**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.

The **neuroprotective effect** of GZK-111 was studied **in vitro** in a 6-OHDA toxicity model in SH-SY5Y cells (cellular model of Parkinson's disease) [19, 31], which demonstrated the neuroprotective effect of CPG previously [6]. GZK-111 was introduced 24 h before 6-OHDA, because pharmacological effects of its metabolite (CPG) were probably caused by BDNF synthesis [12], which took no less than 6 h [32]. When introduced 24 h before the injury, GZK-111 was revealed to have neuroprotective activity at concentrations of 10<sup>7</sup> and 10<sup>6</sup> M (**Figure 4**). The range of active concentrations of CPG was 10<sup>8</sup> –10<sup>5</sup> M in this model [6].

The **neuroprotective activity** of GZK-111 was studied **in vivo** in a model of ischemic stroke caused by transient occlusion of the middle cerebral artery in rats, according to [20]. This model allows to simulate the most common cerebrovascular accident—an extensive ischemic stroke in the middle cerebral artery basin—and provides a reproducible volume of ischemic injury [33]. The compound was administered, i.p., at a dose of 0.5 mg/kg/day for 7 days; the first injection was given 6 h after surgery. The GZK-111 dose was taken accordingly to the results of other pharmacological studies (see **Tables 1** and **2**). The duration of substance introduction corresponds to the period of poststroke treatment in a hospital with consideration to differences in the metabolic rates of animals and humans [34]. The first administration of the compound 6 h after ischemia is explained by preservation of penumbra zone during this period [35], which makes it possible to decrease the volume of ischemic damage. Neurological functions were evaluated 3 and 6 days after surgery using the limb-placing test [21]. The infarct volume was evaluated 7 days after surgery according to [22] with 2,3,5-triphenyltetrazolium chloride staining and computerized image analysis. GZK-111 was found to have pronounced neuroprotective effects, reducing the volume of the infarct zone by 1.6 times (**Figure 5**) and improving the neurological status of animals by approximately 30% (**Figure 6**).

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 [7].

The **nootropic antiamnesic effect** of GZK-111 was evaluated by its ability to prevent impaired reproduction of the conditioned passive avoidance reflex (passive avoidance reaction) in rats caused by electroconvulsive shock (ECS) by the Ader method [24]. The test is based on the inborn hole exploratory behavior of rodents.

*The investigation of GZK metabolism in blood plasma by HPLC method. (A) Standards: (1) cyclo-Lprolylglycine, (2) N-phenylacetyl-glycyl-L-proline, and (3) N-phenylacetyl-glycyl-L-proline ethyl ester;*

*(B) control blood plasma; (C) blood plasma + GZK-111, 10-h incubation.*

*Glyproline Pro-Ampakine with Neuroprotective Activity DOI: http://dx.doi.org/10.5772/intechopen.91192*

**Figure 2.**

**261**

#### **Figure 1.** *The scheme of synthesis of N-phenylacetyl-glycyl-L-proline ethyl ester enantiomers.*

*Glyproline Pro-Ampakine with Neuroprotective Activity DOI: http://dx.doi.org/10.5772/intechopen.91192*

**3.2 Biotransformation of GZK-111 to CPG**

*Neuroprotection - New Approaches and Prospects*

**3.3 Pharmacological effects of GZK-111**

range of active concentrations of CPG was 10<sup>8</sup>

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**.

Based on the previously established pharmacological effects of CPG, the pharmacological activity of GZK-111 (viz., neuroprotective, antiamnesic, antihypoxic,

The **neuroprotective effect** of GZK-111 was studied **in vitro** in a 6-OHDA toxicity model in SH-SY5Y cells (cellular model of Parkinson's disease) [19, 31], which demonstrated the neuroprotective effect of CPG previously [6]. GZK-111 was introduced 24 h before 6-OHDA, because pharmacological effects of its metabolite (CPG) were probably caused by BDNF synthesis [12], which took no less than 6 h [32]. When introduced 24 h before the injury, GZK-111 was revealed to have neuroprotective activity at concentrations of 10<sup>7</sup> and 10<sup>6</sup> M (**Figure 4**). The

