RU2756449C2 - Linear substituted glyproline with antidepressant and analgesic effect - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: invention relates to medicine, in particular to pharmacology, and concerns new application of known linear substituted glyproline – ethyl ether of N-phenylacetylglycyl-L-proline (GZK-111) as analgesic agent. It is established that N-phenylacetylglycyl-L-proline has both antidepressant and analgesic effect.

EFFECT: new application of linear substituted glyproline.

1 cl, 1 dwg, 2 tbl, 3 ex

Description

Scope of invention

The invention relates to medicine, in particular to pharmacology, and concerns a new use of linear substituted glyproline - ethyl ester of N-phenylacetylglycyl-L-proline (GZK-111) - as an antidepressant and analgesic agent.

State of the art

According to the World Health Organization, approximately 4-5% of the world's population suffers from depression, while the risk of developing clinical depression is 15-20%. Depression is a chronic relapsing disease, with recurrent episodes occurring in about 60% of patients (Ayso-Mateos JL et al. Br J Psychiatr. 2001; 179: 308-16). The interplay of chronic pain and depression has been well studied in population-based studies and in primary care and specialized clinical sample models. So, out of 18,980 examined in European countries, 4% had symptoms that meet the DSM-IV criteria, among them 43.3% of patients indicated the presence of at least one symptom of pain, while among those who did not have signs of depressive disorders, they complained about pain. only 16.1% (Ohayon MM. J Clin Psychiatry. 2004; 65 suppl 12: 5-9).

According to the glutamate hypothesis, with depression there are disturbances in the glutamatergic system, in particular, affecting AMPA receptors, which are involved in the transmission of rapid excitatory signals in the synapses of the central nervous system, which is confirmed by the following facts. Firstly, knockout mice for the GluAl gene exhibit increased sensitivity to the development of depression (Chourbaji S et al. The FASEB J. 2008; 22 (9): 3129-3134), and secondly, antidepressants are able to increase the expression of GluAl and GluA2 subunits AMPA receptors in the prefrontal cortex and nucleus accumbens (Tan C.-H et al. Exper. Br. Res. 2006; 170 (4): 448-456), and finally AMPA receptor modulators that increase the functional activity of these receptors show antidepressant properties (Skolnick P. Biol. Psych. 2008; 63 (4): 347-348). According to pharmacological studies, ketamine and ampakines, which increase glutamatergic transmission through activation of AMPA receptors, relieve depression symptoms in the presence of chronic pain (Le AM et al. Anesthesiology. 2014; 121 (5): 1080-1090), which confirms the involvement of AMPA- receptors in the regulation of depression, pain and depression in chronic pain conditions.

BDNF is known to play a central role in the pathogenesis of depression (Jiang C, Salton R. Transl Neurosci. 2013; 1: 46-58; Autry AE, Monteggia LM. Pharmacological Reviews. 2012; 2: 64: 238-258). Binding of BDNF to TrkB receptors promotes the formation of long-term potentiation and de novo neurogenesis. A decrease in BDNF content was found in patients with depressive disorders, and, conversely, an increase in BDNF level was recorded in patients who underwent successful pharmacotherapy with antidepressants (Karege F et al. Psychiatry Res. 2002; 109 (2): 143-148; Boulle F et al. Mol Psych 2012; 17 (6): 584-596).

