any parameter. When determining the parameters of racks with support, grooming and the number of boluses, the experimental groups did not show reliable differences.
The behavior of animals that were administered kisspeptin-10 in doses of 1 and 10 μg did not change significantly in any parameter. Intraperitoneal administration of 100 μg kisspeptin-10 is characterized by a reliable increase in horizontal activity, vertical racks, exploration of holes and crossed squares (p≤0.05), compared with intact mice. The remaining parameters did not show reliable differences from the intact group.
Thus, emotional-exploratory and motor behavior after intranasal administration of kisspeptin-10 changed reliably and dose-dependently, compared with the intact group. Intraperitoneal administration of kisspeptin-10 at a dosage 10 times higher than the dosage for intranasal administration had a significantly smaller effect on the behavior of animals, indicating the effectiveness of the intranasal method of delivering kisspeptin-10 to the CNS at lower doses compared to peripheral methods of administration.

Figure 3 - Results of the “Open Field” test according to the indicators of sniffing (I), locomotion (II), vertical racks (III) and racks with emphasis (IV)

1. Intact - an intact group of animals without influence,
2. Phys.r. IP - intraperitoneal administration of a physiological solution
3. Phys.r. IN - intranasal administration of physiological solution
4. Kisspeptin IN 0.1-intranasal administration of Kisspeptin-10 in a dosage of 0.1 μg
5. Kisspeptin IN 1-intranasal administration of Kisspeptin-10 in a dosage of 1 μg
6. Kisspeptin IN 10-intranasal administration of Kisspeptin-10 in a dosage of 10 μg
7. Kisspeptin IP 1- intraperitoneal administration of Kisspeptin-10 in a dosage of 1 μg
8. Kisspeptin IP 10- intraperitoneal administration of Kisspeptin-10 in a dosage of 10 μg
9. Kisspeptin IP 100- intraperitoneal administration of Kisspeptin-10 in a dosage of 100 μg

Note: * - p≤0.05, ** - p≤0.01, differences between intact and experimental animals

Figure 4 - Results of the Open Field test according to the indicators of the study of minks (I), crossed squares (II), the number of pain (III) and grooming (IV)

1. Intact - an intact group of animals without influence,
2. Phys.r. IP - intraperitoneal administration of a physiological solution
3. Phys.r. IN - intranasal administration of physiological solution
4. Kisspeptin IN 0.1-intranasal administration of Kisspeptin-10 in a dosage of 0.1 μg
5. Kisspeptin IN 1-intranasal administration of Kisspeptin-10 in a dosage of 1 μg
6. Kisspeptin IN 10-intranasal administration of Kisspeptin-10 in a dosage of 10 μg
7. Kisspeptin IP 1- intraperitoneal administration of Kisspeptin-10 in a dosage of 1 μg
8. Kisspeptin IP 10- intraperitoneal administration of Kisspeptin-10 in a dosage of 10 μg
9. Kisspeptin IP 100- intraperitoneal administration of Kisspeptin-10 in a dosage of 100 μg

Note: * - p≤0.05, ** - p≤0.01, differences between intact and experimental animals

DISCUSSION
The work demonstrates for the first time the fundamental possibility of delivering kisspeptin-10 to the central nervous system using intranasal administration, as well as the dose-dependence of the studied effect of the drug. This is confirmed by an increase in horizontal and vertical motor activity (an increase in the number of crossed squares, sniffing, and exploration of holes) in the Open Field test after the intranasal administration of 1 μg of the studied substance compared to the intact group. With an increase in the concentration of kisspeptin-10 to 10 μg, horizontal activity and stances with support change reliably in addition to the above indicators (p≤0.01). While intraperitoneal administration of kisspeptin-10 at doses of 1 and 10 μg did not demonstrate reliable differences, and the administration of 100 μg affected the indicators of the burrow, crossed squares, vertical stands and horizontal activity compared to intact mice (p≤0.05).
Thus, testing in the open field demonstrated a reliable increase in exploratory and motor activity in rats after intranasal administration of the test substance and an increase in the effect with an increase in the dose. The group of rats after intraperitoneal administration of the substance showed reliable differences in the test for 4 indicators out of 8, while intranasal administration at a dosage 10 times less showed changes in 5 indicators out of 8 with greater reliability.
In the "Elevated Plus Maze" test, with intranasal administration of kisspeptin-10 0.1 μg, no reliable changes were observed. When 1 μg was administered intranasally, a reliable increase in two parameters was noted: the time spent in the open arms and the number of hangings from the sleeve (p≤0.05) compared to intact mice. After the administration of 10 μg of the test substance, changes were observed in three parameters: the number of runs between the arms, the time in the open arms and the number of hangings from the sleeve (p≤0.01), indicating a dose-dependent effect of kisspeptin-10. The most effective concentration was 10 μg.
Intraperitoneal administration of 1 and 10 μg of kisspeptin-10 did not affect the behavior of animals, but the administration of 100 μg caused an increase in the time spent in the open arms, the number of hangings and the number of runs between the arms (p≤0.05), these effects are comparable to the intranasal administration of kisspeptin at a dosage of 1 μg. The increase in these parameters is mediated by a decrease in anxiety and/or an increase in exploratory activity in a group of rats after intranasal administration of kisspeptin-10.
For the other parameters assessed, time in closed arms and the number of grooming acts, no reliable differences were found between the groups.
Thus, kisspeptin delivered by the intranasal method of delivery penetrates the central nervous system, as a result of which it reliably changed behavior in the elevated plus maze test. The effects of kisspeptin-10 with intraperitoneal administration are comparable to intranasal administration of kisspeptin at a dosage of 1 μg. The greatest effect is caused by intranasal administration of kisspeptin at a dosage of 10 μg.
In the "Sexual Motivation" test, both parameters under study demonstrated changes in a group of mice after the intranasal method of delivery of kisspeptin-10. Intranasal administration of the pharmacological agent significantly reduced the latency time and increased the number of attempts to reach the female by the male at the septum at a dose of 1 μg (p≤0.05) and 10 μg (p≤0.01), respectively, compared with the intact group.
As for intraperitoneal administration, significant changes were observed only after the administration of 100 μg and only for one indicator. A decrease in latency time was observed (p≤0.05) compared with the intact group of mice. The effect of kisspeptin after peripheral administration may be associated with its indirect 

