The Antidepressant-Like Activity Of The New Tetrapeptide Neuroprotector Kk-1 , Homologous Of Acth 15-18 Sequence ( An Experimental Study )

The aim of study is to evaluate antidepressant-like activity of the new peptidergic neuroprotector acetyl-(D-Lys) -Lys-Arg-Arg-amide, homologous of ACTH15-18 primary amino acids sequence, that demonstrates nootropic and neuroprotective properties. Using Porsolt swimming test (PST) efficacy of tetrapeptide neuroprotector KK-1 at a single dose of 0.02 mg/kg was investigated on 16 white random bred male rats (body mass equaled 180-220 grams). Imipramine (15 mg/ kg i.p.) was used as a reference drug. Then depression was induced in these rats by reserpine (4 mg/kg, i.p.). The KK-1 (intranasally, i.n.) and imipramine were administered once a day during 3 days until the reserpineinduced depression was reproduced. The indices of rats behavior under the conditions of open-field test (OFT) and PST were evaluated. The influence of both drugs on specific reserpine-induced depressions symptoms (hypothermia and blepharoptosis) was also registered. The results were processed statistically. The tetrapeptide neuroprotector KK-1 reduced immobilization time of rats at PST (statistically significant differences compared with control group), exceeding efficacy of reference drug imipramine. Normalizing of locomotor and exploratory activity in the OFT, decreasing indices of rats helplessness behavior in PST by tetrapeptide neuroprotector KK-1 demonstrates its antagonism with depressive action of reserpine. The tetrapeptide KK-1 showed antidepressant-like action both in intact rats and in rats with the model of reserpineinduced depression. It reduced specific symptoms of depression – hypothermia and blepharoptosis, exceeding the activity of reference drug imipramine. DOI : 10.14302/issn.2476-1710.JDT-16-1346 Corresponding author: Master of Pharmacy, post-graduate student of the department of pharmacology of National University of Pharmacy. Address: Ukraine, Kharkiv, Kulykivska-street, 12. Tel. +380 99 962 13 83, email: roman.deyko@gmail.com


Introduction.
Organic and functional disorders of CNS are widespread in population and often are accompanied with depressive symptomatic [1].The much of these cases neuroprotective therapy are needed.The neuroprotectors are relatively new pharmacological class of drugs improving CNS function and protecting neurons from damaging factors [2].Peptidergic substances are perspective group of neuroprotectors.
Adrenocorticotropine derivatives, brain-derived neurotrophic factor as well as drugs developed on their basis have pleiotropic mechanism of protective action and demonstrate advantageous psychotropic properties.
The depressive disorders are determined largely by monoaminergic transmission deficit and are seen at bipolar disorder and numerous CNS disorders of diverse etiology [3].Numerous CNS disorders such as acute cerebrovascular diseases are followed by secondary brain tissue injuries and impairment of patient mental health.In clinical picture of vascular and traumatic brain injuries the depressive symptomatic often take a place [1].The neuroprotectors with pleiotropic mechanism of action influencing on several neuromediator systems of the brain are perspective as depressive disorders correctors.
A natural relation between ability of numerous psychotropic drugs to modulate the high mental functions and to protect neurons from damage is known.
Furthermore, the neuroprotective properties of these drugs at cerebrovascular and neurodegenerative diseases are well known [8].
It's well known that many neuropeptides [9,10], as well as drugs created on their basis [11,12] show the antidepressant-like properties under the conditions of experimental or clinical trials.In the recent literature data the reviews of psychopharmacologic profile of many peptide neuroprotectors are presented.
Our attention was attracted by series of new peptidergic neuroprotectors.In the basis of their structure underlies the primary amino acids sequence of Since, the tetrapeptide КК-1 may function in CNS as an ACTH antagonist, it is appropriate to suppose the antidepressant-like properties of its.Earlier we have tested КК-1 on mice in the tail suspension test [18].
There was shown the absence of influence of KK-1 at the dose of 0.02 mg/kg on the general time of immobilization of mice.But this test is considered to be less sensitive than Porsolt swimming test (PST).
Furthermore, the different sensitivity of animals' species (rats and mice) to the expected antidepressant-like action of neuropeptide KK-1 should not to be excluded.
The aim of the study is to find out the efficacy of new tetrapeptide neuroprotector KK-1 on the model of reserpine-induced depression in rats.

Experimental Procedures.
The experiment was carried on 16 white random bred sexually mature male rats weighing 180-220 g.

Results.
The results of OFT are presented in table 1.
As seen from the table 1, the investigated tetrapeptide KK-1 decreased locomotor and exploratory activity of intact animals in OFT as well as reduced their emotional reactions.Imipramine showed a tendency to decrease these activity markers of the intact animals.
The level of their emotional reactions was increased under the conditions of imipramine administration (in 2.9 The influence of the studied drugs on the rats' rectal temperature is shown in figure 1.
As seen from figure 1, the basic temperature level of rats of all 3 groups is comparable and corresponds to normal index of these animals' species.