The **neuroprotective activity** of GZK-111 was studied **in vivo** in a model of ischemic stroke caused by transient occlusion of the middle cerebral artery in rats, according to [20]. This model allows to simulate the most common cerebrovascular accident—an extensive ischemic stroke in the middle cerebral artery basin—and provides a reproducible volume of ischemic injury [33]. The compound was administered, i.p., at a dose of 0.5 mg/kg/day for 7 days; the first injection was given 6 h after surgery. The GZK-111 dose was taken accordingly to the results of other pharmacological studies (see **Tables 1** and **2**). The duration of substance introduction corresponds to the period of poststroke treatment in a hospital with consideration to differences in the metabolic rates of animals and humans [34]. The first administration of the compound 6 h after ischemia is explained by preservation of penumbra zone during this period [35], which makes it possible to decrease the volume of ischemic damage. Neurological functions were evaluated 3 and 6 days after surgery using the limb-placing test [21]. The infarct volume was evaluated 7 days after surgery according to [22] with 2,3,5-triphenyltetrazolium chloride staining and computerized image analysis. GZK-111 was found to have pronounced neuroprotective effects, reducing the volume of the infarct zone by 1.6 times (**Figure 5**) and improv-

ing the neurological status of animals by approximately 30% (**Figure 6**).

bilateral common carotid arteries occlusion in rats [7].

*The scheme of synthesis of N-phenylacetyl-glycyl-L-proline ethyl ester enantiomers.*

**Figure 1.**

**260**

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

–10<sup>5</sup> M in this model [6].

anxiolytic, antidepressant-like, and analgesic effects) was studied.

#### **Figure 2.**

*The investigation of GZK metabolism in blood plasma by HPLC method. (A) Standards: (1) cyclo-Lprolylglycine, (2) N-phenylacetyl-glycyl-L-proline, and (3) N-phenylacetyl-glycyl-L-proline ethyl ester; (B) control blood plasma; (C) blood plasma + GZK-111, 10-h incubation.*

The **nootropic antiamnesic effect** of GZK-111 was evaluated by its ability to prevent impaired reproduction of the conditioned passive avoidance reflex (passive avoidance reaction) in rats caused by electroconvulsive shock (ECS) by the Ader method [24]. The test is based on the inborn hole exploratory behavior of rodents.

**Figure 3.** *The scheme of GZK-111 metabolism.*

**Figure 4.**

*The neuroprotective effect of GZK-111 in a 6-OHDA toxicity model, SH-SY5Y cells. Notes: \*p < 0.05 comparison with the control, ^p < 0.05 comparison with the damage group (Kruskal-Wallis ANOVA test with Dunn's post hoc).*

An animal learns not to enter the dark compartment using electric pain stimulation. Once the animal is subjected to EKS after training, the memorial trail is erased, and the animal again enters the dark chamber. Nootropic activity is defined as the ability to decrease the amnesic effect of ECS. GZK-111 was demonstrated to have antiamnesic activity at doses of 0.1, 0.5, and 1.0 mg/kg, i.p. (see **Table 1**), i.e., at least in the same dose range as CPG. Unlike the L-isomer, the D-stereoisomer of GZK-111, ethyl ester of *N*-phenylacetyl-glycyl-*D*-proline (GZK-121), had no antiamnesic activity.

exhibit an antihypoxic effect at a dose of 0.5 mg/kg, significantly increasing the life expectancy of animals (see **Table 2**). The stereoisomer GZK-121 at doses of 0.1, 0.5,

*The effects of GZK-111 and GZK-121 in normobaric hypoxia test with hypercapnia in mice, compared to*

L-CPG [6] 20.9 � 0.6 20.2 � 0.8 24.0 � 0.7\* 25.0 � 1.6\*\* D-CPG [37] 21.8 � 1.7 23.3 � 1.5 22.7 � 0.5 22.3 � 1.4 GZK-111 27.1 � 0.9 25.2 � 0.5 30.2 � 0.7\* 26.2 � 0.3 GZK-121 25.8 � 0.8 23.9 � 0.7 26.0 � 0.7 23.5 � 0.8