influence on sexual behavior or penetration of part of the substance into central structures. Thus, it can be assumed that kisspeptin-10, when administered intranasally, penetrated the brain and exerted a central effect, due to which it caused an increase in sexual motivation in mature male rats in both parameters. Kisspeptin-10 significantly increased the preference of male rats for females, while intraperitoneal administration of kisspeptin-10 showed a change in one of the parameters. The results of the present study demonstrate the possibility of delivering kisspeptin to the brain after intranasal administration.
Thus, in each of the three behavioral tests, reliable changes were observed after intranasal administration of kisspeptin-10, which showed a dose-dependent effect. While the indicators after intraperitoneal administration of kisspeptin-10 practically did not cause changes in behavior at doses of 1 and 10 μg. Increasing the dosage to 100 μg intraperitoneally showed a reliable change in behavior, but not as strong as after intranasal administration of the substance in an amount of 10 μg. In summary, it can be assumed that the intranasal method of delivery can be considered as a method for delivering the studied substances to the CNS, bypassing the BBB and deserves further study. The intranasal method of delivery required lower doses of the pharmaceutical agent to achieve effects.

CONCLUSION
The intranasal route of delivery has many advantages and deserves further study. One of the main advantages of the method is the bypass of the BBB. In the present study, the central effect of kisspeptin-10 on the brain after intranasal administration was demonstrated for the first time, in comparison with groups of animals after intraperitoneal administration, as well as the introduction of saline by similar routes, and the dose dependence of the observed effect was shown. Stable and dose-dependent effects of kisspeptin-10 on the behavior of rats after intranasal administration were obtained. Intranasal kisspeptin-10 caused a reliable increase in sexual motivation, an increase in horizontal and vertical motor activity, a decrease in stress and an increase in exploratory activity in sexually mature male rats. Based on the obvious effects of kisspeptin-10 after intranasal administration in each test, it can be assumed that kisspeptin-10 penetrated the brain, bypassing the BBB and exerted a central effect. The data confirm the potential and significance of this method of delivery for the regulation of sexual behavior and for influencing processes of an emotional nature (for example, reducing anxiety).
The second advantage of the intranasal route of delivery is the low dosage of the active substance. In this study, 3 doses of kisspeptin-10 were used intranasally, which are 10 times less than intraperitoneal doses. Even a high concentration of the substance did not show significant changes in behavior with peripheral administration, in contrast to intranasal administration. The creation of intranasal drugs for the treatment of CNS diseases is a promising direction of modern pharmacology, but there are a number of problems that limit its use, including the impossibility of intranasal administration of some drugs, the difficulty in ensuring a stable concentration of intranasal drugs, and the difficulty of precise dosing. In this regard, there is a great need for fundamental research into the intranasal delivery method.

About the authors

Mariya Litvinova

Author for correspondence.
Email: litvinova-masha@bk.ru
ORCID iD: 0000-0002-2924-7475

Andrei A. Lebedev

Institute of Experimental Medicine

Email: aalebedev-iem@rambler.ru
ORCID iD: 0000-0003-0297-0425
SPIN-code: 4998-5204

MD, Dr. Sci. (Biology), Professor

Russian Federation, 12, Akademika Pavlova st., Saint Petersburg, 197022

Evgeny R. Bychkov

Institute of Experimental Medicine

Email: bychkov@mail.ru
ORCID iD: 0000-0002-8911-6805
SPIN-code: 9408-0799

MD, Cand. Sci. (Medicine)

Russian Federation, Saint Petersburg

Petr D. Shabanov

Institute of Experimental Medicine; Kirov Military Medical Academy

Email: pdshabanov@mail.ru
ORCID iD: 0000-0003-1464-1127
SPIN-code: 8974-7477

MD, Dr. Sci. (Medicine), Professor

Russian Federation, 12, Akademika Pavlova st., Saint Petersburg, 197022; 6, Akademika Lebedeva st., Saint Petersburg, 194044

References