As a result of many years of fundamental research at the V.V. Zakusov, a putative peptide prototype of the classical nootropic drug piracetam - cyclo-L-prolylglycine (CPG), which was later discovered in the brain of rats as an endogenous compound, was constructed (Gudasheva T.A. et al. Chem.-Pharm. J. 1991; 6 : 25: 12-16; Gudasheva T.A. et al. FEBS Letters. 1996; 391: 149-152). According to pharmacological studies, CPH regulates memory and response to emotional stress (Gudasheva T.A. et al. Bull Exp Biol. And Med. 1999; 10 (128): 411-413; Gudasheva T.A. and et al.Bull Expert Biol. and Med. 2001; 5 (131): 545-550; Seredenin S.B. et al.Bull Expert Biol. and Med. 2002; 4 (133): 417-419) , has antihypoxic (Kolyasnikova K.N. et al. Exp. and Clin. Pharmacol. 2012; 75: 9: 3-6), neuroprotective (Povarnina P.Yu. et al. Bulletin Expert Biol. Med. 2015; 11: 160: 600-603), analgesic (Ferro JN et al. Clin. Exp. Pharmacol. Physiol. 2015; 42 (12): 1287-1295) and antidepressant action (Abdullina A.A. et al. Exp. And wedge pharmacol. 2018; 81 (S): 8). CPH has a positive modulating effect on glutamate AMPA receptors (Gudasheva T.A. et al. DAN. 2016; 1 (471): 106-108) and stimulates the synthesis of brain derived neurotrophic factor (BDNF) (Gudasheva T.A. A. et al. DAN. 2016; 4: 469: 492-495).

In continuation of the research, a linear substituted glyproline was designed and synthesized - an ethyl ester of N-phenylacetylglycyl-L-proline (compound GZK-111) containing the amino acid sequence Gly-Pro with a high proportion of cisoid peptide bond (Ovchinnikov YuA and Ivanov VT, 1975; Baldoni HA et al. J. Molec. Struct. (Theochem). 2000; 500: 97-111), which promotes the cyclization of the molecule (Rydon HN et al. J. Chem. Soc. 1956; 3642-3650; Manabe S. et al. Med Chem. Commun. 2013; 4: 792-796). According to the hypothesis put forward, the compound GZK-111 undergoes biotransformation with the formation of CPH and exhibits a spectrum of effects characteristic of the latter (Gudasheva T.A. et al. Chem.-Pharm. J. 2016; 11 (50): 3-8), which confirmed by pharmacological studies that proved the presence of nootropic, anxiolytic, antihypoxic and neuroprotective activity in the pharmacological profile of GZK-111 in the dose range of 0.1-1.5 mg / kg (Gudasheva T.A. et al. Chem.-Pharm. zh. 2016 ; 11: 50: 3-8; Kolyasnikova K.N. et al. Chem.-Pharm. J. 2018; 52 (6): 13-17).

The neuropsychotropic effect of the GZK-111 metabolite, CPG, which is a positive modulator of AMPA receptors, is presumably realized by the BDNF-ergic mechanism (Gudasheva TA et al. DAN. 2016; 4: 469: 492-495), which suggests the presence of GZK- 111 antidepressant activity.

Previously, analgesic activity was shown in in vivo experiments for the ACTH _4-10 fragment (Met-Glu-His-Phe-Arg-Trp-Gly) at a dose of 0.5 mg / kg and its analogue Semax (Met-Glu-His-Phe-Pro -Gly-Pro), while Semax exhibited more pronounced antinociceptive activity in the dose range of 0.015-0.5 mg / kg, apparently due to the replacement of the C-terminal amino acid residues in ACTH _4-10 with the Pro-Gly-Pro sequence (Ivanova DM et al Bull Exp Biol Med 2007; 143 (1): 5-8). AMPA receptors are known to play a central role in the delivery of pain stimuli, with the positive allosteric modulators of AMPA receptors ampakines exhibiting analgesic properties (Sun Y et al. Behav Brain Res. 2017. 334: 1-5). According to the works of JN de Souza Ferro et al. (2015), CPH increases the pain response threshold and has an anti-inflammatory effect, which may be due to its positive modulating effect on glutamate AMPA receptors (Gudasheva T.A. et al. Reports of the Academy of Sciences. 2016; 1 (471): 106- 108). The totality of the presented data suggests the presence of analgesic properties in the compound GZK-111.

Currently, there is no information on the antidepressant and analgesic properties of GZK-111, characteristic of CPH.

The essence of the invention

The essence of the invention consists in the use of GZK-111 as an antidepressant and analgesic agent.

The purpose of the invention is the development of new antidepressants and analgesics. This goal was achieved by investigating the pharmacological effects of GZK-111 in in vivo experiments in the Porsolt forced swimming test and in the hot plate test.