**Compound Dose, mg/kg, i.p. (n = 10) Latent period, s Effect, %**

L-CPG [3] 0.05 91 � 34 19 � 8° 25 � 7 +8

D-CPG [36] 0.1 113 � 12 48 � 11° 27 � 8\* �32\* GZK-111 0.1 180 � 0 129 � 28° 169 � 10\* +76\*

GZK-121 0.5 180 � 0 95 � 31° 133 � 23 +44

*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*

*\*р < 0.05 comparison with the amnesia group (Mann-Whitney U test with a Bonferroni correction).*

*The effects of GZK-111 and GZK-121 in passive avoidance reaction test, compared to CPG.*

**Control Amnesia Amnesia + compound**

0.1 91 � 34 19 � 8° 73 � 26\* +75\* 1.0 91 � 34 19 � 8° 43 � 19\* +33\*

0.5 180 � 0 100 � 25 176 � 6\* +95\* 1.0 180 � 0 129 � 28° 179 � 2\* +98\*

LPcontrol�LPamn � 100%*: where AA% is antiamnesic*

**Control 0.1 0.5 1.0 Life expectancy, min**

The **anxiolytic activity** of GZK-111 was studied in rats at doses of 0.75, 1.5, and 3.0 mg/kg, i.p., using the elevated plus maze test (EPM) according to Pellow [26]. This test is the primary model for anxiolytic activity detection; it is based on the conflict between rodents' inherent fear of open spaces and natural exploratory behavior. A significant anxiolytic effect was observed at a dose of 1.5 mg/kg. GZK-111 increased the time rats spent in the open arms by 12 times relative to the control, which is comparable with the effect of CPG. The enantiomer, GZK-121, was

and 1.0 mg/kg showed no antihypoxic activity in this test.

*Notes: The outbred white male mice were used in the experiments;*

*\*\*p < 0.01 comparison with the control (Mann-Whitney U test).*

*n, number of animals; \*p < 0.05;*

*Notes: n, number of animals; °р < 0.05 comparison with the control;*

**Table 2.**

**Table 1.**

*CPG.*

**263**

**Compound Dose, mg/kg, i.p. (n = 10)**

*Antiamnesic activity (AA) was calculated as follows:* AA% <sup>¼</sup> LPtest�LPamn

*chamber in animals administered with 0.9% NaCl without amnesia.*

*Glyproline Pro-Ampakine with Neuroprotective Activity DOI: http://dx.doi.org/10.5772/intechopen.91192*

inactive at a dose of 1.5 mg/kg (see **Table 3**).

Thus, GZK-111 possesses an antiamnesic activity similarly to CPG, and its effect is stereospecific, like that of CPG. However, if the *D*-enantiomer of CPG exhibits a pro-amnesic effect, the *D*-enantiomer of GZK-111 (GZK-121) has no activity. The possible explanation is that *D*-CPG blocks the receptor, while GZK-121 is not metabolized to CPG at all.

The **antihypoxic activity** of GZK-111 was studied in the normobaric hypoxia test with hypercapnia ("canned" hypoxia) in mice, which is the simplest method to assess antihypoxic activity [25]. GZK-111 was administered at doses of 0.1, 0.5, and 1.0 mg/kg, i.p., 60 min before test. Likewise CPG, the compound was found to

#### *Glyproline Pro-Ampakine with Neuroprotective Activity DOI: http://dx.doi.org/10.5772/intechopen.91192*


*Notes: n, number of animals;*

*°р < 0.05 comparison with the control;*

*\*р < 0.05 comparison with the amnesia group (Mann-Whitney U test with a Bonferroni correction).*

*Antiamnesic activity (AA) was calculated as follows:* AA% <sup>¼</sup> LPtest�LPamn LPcontrol�LPamn � 100%*: 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.*

#### **Table 1.**

*The effects of GZK-111 and GZK-121 in passive avoidance reaction test, compared to CPG.*


