The effects of GZK-111 and GZK-121 in passive avoidance reaction test, compared to CPG.
Previously it was shown that neuropeptide cyclo-L-prolylglycine (CPG) is a positive modulator of AMPA receptors, which increases BDNF level in neuronal cell cultures. The spectrum of CPG’s pharmacological effects corresponds to that of BDNF. Dipeptide N-phenylacetyl-glycyl-L-proline ethyl ester (GZK-111) was designed and synthesized as a linear analog of CPG. The aim of the present work was to reveal the pharmacological profile of GZK-111. Dipeptide GZK-111 was shown to metabolize into CPG in vitro and increased cell survival by 28% at concentrations of 10-7–10-6 M in a Parkinson’s disease cell model. In a model of cerebral ischemia, GZK-111, at a dose of 0.5 mg/kg, i.p., was found to have neuroprotective effects, reducing the cerebral infarct volume by 1.6 times. Similar to CPG, GZK-111, at the range 0.1–1.0 mg/kg, i.p., possessed a stereospecific antiamnesic activity. A significant anxiolytic effect was observed at a dose of 1.5 mg/kg. GZK-111, at the range 0.5–4.0 mg/kg, i.p., demonstrated analgesic activity. GZK-111, at a dose of 10 mg/kg/7 days, i.p., possessed antidepressant-like activity. So, the neuroprotective, nootropic, antihypoxic, anxiolytic, antidepressant-like, and analgesic effects of GZK-111 were revealed. Thus, GZK-111 can be considered as a pharmacologically active pro-ampakine with a BDNF-ergic mechanism of action.
- glyproline GZK-111
- neuroprotective activity
CPG is a hydrophilic compound. The task to create an amphiphilic CPG prodrug with improved pharmacokinetic properties, converted to active molecule in the brain, was established to increase drug passage through biological membranes, including the blood-brain barrier. Two variants of substituted dipeptides, based on the Pro-Gly or Gly-Pro sequence, could be used for this purpose. We selected the second one (i.e., Gly-Pro), following the known information that an imide bond with proline in Gly-Pro dipeptide sequence increased the proportion of the cisoid peptide bond [13, 14], which, in turn, promoted cyclization of the dipeptide [15, 16].
In this work, we synthesized substituted glyproline
2. Materials and methods
2.1 Chemical experimental part
The chemical reagents used in the synthesis were obtained from commercial suppliers and used without purification. All solvents were dried and purified by standard procedures if required. Melting points were measured in open capillary tubes using OptiMelt melting point apparatus (Stanford Research Systems, USA). The structures of the compounds were confirmed by elemental analysis and 1H NMR spectroscopy. The NMR spectra were obtained on a Bruker Fourier 300 (Bruker, Germany) spectrometer using tetramethylsilane as an internal standard. The NMR peaks were designated as follows: s, singlet; d, doublet; t, triplet; and m, multiplet. Microanalyses for C, H, and N agreed with calculated values within 0.4%. Specific optical rotations were recorded by automatic polarimeter ADP 440 (Bellingham + Stanley Ltd., England). The TLC was carried out on Merck silica gel 60 F 254 plates with spot visualization by iodine vapor or UV light.
2.2 Biological experimental
2.2.1 Study of GZK-111 metabolism
2.2.2 In vitro pharmacological study
At the end of the experiment, the culture medium was replaced with MTT solution (0.5 mg/ml) and incubated for 30 min at 37°C. Then, a MTT solution was removed from the wells, and DMSO was added to dissolve the formazan. Absorbance was measured at 600 nm using a 96-well plate reader Multiscan (Thermo, USA).