The technical result of the invention is the antidepressant and analgesic effects of GZK-111.

Animals. The experiments were carried out on white outbred male mice (n = 160) with a body weight of 18-20 g and inbred male mice of the C57B1 / 6 line (n = 50) with a body weight of 18-22 g (Federal State Budgetary Scientific Institution "Scientific Center for Biomedical Technologies Federal Medical and Biological Agency ", branch" Stolbovaya "). The animals were kept 15 individuals in a T / 3 standard cage in the vivarium of the V.V. Zakusova "(temperature 21-23 ° C, relative air humidity 40-60%) in natural light and free access to water and briquetted feed for 10 days before testing. The organization and implementation of the work was carried out in accordance with the order of the Ministry of Health of Russia No. 199 dated April 01, 2016 "On the approval of the rules of good laboratory practice." The animals were kept in accordance with SP 2.2.1.3218-14 "Sanitary and Epidemiological Requirements for the Arrangement, Equipment and Maintenance of Experimental Biological Clinics (Vivariums)" dated August 29, 2014 No. 51. The experiments were approved by the Commission on Biomedical Ethics of the V.V. Zakusov ".

Preparations. GZK-111 (a substance synthesized at the VV Zakusov Research Institute of Pharmacology) in doses of 0.01-20.0 mg / kg, intraperitoneally (i.p.), was injected at the rate of 0.1 ml / 10 g of animal weight. Control animals received water for injection. A classic tricyclic antidepressant amitriptyline was used as a reference drug, which was diluted in water and injected intravenously at a dose of 10 mg / kg. The choice of the dose of the reference drug is based on literature data (Takamori K et al. Pharmacology. 2001; 3: 63: 147-53).

Methodology

The Porsolt forced swim test was used to evaluate antidepressant activity (Porsolt R.D. et al. Eur. J. Pharmacol. 1978; 47: 379-391). Mice were placed in cylinders (30 cm high and 10 cm in diameter) filled two-thirds with water at a temperature of 22 ° C for 5 min, during which the time of maintaining the characteristic posture of immobilization (refusal from active-defensive and exploratory behavior) was assessed. The behavior of the animals was recorded using a video camera. The video recording of the experiment was processed in a semi-automatic mode using the RealTimer software (NPK Open Science LLC). A decrease in the duration of immobilization was regarded as a manifestation of antidepressant activity.

The hot plate test was used to assess the nociceptive response. The latent reaction time (licking a hind paw or jumping) was recorded using an Ugo Basile analgesimeter (Italy). Animals were selected 1-2 h before the start of the experiment on the basis of basic reactivity under the conditions of this experimental model, excluding mice that remained on the plate heated to 55 ± 0.5 ° C for longer than 10 s. A latency period of 20 s (maximum exposure time) was regarded as 100% analgesia. The time of the onset of the reaction in mice was recorded 30, 60, 90, and 120 min after a single injection of the studied drugs. The results obtained were expressed as the maximum possible effect (MPE) in%. MVE = (latency period of reaction after drug administration minus background latency period of reaction) / (maximum exposure time minus background latency period of reaction) X 100%

Statistics. The results were processed using the Student's t-test. The critical level of significance is α = 0.05.

The invention is illustrated by the following examples

Example 1. Obtaining GZK-111

Receiving GZK-111

C ₆ H ₅ -CH ₂ -C (O) -Gly-OH. To a solution of 7.5 g (100 mmol) of glycine in 25 ml of 4 M NaOH at -10 ° C with vigorous stirring were added 13.24 ml (100 mol) of phenylacetyl chloride and 25 ml of 4 M NaOH. Then the reaction mixture was stirred with cooling for 30 min, then the external cooling was removed and the impurities and unreacted acid chloride were extracted with ethyl acetate (3 × 15 ml). Then the solution was acidified with 4 M HCl to pH 2-3 according to a universal indicator, the precipitate that formed was filtered off, washed with water, and dried in air for a day. 5.83 g (30%) of N-phenylacetylglycine were obtained; t. pl. 143-144 ° C; Rf 0.8 (A). PMR spectrum (DMSO-d6 + CF3COOD) δ, ppm: 3.47 (s, 2H, CH ₂ Ar), 3.76 (d, J 6.0 Hz, 2H, CH ₂ Gly), 7, 27 (m, 5H, ArH), 8.40 (t, J 6.0 Hz, 1H, NH Gly). (Young DW et al. Eur. J. Biochem. 1977; 75: 133-147) mp. 138-143 ° C.