*Notes: The outbred white male mice were used in the experiments;*

*n, number of animals;*

*\*p < 0.05;*

*\*\*p < 0.01 comparison with the control (Mann-Whitney U test).*

#### **Table 2.**

An animal learns not to enter the dark compartment using electric pain stimulation. Once the animal is subjected to EKS after training, the memorial trail is erased, and the animal again enters the dark chamber. Nootropic activity is defined as the ability to decrease the amnesic effect of ECS. GZK-111 was demonstrated to have antiamnesic activity at doses of 0.1, 0.5, and 1.0 mg/kg, i.p. (see **Table 1**), i.e., at least in the same dose range as CPG. Unlike the L-isomer, the D-stereoisomer of GZK-111, ethyl ester of *N*-phenylacetyl-glycyl-*D*-proline (GZK-121), had no

*The neuroprotective effect of GZK-111 in a 6-OHDA toxicity model, SH-SY5Y cells. Notes: \*p < 0.05 comparison with the control, ^p < 0.05 comparison with the damage group (Kruskal-Wallis ANOVA test with*

Thus, GZK-111 possesses an antiamnesic activity similarly to CPG, and its effect is stereospecific, like that of CPG. However, if the *D*-enantiomer of CPG exhibits a pro-amnesic effect, the *D*-enantiomer of GZK-111 (GZK-121) has no activity. The possible explanation is that *D*-CPG blocks the receptor, while GZK-121 is not

The **antihypoxic activity** of GZK-111 was studied in the normobaric hypoxia test with hypercapnia ("canned" hypoxia) in mice, which is the simplest method to assess antihypoxic activity [25]. GZK-111 was administered at doses of 0.1, 0.5, and 1.0 mg/kg, i.p., 60 min before test. Likewise CPG, the compound was found to

antiamnesic activity.

**Figure 3.**

**Figure 4.**

**262**

*Dunn's post hoc).*

*The scheme of GZK-111 metabolism.*

*Neuroprotection - New Approaches and Prospects*

metabolized to CPG at all.

*The effects of GZK-111 and GZK-121 in normobaric hypoxia test with hypercapnia in mice, compared to CPG.*

exhibit an antihypoxic effect at a dose of 0.5 mg/kg, significantly increasing the life expectancy of animals (see **Table 2**). The stereoisomer GZK-121 at doses of 0.1, 0.5, and 1.0 mg/kg showed no antihypoxic activity in this test.

The **anxiolytic activity** of GZK-111 was studied in rats at doses of 0.75, 1.5, and 3.0 mg/kg, i.p., using the elevated plus maze test (EPM) according to Pellow [26]. This test is the primary model for anxiolytic activity detection; it is based on the conflict between rodents' inherent fear of open spaces and natural exploratory behavior. A significant anxiolytic effect was observed at a dose of 1.5 mg/kg. GZK-111 increased the time rats spent in the open arms by 12 times relative to the control, which is comparable with the effect of CPG. The enantiomer, GZK-121, was inactive at a dose of 1.5 mg/kg (see **Table 3**).

**Figure 5.**

*GZK-111 reduces the cerebral infarct volumes in rats with experimental stroke. Notes: Morphometric measurements of infarct volume were performed using TTC staining. The data are presented as mean SEM. \*p = 0.05 compared to the "stroke" group (Mann-Whitney U test).*

The tricyclic antidepressant amitriptyline (10.0 mg/kg, i.p.) was used as a comparison drug. GZK-111 administered at a dose of 10 mg/kg for 7 days was established to statistically significantly reduce the immobility time by 11% (**Table 4**), which is

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**).

Recently, the antidepressant activity of started CPG was also discovered in the

The **analgesic properties** of GZK-111 were determined in the hot plate test in inbred male C57Bl/6 mice [39] at acute i.p. administration at doses of 0.5, 1.0, 2.0, and 4.0 mg/kg. This test was widely used to study the pain sensitivity in rodents at supraspinal level, and the effectiveness of analgesics evaluates the pain response on paw pad contact with a hot surface; the time until the hind paw pulling and/or jumping is measured. The average baseline nociceptive response for C67Bl/6 mice

p = 0,36,098) 30 min after administration at each of the doses studied. At a dose of 2.0 mg/kg, GZK-111 statistically significantly increased the latent period of the nociceptive reaction (F(4, 44) = 3.1489, p = 0.02319) 60 min (p < 0.01) and 120 min (p < 0.05) after administration, compared with the control group. At a

forced swimming test in mice (**Table 5**) [9] and in an experimental model of

comparable to the effect of amitriptyline at a dose of 10 mg/kg.

