Antidrug effects of GTS201 dipeptide, an imitation of the second bird BDNF, in morphine-addicted rats

Толық мәтін

BACKGROUND

Previously, at the V.V. Zakusov Research Institute of Pharmacology, dipeptide dimeric mimetics of loops 1, 2, and 4 of the brain-derived neurotrophic factor (BDNF) were created, namely, bis-monosuccinyl-methionyl-serine heptamethylenediamide (GSB-214), bis-hexanoyl-seryl-lysine hexamethylenediamide (GTS-201), and bis-monosuccinyl-seryl-lysine hexamethylenediamide (GSB-106), respectively. Studies have found that GTS-201, a mimetic of BDNF loop 2, activates the specific receptor TrkB and the MAPK/Erk kinase pathway, does not affect the PI3K/Akt signaling pathway, and has neuroprotective activity similar to BDNF in in vitro experiments, in an oxidative stress model, and in the culture of HT-22 line hippocampal neurons [1, 2].

For the first time, this study examined the effect of the dipeptide GTS-201 on various behavioral indicators of laboratory white rats during the development of dependence and withdrawal syndrome (WS) of morphine. Data were compared with the corresponding indicators for GSB-106, one of the promising and previously studied peptide compounds, in terms of possible correction of opiate addiction [3, 4].

This study aimed to investigate the effect of GTS-201 on various behavioral indicators of laboratory white rats during the development of dependence and WS of morphine.

RESEARCH METHODS

The experiments were performed on 80 outbred male rats weighing 240–260 g. The animals were obtained from the Stolbovaya nursery. Before the start of the experiment, they were kept for 1 week in standard plastic cages, with eight rats each, in a vivarium under natural light and air temperature of 21–23°C. Granulated food and water were given ad libitum.

The experiments were performed in accordance with the requirements imposed in the Order of the Ministry of Health of the Russian Federation dated April 1, 2016, No. 199n “On approval of the Rules of Good Laboratory Practice” and Decisions of the Council of the European Economic Union No. 81 “On approval of the Rules of Good Laboratory Practice of the Eurasian Economic Union in the Area of Circulation of Medicines.” All animal procedures were approved by the bioethical commission of the V.V. Zakusov Research Institute of Pharmacology, with the decision of the ethical committee in Protocol No. 7 of May 16, 2022.

To reduce the stress level and adapt to the experimental conditions, the animals were subjected to a handling procedure for 3–4 days before the experiment started. One day before the start of the experiment, the rats were placed in an experimental room with natural lighting changes and air temperature maintained within 23 ± 1.0°C. The animals were weighed every other day for 12 days, starting from day 1 of the administration of morphine and GTS-201. The average bodyweight changes in groups were calculated according to the number of days of the experiment.

Morphine hydrochloride was administered in doses of 10–20 mg/kg, and μ-opioid receptor agonist (Chimkent Pharmaceutical Plant), naloxone hydrochloride, an antagonist of µ-opioid receptors (DuPont DeNemours Int. S.A., Switzerland), was injected at a single dose of 1 mg/kg to provoke WS of morphine. GTS-201, hexamethylenediamide bis(N-hexanoyl-L-seryl-L-lysine), a mimetic of BDNF loop 2 (V.V. Zakusov Research Institute of Pharmacology), was administered in doses of 1 and 5 mg/kg. The substances were dissolved in distilled water and administered extempore intraperitoneally at a dose of 1 mL/1 kg of animal bodyweight.

Four experimental groups were formed: group 1 received chr. morphine + naloxone (active control group), group 2 received chr. morphine + GTS-201 (1) + naloxone, group 3 received chr. morphine + GTS-201 (5) + naloxone, and group 4 received chr. morphine + GTS-201 (1 × 5) + naloxone (where chr. indicated the administration of morphine for 5 days; (1) and (5) were doses of substances in mg/kg, (1 × 5) indicated chronic administration of the peptide within 5 days).

Morphine addiction was induced in rats according to a previously developed scheme, i.e., morphine was administered in increasing doses, from 10 to 20 mg/kg, two times a day, with an interval of 8 h, for 5 days [5]. GTS-201 was administered in single doses of 1 or 5 mg/kg 30 min before morphine administration on day 5 of the experiment or daily in one of the groups for 5 days in the morning 30 min before morphine administration. The animals were assessed for the presence of specific signs of morphine WS on experiment day 5 for 5 min in an “open field” (lit round arena) 15 min after the administration of the opiate receptor antagonist naloxone. The control group received daily injections of distilled water for 5 days according to the same regimen as animals from the experimental groups; they received naloxone at a dose of 1 mg/kg on day 5 of the experiment, before testing. Behavioral reactions, namely, locomotor activity, stances, grooming, defecation, and specific signs of morphine WS (up to 18 indicators), were registered in all groups. Discrete signs of withdrawal (such as diarrhea and episodes of shaking and grinding of teeth) were assessed quantitatively and alternatively, and the rest were evaluated in an alternative form, according to the “yes/no” principle. The total index (TI) of WS severity for each animal and the average values for the experimental and control groups were calculated based on alternative signs with the maximum possible TI value of 18 points. The average value of the WS severity in group 1 was 100%.