HL-Pro-OC ₂ H ₅ HCl. To 60 ml of absolute ethanol, cooled to -20 ° C, 2.54 ml (35 mmol) of thionyl chloride was added dropwise and 2.0 g (17.5 mmol) of L-proline were added in portions. Then the reaction mixture was stirred for 2 h at -5 ° C and for 2 h at room temperature. The solvent was removed in vacuo; this operation was repeated twice, each time adding 30 ml of absolute ethanol. There was obtained 2.4 g (77%) of L-proline ethyl ester hydrochloride in the form of an oil. [α] 23D -43 ° (c 3, ethanol); Rf 0.75 (B). PMR spectrum (DMSO-d6) δ, ppm: 1.19 (m, 3H, CH ₃ CH ₂ O), 1.8-2.1 (m, 4H, СγН ₂ Pro, СβН ₂ Pro) , 3.2 (m, 2H, CδH ₂ Pro), 4.2 (m, 2H, CH ₃ CH ₂ O), 4.5 (m, 1H, CαH Pro), 9.9 (br.s, 1H , NH) (Nazarova L.S. et al. Chem.-Pharm. J. 1984; 18 (12): 1445-1448), oil; [α] 23D -44.8 ° (c 3.03, ethanol).

C ₆ H ₅ -CH ₂ -C (O) -Gly-L-Pro-OEt (GZK-111). 1.93 g (10 mmol) of N-phenylacetylglycine was dissolved in 10 ml of dry DMF. At -10 ° C with vigorous stirring, 1.35 ml (10 mmol) of isobutyl chloroformate and 1.1 ml (10 mmol) of N-methylmorpholine were added simultaneously. After stirring for 2-3 minutes, a solution of 1.79 g (10 mmol) of proline ethyl ester hydrochloride and 1.1 ml (10 mmol) of N-methylmorpholine in 15 ml of DMF was added dropwise. The reaction mixture was stirred for 30 min at -10 ° C, then the external cooling was removed and stirred for 1 h at room temperature. The precipitate was filtered off, the filtrate was evaporated in vacuum, the residue was dissolved in 25 ml of CHCl ₃ , the solution was successively washed with 3% NaHCO ₃ (3 × 7 ml), water (3 × 7 ml), 1 M HCl solution (3 × 7 ml) and again water (3 × 7 ml), dried over sodium sulfate, the desiccant was filtered off, the filtrate was evaporated on a rotary evaporator. 2.87 g (90%) of a chromatographically homogeneous product were obtained in the form of an orange oil, crystallized from a mixture of ethyl acetate and diethyl ether. m.p. 111-112 ° C; [a] 23D -90.0 ° (c 1, water); Rf = 0.80 (B). PMR spectrum (DMSO-d6) δ, ppm: 1.2 (m, 3H, CH ₃ CH ₂ O), 1.8-2.1 (m, 4H, СγН ₂ Pro, СβН ₂ Pro) , 3.5 (s, 2H, CH ₂ ArH), 3.6 (m, 2H, СδН ₂ Pro), 3.85-4.0 (2 dd, J 17.6 Hz, J 5.6 Hz, 2H , СαН ₂ Gly), 4.2 (m, 2H, CH ₃ CH ₂ O), 4.5 (dd, J 4.0 Hz, J 8.5 Hz, 1H, СαН Pro), 6, 4 (br. T, J 5.6 Hz, 1H, NH), 7.3 (m, 5H, ArH). Found,%: C 64.27; H 7.05%; N 8.63. C ₁₇ H ₂₂ N ₂ O ₄ . Calculated,%: C 64.13; H 6.97; N 8.80.