. Influence of peptide neuroprotector КК-1 and reference drug imipramine on the indices of intact animals and animals with reserpine-induced depression in open-field test
Note.I -intact animals, R -reserpine-depressed animals; р≤0.05: * -compared with control group, ^ -compared with imipramine group, ® -between the indices of the same rats before and after reserpine-induced depression.The changes of indices (in %) of the same rats before and after reserpine-induced depression are given in brackets.

Indices
The

adrenocorticotropine 15 -
18 (Lys-Lys-Arg-Arg, ACTH[15][16][17][18].As it's known from the literature data, ACTH 15-18 is minimal fragment of ACTH capable to bind with melanocortin receptors (MCRs), that plays role of antagonist of the cerebral and peripheral MCRs[13].Moreover, the sequence Lys-Lys-Arg-Arg is presented in the structures of some neurotrophic factors, such as nerve growth factor and brain-derived neurotrophic factor[14].Taking into account the physiologically role of ACTH 15-18 , on its basis at the State Research Institute of Highly Pure Biopreparations (Russia, Saint-Petersburg) was synthetized the series of modified tetrapeptides with general formula acetyl-Lys-Lys-Arg-Arg-amide[15].The some amino acids were inserted in tetrapeptide chain as unnaturally D-forms (D-Lys or/and D-Arg).It has improved their resistance to the blood peptidases action and allowed to administrate these drugs only once a day.Subsequently under the conditions of pre-clinical studying were established their pronounced neuroprotective and nootropic properties[16,17].The leader among 10 investigated peptides according to the neuroprotective, anti-hypoxic, nootropic, anti-alcoholic and other protective properties is tetrapeptide КК-1 (acetyl-(D-Lys)-Lys-Arg-Arg-amide).
) and exploratory activity (number of vertical uprights and surveys holes), emotional reactions and their vegetative accompaniment (fecal boli, urinations and grooming acts) at open-field test (OFT) for rats were registered for 3 min [19, 20].The animals depressivity indices (latent time of immobilization, number of immobilizations acts and their general time) were evaluated at PST for 6 min (24 hours before testing at PST the rats were pretested under the same conditions for 15 min) [20, 21].During the next 2 days the medicines were administered at the same doses once a day, last time -30 min before reserpine administration.Reserpine (Sigma, USA) was administered per os on the 3 rd day (2 nd stage of experiment) at a dose of 4 mg/kg as suspension stabilized with tween-80.After 6-7 h behavioral indices of all experimental groups were evaluated at OFT and PST.The rectal temperature was measured with electronic thermometer Microlife MT 1951 (Switzerland), the blepharoptosis degree was rated using 4-point scored scale (from 1 to 4) [19] and relative quantify (%) of animals in the group with maximally expressed blepharoptosis.Statistical analysis was performed using one-way analysis of variances.Fisher angular transformation of φ was used when the results were determined in alternative manner (presence or absence of characteristic).The significance of intragroup differences were determined by Ť-Wilcoxon's criterion.Inter-and intragroup differences were considered statistically significant at p<0.05.
pramine on the indices of intact animals and animals with reserpine-induced depression under the conditions of Porsolt swimming test Note.I -intact animals, R -the animals with reserpine-induced depression; * -р≤0.05 compared with control group; The changes of indices (in %) of the same rats before and after reserpine-induced depression are given in brackets.

Freely Available Online Journal of Depression and Therapy CC-license DOI : 10.14302/issn.2476-1710.JDT-16-1346
KK-1 group (0.02 mg/kg i.n, n=5).25-30 min before experiment beginning the measuring of rats' rectal temperature and administration of the appropriate drug were done.The control group animals received 0.9% saline i.p. and i.n.Then the locomotor activity (number of crossed squares times, р<0.05 compared with control group).
of intact animals), and by tendency to increment in emotional reactions level in 1.9 times.The sum of all indices at OFT was decreased by 97% (р<0.05).Under the conditions of imipramine use the level of these disorders fractionally decreased.Locomotor activity was reduced by 79.2% (р<0.05 compared with index of intact animals), exploratory activity was decreased by 76.5% (р<0.05 compared with value of intact animals).As shown intable 2, according to the results of PST tetrapeptide neuroprotector KK-1 demonstrated antidepressant-like properties both in intact animals and in the animals with reserpine-induced depression.The antidepressant-like properties of Freely Available Online Journal of Depression and Therapy CC-license DOI : 10.14302/issn.2476-1710.JDT-16-1346 Vol-1 Issue 3 Pg.no.-13 http://www.openaccesspub.org/statistically significant antidepressant-like effect.As in case with intact animals, KK-1 increased latent time of immobilization in 4.5 times (р<0.05compared with control group), decreased number of immobilizations episodes in 2.6 times (р<0.05compared with control group) and general time of immobilizations in 3.6 times (р<0.05compared with control group).

Table 1
Blepharoptosis is also among the symptoms of the depression developed.