To assess the effect of GTS-201 on the behavior of morphine-dependent rats in the elevated plus maze (EPM) test, a standard configuration was used, with an arm length of 50 cm, width of 14 cm, central platform of 14 × 14 cm, and height of sides of closed arms of 15 cm. At 24 h after the withdrawal of morphine injections, standard behavioral indicators were recorded for 5 min, namely, the number of entries into the open arms, time spent in the open arms in seconds, and number of entries into the closed arms.

Tactile thresholds were assessed in rats using the von Frey test using a standard set of filaments from Ugo Basile (Italy), which can exert graded pressures on the plantar surface of the hind paws of rats. Each measurement with a separate filament was performed at least three times, the animals’ response to pressure was assessed, and strength was presented in grams. For each group, tactile thresholds were measured at baseline, before the use of substances, and 24 h after morphine withdrawal and last administration of the GTS-201 dipeptide.

Statistical analysis was performed using the analysis of variance (ANOVA) test, Mann–Whitney U-test, and Duncan test to compare differences between the groups. Data were presented as mean values ± error of the mean (m ± SEM), as well as percentage relative to the initial level of values in each test. Data with p values < 0.05 were assessed as statistically significant.

RESEARCH RESULTS AND DISCUSSION

Changes in animal bodyweights were recorded by groups for 12 days. Statistically significant changes in this indicator were noted from day 5, from the start of morphine administration. In groups 1 and 2 by this day, the average decrease in bodyweight was 11% (p < 0.05) from the initial level. After morphine withdrawal, on day 7 of the experiment, the maximum decreases in bodyweights in groups 1, 2, and 3 were 17.8%, 16.9%, and 14.6%, respectively (p < 0.01). When administered chronically, GTS-201 blocked a decrease in bodyweight in morphine-dependent rats in group 4 (Table 1).

 

Table 1. Weight changes of rat body after administration of morphine and GTS-201 compound (m ± S.E.M.)

Таблица 1. Изменение массы тела крыс под влиянием морфина и соединения ГТС-٢٠١ (m ± S.E.M.)

Days of experiment	Bodyweight of rats (g) in the experimental groups
	Group 1		Group 2		Group 3		Group 4
	gram	%	gram	%	gram	%	gram	%
1	273.5 ± ٤.٩	١٠٠	282.5 ± ٢.٥	١٠٠	272.0 ± ٤.٦	١٠٠	283.0 ± ٣.٣	١٠٠
3	260.5 ± ٦.٢	95.2 ± ١.٤	271.5 ± ٣.٦	96.1 ± ١.٤	261.5 ± ٣.٩	96.2 ± ١.٤	272.5 ± ٥.٦	96.2 ± ١.٢
5	243.5 ± ٥.٢	89.0* ± ٠.٦	251.5 ± ٢.١	89.0* ± ٠.٥	254.5 ± ٤.٦	93.5 ± ٠.٩	261.0 ± ٦.٧	92.1 ± ١.٧
7	224.8 ± ٤.٣	82.2** ± ١.١	234.7 ± ٢.٣	83.1** ± ٠.٩	232.3 ± ٣.٨	85.4** ± ١.٢	264.6 ± ٤.٦	93.5# ± ١.٥
12	266.4 ± 4.8	97.4 ± 1.3	273.2 ± ١.٨	96.7 ± ٠.٨	263.8 ± ٤.٢	97.0 ± ٠.٩	277.9 ± ٣.٩	98.2 ± ١.٣

Note. *P < 0.05; **P < 0.01 when comparing bodyweight values on days 5 and 7 with values on day 1 of the experiment. ^#P < 0.05 when comparing groups 1 and 4 on day 7 of the experiment. The groups are described in the Methods section.

Примечание. ^*P < 0,05; ^**P < 0,01 при сравнении величин массы тела в ٥-й и ٧-й дни с значениями в ١-й день эксперимента. ^#P < 0,05 при сравнении гр. ١ и гр. ٤ в ٧-й день эксперимента. Описание групп см. в разделе «Методы исследования».

 

Tactile thresholds in rats were markedly reduced in all groups after morphine withdrawal. However, after the administration of GTS-201, a partial restoration of tactile thresholds was note, most pronounced for group 2 at 25.6% (p < 0.05) and group 3 at 20.1% (p < 0.01) and to a lesser extent for group 4 at 12.5% (p < 0.05) of the initial values. These values were statistically significantly different from the average threshold values in group 1 (control) (Table 2).