*The effects of GZK-111 and GZK-121 in EPM test in rats, compared to CPG.*

*n, number of animals; m, number of entries into the open arms of the maze;*

*\*\*p < 0.001 comparison with the control (Mann-Whitney U test).*

*\*p < 0.05;*

**Table 3.**

**265**

**Compound Dose, mg/kg, i.p. (n = 10) Number of entries into the**

*Glyproline Pro-Ampakine with Neuroprotective Activity DOI: http://dx.doi.org/10.5772/intechopen.91192*

**open arms**

0.1 1.79\* 895\* 13.8\* 641\* 1.0 0.25 125 1.46 68 Control 2 0.58 100 6.38 100 0.05 1.60\*\* 276\*\* 57.4\*\* 900\*\* 0.2 1.50 259 19.3 303

0.05 0.34 170 3.87 180 0.1 0.26 132 2.58 120

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

1.5 0.2 0.2 100 4.7 1.5 114

L-CPG [4] Control 1 0.20 100 2.15 100

D-CPG [4] Control 0.20 100 2.15 100

GZK-111 Control 0.9 0.6 100 4.8 2.5 100

GZK-121 Control 0.2 0.2 100 4.1 1.5 100

*Notes: Number of entries into the open arms and time spent in the open arms were taken as 100% for control animals.*

**Time spent in the open arms**

**m % s%**

learned helplessness in rats [10] at i.p. administration for 14 days.

was 8.2 0.2 s. GZK-111 showed no analgesic effect (F(4, 44) = 11,163,

#### **Figure 6.**

*GZK-111 improves neurological status in rats with experimental stroke. Notes: Neurological status is given for the injured side of the body (which is the opposite to the ischemic lesion) in the limb-placing test. The data are presented as mean SEM. \*p = 0.003 compared to the "sham" group, #p = 0.01 compared to the "stroke" group (Mann-Whitney U test).*

The **antidepressant-like effect** of GZK-111 was studied in the forced swimming test [27], one of the most widely used for this purpose [38]. The test is based on the ability of antidepressants to reduce the time of immobility in case of unavoidable swimming of the animal in a cylinder with water. The study was performed in mice at daily i.p. administration at a dose range of 0.01–20.0 mg/kg within 7 or 14 days.


#### *Glyproline Pro-Ampakine with Neuroprotective Activity DOI: http://dx.doi.org/10.5772/intechopen.91192*

*Notes: Number of entries into the open arms and time spent in the open arms were taken as 100% for control animals. n, number of animals; m, number of entries into the open arms of the maze;*

*\*p < 0.05;*

*\*\*p < 0.001 comparison with the control (Mann-Whitney U test).*

#### **Table 3.**

*The effects of GZK-111 and GZK-121 in EPM test in rats, compared to CPG.*

The tricyclic antidepressant amitriptyline (10.0 mg/kg, i.p.) was used as a comparison drug. GZK-111 administered at a dose of 10 mg/kg for 7 days was established to statistically significantly reduce the immobility time by 11% (**Table 4**), which is comparable to the effect of amitriptyline at a dose of 10 mg/kg.

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**).

Recently, the antidepressant activity of started CPG was also discovered in the forced swimming test in mice (**Table 5**) [9] and in an experimental model of learned helplessness in rats [10] at i.p. administration for 14 days.