2.2.3 In vivo pharmacological studies
In vivo studies were performed in outbred male rats weighing 200–270 g (nootropic and a anxiolytic activity), in outbred male mice weighing 25–28 g (antihypoxic and antidepressant-like activity), and in C57Bl/6 male mice weighing 25–29 g (analgesic activity), received from the Stolbovaya Branch of the Scientific Center of Biomedical Technologies of the Federal Medical Biological Agency (FMBA) of Russia, and in Wistar male rats weighing 200–250 g (neuroprotective activity) received from the Andreevka Branch of the Scientific Center of Biomedical Technologies of the FMBA of Russia. The animals were kept in vivarium under natural circadian light/dark cycles with free access to standard granular feed and water. The study complied with the requirements of Order of the Ministry of Health of the Russian Federation No. 199 “On Approval of the Rules of Good Laboratory Practice” and Decision of the Council of the Eurasian Economic Commission No. 81 “On Approval of the Rules of Good Laboratory Practice of the Eurasian Economic Union in the Area of Circulation of Medicines.” All manipulations with animals were approved by the Bioethical Commission of the Zakusov Research Institute of Pharmacology. The experiments were carried out from 10 to 16 pm. The test substances were dissolved in saline or in distilled water and administered, i.p. The animals of the control groups were injected with saline or with distilled water, respectively.
2.2.4 Neuroprotective activity
The rats were anesthetized with an i.p. injection of chloral hydrate (350 mg/kg) as a 5% solution in saline. The right common carotid artery, internal carotid artery, and external carotid artery were surgically exposed. A nylon suture (0.25 mm in diameter) with a silicon-coated tip was inserted from the external carotid artery into the internal carotid artery and then to the circle of Willis to occlude the origin of the middle cerebral artery. After 1 h of MCAO, the suture was carefully removed to induce reperfusion. Sham-operated rats (n = 6) underwent identical surgery except that the suture was not inserted. During the surgery the body temperature was maintained at 37.0 ± 0.5°C using a heating pad. Three rats received MCAO without any neurological deficits observed after awakening was excluded.
Passive avoidance reaction was developed in rats in a certified Lafayette Instrument Co installation (USA) according to the method of  using a single training procedure. The illuminated start platform (25 × 7 cm) was connected to a dark 40 × 40 × 40 cm chamber equipped with an electrified floor through a square guillotine door. The animal was placed on the start platform with its tail to a dark chamber. When governed by the hole exploratory behavior, the rat found the entrance and passed into the dark compartment; the hole was closed. In the dark chamber, eight unavoidable electric pain stimuli were applied through the floor to the rat (the training current was 0.45 mA, the duration of each pulse was 1 s, and the interval between consecutive pulses was 2 s). Immediately after this, the rat was removed from the dark chamber and subjected to EKS (250 V, 120–122 mA, 0.1 s) applied transcorneally using a certified Harvard apparatus (Germany). After 24 h, the animal was again placed on the illuminated platform for learning test. The latent period of the first animal entry into the dark chamber was recorded. Antiamnesic activity (AA) was calculated as Eq. (1):
where AA% is antiamnesic activity, LPtest is the average latent period of entry into the chamber in the animals administered with the test compound and subjected to amnesia, LPamn is the average period of entry into the chamber in the animals administered with 0.9% NaCl and subjected to amnesia, and LPcontrol is the average latent period of entry into the chamber in animals administered with 0.9% NaCl without amnesia.
3. Results and discussions
3.1 Synthesis of GZK-111
3.2 Biotransformation of GZK-111 to CPG
Synthesized GZK-111 was subjected to biotransformation in the presence of plasma enzymes to show the fundamental possibility of its conversion to CPG. The initial plasma contained endogenous CPG at a concentration of 1 μM, according to RP HPLC. Upon GZK-111 incubation with plasma at 37°C for 10 h, an increase of the CPG peak and appearance of a peak corresponding to the retention time of the compound with an open carboxyl group, N-phenylacetyl-glycyl-L-proline, were observed (see Figure 2). Thus, CPG is actually formed from GZK-111 in the presence of blood plasma enzymes. The scheme of GZK-111 metabolism is shown in Figure 3.