 

Table 2. Tactil thresholds changes in morphine-depended rats in von Frey’s test (m ± S.E.M.)

Таблица 2. Изменения тактильных порогов у морфин-зависимых крыс в тесте von Frey (m ± S.E.M.)

Experimental groups	Baseline values		After withdrawal
	Gram	%	Gram	%
Group 1	0.72 ± 0.06	100	0.05 ± 0.005 P = 0.004##	7.67 ± 0.93
Group 2	0.82 ± 0.09	100	0.18 ± 0.04 P = 0.02#	25.56 ± 7.04 P = 0.02*
Group 3	0.88 ± 0.06	100	0.17 ± 0.03 P = 0.02#	20.10 ± 2.70 P = 0.004**
Group 4	1.02 ± 0.14	100	0.13 ± 0.03 P = 0.01##	12.47 ± 1.98 P = 0.04*

Notes. ^#P < 0.05; ^##P < 0.01 between the initial values of tactile thresholds and their values after morphine withdrawal; ^*P < 0.05; ^**P < 0.01 between the values of tactile thresholds for group 1 in comparison with the morphine-dependent groups receiving GTS-201. The ANOVA test, Mann–Whitney U-test, and Duncan test were used to compare between-group differences.

Примечания. ^#P < 0,05; ^##P < 0,01 между исходными величинами тактильных порогов и их значениями после отмены морфина; ^*P < 0,05; ^**P < 0,01 между величинами тактильных порогов для группы 1 в сравнении с группами морфин-зависимых животных, получавших дипептид ГТС-201. Тест ANOVA, Mann–Whitney U-test и Duncan test для сравнения различий между группами.

 

When studying the effect of GTS-201 on the behavior of morphine-dependent rats in the EPM test, no significant differences in the number of entries into open arms and the time spent in them were noted between active control group 1 and group 2. A moderately pronounced tendency was noted toward a decrease in these indicators when comparing groups 1 and 3 (p = 0.1) (Table 3).

 

Table 3. Effect of dipeptide GTS-201 on behavior of morphine-depended rats in elevated plus maze test (m ± S.E.M.)

Таблица 3. Влияние дипептида ГТС-201 на поведение зависимых от морфина крыс в тесте приподнятого крестообразного лабиринта (m ± S.E.M.)

Groups of animals	Entries into open arms	Time spent in open arms (s)	Entries into closed arms
Group 1	2.4±0.74	25.2±7.20	4.3±0.96
Group 2	2.0±0.39 P = 0.58*	27.6±5.90 P = 0.77*	3.7±0.75 P = 0.58
Group 3	1.1±0.27 P = 0.10*; 0.22#	11.6±3.37 P = 0.10*; 0.14#	3.5±0.54 P = 0.50*; 0.85#

Note. Statistical differences between and within each group were not significant.

Примечание. Статистические различия между и внутри каждой группы не были значимы.

 

When studying the effect of GTS-201 on behavioral, somatic, and neurological indicators of animal behavior after morphine withdrawal, significant changes in the pronouncement of individual signs of WS were noted. The incidence of diarrhea decreased significantly in all groups that received the peptide. In group 3, the maximum effect was noted, that is, diarrhea decreased by 71.0% (p < 0.001), convulsions by 83.3% (p < 0.05), escape attempts by 71.4% (p < 0.01), and vocalization by 62.5% (p < 0.05). In group 2, GTS-201, which was administered at a single dose of 1 mg/kg, eliminated completely escape attempts, whereas in group 4, the same dose of peptide administered chronically arrested completely convulsive reactions in rats. Despite significant changes in individual parameters, the TI of morphine WS for the groups that received morphine chronically did not change statistically significantly compared with that in the active control group, with values of 7.3 ± 0.36 points (100%) in the control group and 6.2–6.5 points (84.9%–89.0%) in groups 2, 3, and 4 (p > 0.05; Table 4).

 

Table 4. Effect of dipeptide GTS-201 on behavior indexes of withdrawal syndrome of morphine in rats (m ± S.E.M.)

Таблица 4. Влияние дипептида ГТС-201 на поведенческие показатели синдрома отмены морфина у крыс (m ± S.E.M.)