The **analgesic properties** of GZK-111 were determined in the hot plate test in inbred male C57Bl/6 mice [39] at acute i.p. administration at doses of 0.5, 1.0, 2.0, and 4.0 mg/kg. This test was widely used to study the pain sensitivity in rodents at supraspinal level, and the effectiveness of analgesics evaluates the pain response on paw pad contact with a hot surface; the time until the hind paw pulling and/or jumping is measured. The average baseline nociceptive response for C67Bl/6 mice was 8.2 0.2 s. GZK-111 showed no analgesic effect (F(4, 44) = 11,163, p = 0,36,098) 30 min after administration at each of the doses studied. At a dose of 2.0 mg/kg, GZK-111 statistically significantly increased the latent period of the nociceptive reaction (F(4, 44) = 3.1489, p = 0.02319) 60 min (p < 0.01) and 120 min (p < 0.05) after administration, compared with the control group. At a

The **antidepressant-like effect** of GZK-111 was studied in the forced swimming test [27], one of the most widely used for this purpose [38]. The test is based on the ability of antidepressants to reduce the time of immobility in case of unavoidable swimming of the animal in a cylinder with water. The study was performed in mice at daily i.p. administration at a dose range of 0.01–20.0 mg/kg within 7 or 14 days.

*GZK-111 improves neurological status in rats with experimental stroke. Notes: Neurological status is given for the injured side of the body (which is the opposite to the ischemic lesion) in the limb-placing test. The data are presented as mean SEM. \*p = 0.003 compared to the "sham" group, #p = 0.01 compared to the "stroke"*

*GZK-111 reduces the cerebral infarct volumes in rats with experimental stroke. Notes: Morphometric measurements of infarct volume were performed using TTC staining. The data are presented as mean SEM.*

*\*p = 0.05 compared to the "stroke" group (Mann-Whitney U test).*

*Neuroprotection - New Approaches and Prospects*

**Figure 5.**

**Figure 6.**

**264**

*group (Mann-Whitney U test).*


*Notes: Immobilization time was taken as 100% for control animals.*

*n, number of animals;*

*\*р < 0.05, the statistical significance of the differences relative to control group by the unpaired t-Student criterion.*

**4. Conclusions**

**Figure 7.**

mechanism of action.

**Conflict of interest**

**Funding**

**267**

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

*GZK-111 influences the pain response threshold during thermal stimulation in C57BL/6 mice. X-axis, time of the effect development after substance administration (min); y-axis, maximum possible effect (MPE), %. \*p < 0.05; \*\* p < 0.01, statistically significant in relation to the corresponding control; according to Duncan*

*criterion, the number of animals in groups n = 10; data are presented as mean values.*

*Glyproline Pro-Ampakine with Neuroprotective Activity DOI: http://dx.doi.org/10.5772/intechopen.91192*

The authors declare no conflict of interest, financial or otherwise.

This work was supported by RFBR (project 20-015-00102).

#### **Table 4.**

*The effect of GZK-111 on immobilization time in the Porsolt forced swimming test in mice after 7-day administration.*


*Notes: Immobilization time was taken as 100% for control animals.*

*n, number of animals;*

*\*<sup>р</sup> <sup>&</sup>lt; 0.05, the statistical significance of the differences relative to control group by the unpaired t-Student criterion; # p < 0.05;*

#### **Table 5.**

*The effect of GZK-111 on immobilization time in the Porsolt forced swimming test in mice after 14-day administration, compared to CPG.*

dose of 4 mg/kg, an increase of the reaction thresholds induced by GZK-111 was observed after 90 min (F(4, 44) = 3.8743, p = 0.00881) that persisted for up to 2 h (F(4, 44) = 3.2141, p = 0.02124), compared with the control group (p < 0.05 and p < 0.05, respectively) (**Figure 7**). The data obtained indicate that GZK-111 exhibits dose-dependent analgesic activity, like CPG in rats [40].

*Glyproline Pro-Ampakine with Neuroprotective Activity DOI: http://dx.doi.org/10.5772/intechopen.91192*

#### **Figure 7.**

*GZK-111 influences the pain response threshold during thermal stimulation in C57BL/6 mice. X-axis, time of the effect development after substance administration (min); y-axis, maximum possible effect (MPE), %. \*p < 0.05; \*\* p < 0.01, statistically significant in relation to the corresponding control; according to Duncan criterion, the number of animals in groups n = 10; data are presented as mean values.*

## **4. Conclusions**

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.