3.3 Pharmacological effects of GZK-111
Based on the previously established pharmacological effects of CPG, the pharmacological activity of GZK-111 (viz., neuroprotective, antiamnesic, antihypoxic, anxiolytic, antidepressant-like, and analgesic effects) was studied.
|Compound||Dose, mg/kg, i.p. (n = 10)||Latent period, s||Effect, %|
|Control||Amnesia||Amnesia + compound|
|L-CPG ||0.05||91 ± 34||19 ± 8°||25 ± 7||+8|
|0.1||91 ± 34||19 ± 8°||73 ± 26*||+75*|
|1.0||91 ± 34||19 ± 8°||43 ± 19*||+33*|
|D-CPG ||0.1||113 ± 12||48 ± 11°||27 ± 8*||−32*|
|GZK-111||0.1||180 ± 0||129 ± 28°||169 ± 10*||+76*|
|0.5||180 ± 0||100 ± 25||176 ± 6*||+95*|
|1.0||180 ± 0||129 ± 28°||179 ± 2*||+98*|
|GZK-121||0.5||180 ± 0||95 ± 31°||133 ± 23||+44|
The neuroprotective activity of CPG (1.0 mg/kg, i.p., subchronic) was revealed previously in a model of incomplete global cerebral ischemia induced by permanent bilateral common carotid arteries occlusion in rats .
Thus, GZK-111 possesses an antiamnesic activity similarly to CPG, and its effect is stereospecific, like that of CPG. However, if the
|Compound||Dose, mg/kg, i.p. (n = 10)||Number of entries into the open arms||Time spent in the open arms|
|L-CPG ||Control 1||0.20||100||2.15||100|
|GZK-111||Control||0.9 ± 0.6||100||4.8 ± 2.5||100|
|0.75||1.3 ± 0.5||170||14.8 ± 7.0||180|
|1.50||3.2 ± 1.1||355||58.2 ± 13.3**||1212**|
|3.0||1.0 ± 0.5||132||6.4 ± 2.9||120|
|GZK-121||Control||0.2 ± 0.2||100||4.1 ± 1.5||100|
|1.5||0.2 ± 0.2||100||4.7 ± 1.5||114|
|Experimental groups||Dose, mg/kg, i.p. (n = 10)||Immobilization time, s||Immobilization time compared to the control, %|
|Control||0.0||235.8 ± 11.8||100|
|GZK-111||0.1||240.2 ± 19.8||101|
|GZK-111||0.5||235.3 ± 17.7||100|
|GZK-111||1.0||240.0 ± 22.1||101|
|GZK-111||5.0||253.9 ± 9.2||108|
|Amitriptyline||10.0||218.5 ± 17.9*||93|
|Control||275.3 ± 8.7||100|
|GZK-111||0.01||260.1 ± 7.3||94|
|GZK-111||5.0||268.7 ± 10.2||98|
|GZK-111||10.0||245.5 ± 9.1*||89|
|GZK-111||20.0||259.9 ± 9.2||94|
With a 14-day administration, GZK-111 had a significant antidepressant effect at a dose of 10 mg/kg, i.p., and showed a tendency to decrease immobility time at a dose of 1.0 mg/kg, i.p. (p = 0.08) and 10 mg/kg p.o. (p = 0.06) (Table 5).
|Compound||Dose, mg/kg, i.p. (n = 10)||Immobilization time, s||Immobilization time compared to the control, %|
|GZK-111||Control||180.1 ± 10.7||100|
|0.1||188.4 ± 9.3||105|
|1.0||163.2 ± 11.2 (p = 0.08)||91|
|10.0||157.7 ± 9.2*||88|
|10.0 (p.o.)||160.3 ± 10.4 (p = 0.06)||89|
|CPG ||Control||139 ± 8||100|
|1.0||97 ± 6#||70|
|2.0||101 ± 8#||73|
|Fluoxetine ||10.0||87 ± 9#||63|
Recently, the antidepressant activity of started CPG was also discovered in the forced swimming test in mice (Table 5)  and in an experimental model of learned helplessness in rats  at i.p. administration for 14 days.
Thus, GZK-111 exhibits nootropic, anxiolytic, antihypoxic, antidepressant, neuroprotective, and analgesic effects being characteristic to CPG. The compound is similar to CPG both in the spectrum of activities and in the stereospecificity and its nature. The ampakine CPG identical to endogenous one was proven to form during the fermentolysis of GZK-111. Therefore, GZK-111 can be considered as a “prodrug” of CPG and a pharmacologically active pro-ampakine with a BDNF-ergic mechanism of action.
Conflict of interest
The authors declare no conflict of interest, financial or otherwise.
This work was supported by RFBR (project 20-015-00102).
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