Behavioral traits	Experimental groups
	Group 1	Group 2	Group 3	Group 4
Morphine WS index	7.3±0.36	6.5±0.36 P = 0.45 n.s.	6.2±0.48 P = 0.48 n.s.	6.4±0.47 P = 0.19 n.s.
Diarrhea	3.1±0.27	2.1±0.36*	0.9±0.48***	1.6±0.47**
Posture	0.5±0.16	0.9±0.18	0.8±0.13	0.8±0.13
Ptosis	0.7±0.12	0.6±0.14	0.8±0.13	0.3±0.12
Piloerection	0.8±0.13	0.7±0.16	0.9±0.16	0.8±0.13
Rhinorrhea	0.4±0.10	0.0±0.0	0.4±0.12	0.7±0.14
Dyspnea	0.8±0.13	0.9±0.16	0.9±0.16	0.9±0.16
Writhing	0.4±0.12	0.3±0.12	0.2±0.12	0.1±0.11
Convulsions	0.6±0.16	0.3±0.11	0.1±0.11*	0.0±0.0**
Escape attempts	0.7±0.16	0.0±0.0**	0.2±0.12**	0.7±0.14
Вокализация	0.8±0.13	0.8±0.14	0.3±0.14*	0.6±0.12

Notes. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 between the values of behavioral indicators for group 1 in comparison with those for the morphine-dependent group receiving GTS-201. ANOVA test, Mann–Whitney U-test, and Duncan test were used to compare between-group differences. WS, withdrawal syndrome; n.s., not significant.

Примечание. ^*P < 0,05; ^**P < 0,01; ^***P < 0,001 между величинами поведенческих показателей для группы ١ в сравнении с этими показателями для групп морфин-зависимых животных, получавших дипептид ГТС-٢٠١. Тест ANOVA, Mann–Whitney U-test и Duncan test для сравнения различий между группами. СО — синдром отмены; н.д. — не достоверны.

 

CONCLUSION

The ability of GTS-201, a bis(-N-hexanoyl-L-seryl-L-lysine) hexamethylenediamide, a BDNF loop 2 mimetic, to influence somatic, behavioral, and neurological signs of morphine dependence was examined using laboratory rats. In the EPM test, GTS-201, which was administered at a dose of 5 mg/kg, showed a moderate tendency (p = 0.1) to reduce the number of entries and time spent in the open arms of the maze. The ability of GTS-201 to eliminate or reduce the incidence of certain manifestations of morphine WS in rats, such as diarrhea, vocalization, attempts to escape, and convulsions, has been demonstrated. The single-dose peptide in doses of 1 or 5 mg/kg restored partially the level of tactile threshold in rats, which was reduced after morphine withdrawal. GTS-201, which was administered chronically to animals at a dose of 1.0 mg/kg, restored bodyweight decreases caused by morphine withdrawal. Despite the changes in individual indicators, the TI of morphine WS for different experimental groups did not differ statistically significantly when compared with that of the active control group. A comparison of the results obtained from studying GTS-201 with the corresponding previously presented data for GSB-106, the BDNF loop 4 mimetic, showed a noticeable advantage of the latter in reducing the incidence of behavioral manifestations of morphine WS in rats. Thus, the detected anti-addictive activity of GTS-201 may be mediated by the activation of TrkB receptors and the MAPK/Erk kinase signaling pathway, which does not exclude the participation of opioid receptor mechanisms in the implementation of the registered behavioral phenomena.

ADDITIONAL INFORMATION

Authors’ contribution. Thereby, all authors made a substantial contribution to the conception of the study, acquisition, analysis, interpretation of data for the work, drafting and revising the article, final approval of the version to be published and agree to be accountable for all aspects of the study. The contribution of each author: M.A. Konstantinopolsky, L.G. Kolik, I.V. Chernyakova, N.M. Sazonova — manuscript drafting, writing and pilot data analyses; T.A. Gudasheva — general concept discussion.

Competing interests. The authors declare that they have no competing interests.

Funding source. This study was not supported by any external sources of funding.

Авторлар туралы

Mark Konstantinopolskii

Zakusov Institute of Pharmacology

Email: makonstant.24@mail.ru
ORCID iD: 0009-0002-8375-0167

cand. sci. (biol.)

Ресей, 8, Baltiiskaya street, 126315, Moscow

Larisa Kolik

Zakusov Institute of Pharmacology

Email: glkolik@mail.ru
ORCID iD: 0000-0002-9847-8058

md, dr. sci. (biol.), professor, head of the laboratory

Ресей, 8, Baltiiskaya street, 126315, Moscow

Irina Chernyakova

Zakusov Institute of Pharmacology

Email: 1986567@mail.ru
ORCID iD: 0000-0003-3182-7847

md, cand. sci. (biol.), leading researcher

Ресей, 8, Baltiiskaya street, 126315, Moscow

Nelli Sazonova

Zakusov Institute of Pharmacology

Email: saz-nellya@mail.ru
ORCID iD: 0000-0002-7608-7419

md, cand. sci. (chemic.), senior researcher

Ресей, 8, Baltiiskaya street, 126315, Moscow

Tatyana Gudasheva

Zakusov Institute of Pharmacology

Хат алмасуға жауапты Автор.
Email: tata-sosnovka@mail.ru
ORCID iD: 0000-0002-5185-4474

corresponding member of the russian academy of sciences, md, dr. sci. (biol.), professor

Ресей, 8, Baltiiskaya street, 126315, Moscow

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