Example 2. Antidepressant properties of GZK-111

The experiments were performed on outbred male mice (n = 160). GZK-111 in the dose range 0.01-20 mg / kg, intraperitoneally (ip) in an aqueous solution was injected to mice daily for 7 days or 14 days. Control animals received water for injection in the same regimen. In the first series of experiments, the antidepressant effect of GZK-111 was assessed at doses of 0.1; 0.5; 1.0 and 5.0 mg / kg with 7-day administration, in the second series - at doses of 0.01; 5.0; 10.0 and 20.0 mg / kg with 7-day administration, and in the third series - 0.1; 1.0 and 10.0 mg / kg and 10.0 mg / kg orally for 14 days. Each group consisted of 10 mice.

It was found that the total time of immobilization in control animals averaged 235.8 s in the first experiment, 275.3 s in the second experiment and 180.1 s in the third experiment. With 7-day administration of GZK-111 at a dose of 10 mg / kg, it statistically significantly reduced the immobilization time by 11% (Table 1), the effect of GZK-111 in severity was comparable to the effect of amitriptyline at a dose of 10 mg / kg. When administered for 14 days, GZK-111 had a significant antidepressant effect at a dose of 10 mg / kg, and GZK-111 tended to decrease the immobilization time at a dose of 10 mg / kg (p = 0.08) with parenteral administration and at a dose of 10.0 mg / kg (p = 0.06) after oral administration (Table 2).

Thus, the standard test for assessing depressive behavior revealed a dose-dependent antidepressant activity of GZK-111, which is most pronounced at a dose of 10 mg / kg with parenteral 7-day and 14-day administration. There was a tendency to preservation of antidepressant activity at the maximum effective dose of 10.0 mg / kg after oral administration for 14 days.

Example 3. Analgesic properties of GZK-111

The experiments were performed on inbred male mice of the C57B1 / 6 line (n = 50) GZK-111 at doses of 0.5, 1.0, 2.0, and 4.0 mg / kg, i.p., was injected once at the rate of 0.1 ml / 10 g of animal weight per 30 minutes before the start of testing at the first time point. Control animals received water for injection.

The average baseline level of nociceptive response for C67B1 / 6 mice in the hot plate test was 6.7 ± 0.4 s. Animals with a reaction latency of more than 10 s were not included in the experiment.

When simulating acute somatic pain, it was found that 30 minutes after administration of the drug, the pain response thresholds in the experimental groups did not differ from the control, a pronounced analgesic effect developed 60 minutes after systemic administration of GZK-111 at doses of 1.0 and 2.0 mg / kg (p <0.05 ) and after 60 and 120 min - at doses of 2.0 and 4.0 mg / kg (p <0.05) (Fig. 1). The most pronounced antinociceptive effect of GZK-111 was manifested at a dose of 2.0 mg / kg 60 min after administration. The data obtained indicate that GZK-111 showed dose-dependent analgesic activity, starting from the 60th minute to the 120th minute of observation, statistically significantly (p <0.01, p <0.05) increased the pain response threshold in mice in the test “ hot plate "(Fig. 1).

Thus, for the first time, the central analgesic properties of GZK-111 have been shown when simulating acute somatic pain at the supraspinal level.

Description of drawings

FIG. 1 Effect of GZK-111 on changes in the pain response threshold during thermal stimulation in C57BL / 6 mice

The abscissa shows the development time of the effect after drug administration (min),

the ordinate is the maximum possible effect (MPE),%.

Experimental groups:

"Control" - white column;

"GZK-111 at a dose of 0.5 mg / kg" - light gray column;

"GZK-111 at a dose of 1.0 mg / kg" - gray column;

"GZK-111 at a dose of 2.0 mg / kg" - dark gray column;

"GZK-111 at a dose of 4.0 mg / kg" - black column.

* - p <0.05, ** - p <0.01 - statistically significant in relation to the corresponding control, according to Student's t-test, the number of animals in groups n = 10; data are presented as mean values (mean).

Claims (1)

The use of substituted glyproline - ethyl ester of N-phenylacetylglycyl-L-proline (GZK-111) as an agent with analgesic activity.

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