Assessment of mutagenicity and acute toxicity of linear substituted glyproline – ethyl ester of N-phenylacetylglycyl-L-proline

Introduction. Cyclic glycine-proline has a wide range of pharmacological activities, including analgesic action. Within the framework of the concept of creating a prodrug, a linear substituted glyproline – ethyl ester of N-phenylacetylglycyl-L-proline (GZK-111) was designed and synthesized. A mandatory stage of preclinical research is to study the safety of drug candidates.

The purpose of the work is to evaluate the mutagenicity and acute toxicity of GZK-111.

Materials and methods. When assessing the ability of GZK-111 to induce gene mutations in the Ames test, S.typhimurium strains TA98, TA100, TA1535, TA1537 and combination of E.coli strains pKM101/uvrA were treated with GZK-111 at concentrations of 1.6; 8; 40; 200; 1000, and 5000 μg/ml. When assessing acute toxicity in outbred mice, GZK-111 was administered intraperitoneally once at doses of 500, 1000, 2000 and 3000 mg/kg, followed by recording the terms of development of intoxication and describing the clinical signs for 14 days. Euthanasia and post-mortem examination were performed on the 15th day.

Results. In the Ames test, GZK-111 did not exhibit mutagenicity towards the indicator strains, either with or without metabolic activation. GZK-111 did not cause the death of most experimental animals. The clinical picture of intoxication showed a reversible neurotoxic effect of GZK-111, as well as a dose-dependent decrease in body weight. In the surviving animals, the morphological picture of the internal organs did not differ from that observed in the control group.

Conclusion. Linear substituted glyproline GZK-111 showed no mutagenic activity and can be classified as a relatively harmless compound in toxicity class 6 (classification by KK Sidorov, 1973).

1. Jakubczyk A, Karaś M, Rybczyńska-Tkaczyk K, et al. Current Trends of Bioactive Peptides-New Sources and Therapeutic Effect. Foods. 2020 Jun 29;9(7):846. doi: 10.3390/foods9070846.

2. Perlikowska R. Whether short peptides are good candidates for future neuroprotective therapeutics? Peptides. 2021 Jun;140:170528. doi: 10.1016/j.peptides.2021.170528.

3. Guan J, Harris P, Brimble M, et al. The role for IGF-1-derived small neuropeptides as a therapeutic target for neurological disorders. Expert Opin Ther Targets. 2015 Jun;19(6):785-93. doi: 10.1517/14728222.2015.1010514.

4. Guan J, Gluckman PD. IGF-1 derived small neuropeptides and analogues: a novel strategy for the development of pharmaceuticals for neurological conditions. Br J Pharmacol. 2009 Jul;157(6):881-91. doi: 10.1111/j.1476-5381.2009.00256.x.

5. Gudasheva TA, Boyko SS, Akparov VKh, et al. Identification of a novel endogenous memory facilitating cyclic dipeptide cyclo-prolylglycine in rat brain. FEBS Lett. 1996 Aug 5;391(1-2):149-52. doi: 10.1016/0014-5793(96)00722-3.

6. Povarnina PY, Kolyasnikova KN, Gudasheva TA, et al. Neuropeptide cycloprolylglycine exhibits neuroprotective activity after systemic administration to rats with modeled incomplete global ischemia and in in vitro modeled glutamate neurotoxicity. Bull Exp Biol Med. 2016;160(5):653-655. doi: 10.1007/s10517-016-3241-5.

7. Ferro JN, de Aquino FL, de Brito RG, et al. Cyclo-Gly-Pro, a cyclic dipeptide, attenuates nociceptive behaviour and inflammatory response in mice. Clin Exp Pharmacol Physiol. 2015 Dec;42(12):1287-95. doi: 10.1111/1440-1681.12480.

8. Fan D, Alamri Y, Liu K, et al. Supplementation of Blackcurrant Anthocyanins Increased Cyclic Glycine-Proline in the Cerebrospinal Fluid of Parkinson Patients: Potential Treatment to Improve Insulin-Like Growth Factor-1 Function. Nutrients. 2018 Jun 2;10(6):714. doi: 10.3390/nu10060714.

9. Guan J, Li F, Kang D, et al. Cyclic Glycine-Proline (cGP) Normalises Insulin-Like Growth Factor-1 (IGF-1) Function: Clinical Significance in the Ageing Brain and in Age-Related Neurological Conditions. Molecules. 2023 Jan 19;28(3):1021. doi: 10.3390/molecules28031021.

10. [Koliasnikova KN, Alyaeva AG, Kuznetsova EA. Novel glyproline GZK-111 with neuropsychotropic activity. Farmakokinetika i farmakodinamika = Pharmacokinetics and pharmacodynamics. 2021;(4):18-23. (In Russ.)]. doi:10.37489/2587-7836-2021-4-18-23.

11. Gudasheva TA, Koliasnikova KN, Kuznetsova EA, et al. N-phenylacetyl-glycine showing a similar spectrum of neuropsychotropic activity. Pharmaceutical chemistry journal. 2017;50(11):705-710. doi: 10.1007/s11094-017-1516-4.

12. Kolyvanov GB, Bochkov PO, Litvin AA, et al. Metabolism of a New Dipeptide Neuroprotector in Rats. Bull Exp Biol Med. 2022 Mar;172(5):579-582. doi: 10.1007/s10517-022-05440-3.

13. Litvin AA, Kolyvanov GB, Bochkov PO, et al. Preclinical Pharmacokinetics of GZK-111, a Dipeptide with Neuroprotective Activity. Bull Exp Biol Med. 2022 Jan;172(3):310-313. doi: 10.1007/s10517-022-05400-x.

14. [Alekseev IV, Miroshkina IA, Sorokina AV, et al. Study of acute toxicity of dimeric dipeptide mimetic of neurotrophin-3 in mice. Farmakokinetika i farmakodinamika = Pharmacokinetics and pharmacodynamics. 2025;(1):60-68. (In Russ.)]. doi:10.37489/2587-7836-2025-1-60-68.

15. Flückiger-Isler S, Kamber M. Direct comparison of the Ames microplate format (MPF) test in liquid medium with the standard Ames pre-incubation assay on agar plates by use of equivocal to weakly positive test compounds. Mutat Res. 2012 Aug 30;747(1):36-45. doi: 10.1016/j.mrgentox.2012.03.014.

16. Neumeyer AM, Srivastava S, Holder JL, et al. NNZ-2591 in Children and Adolescents With Phelan-McDermid Syndrome: Single-Group, Open-Label, Phase 2 Trial Results. Neurol Genet. 2025 Dec 23;12(1):e200338. doi: 10.1212/NXG.0000000000200338.

17. Kobylyanskii AG, Zolotarev YA, Andreeva LA, et al. Studying the Toxic Effects of Some Biologically Active Peptides on the Model of Mouse Embryonic Stem Cells. Bull Exp Biol Med. 2017 Oct;163(6):731-736. doi: 10.1007/s10517-017-3891-y.

18. [Kovalenko LP, Smol'nikova NM, Alekseeva SV, et al. Preclinical characterization of the toxicity of noopept. Eksperimental'naya i klinicheskaya farmakologiya. 2002;65(1):62-64. (In Russ.)]. doi: 10.30906/0869-2092-2002-65-1-62-64.

19. [Sorokina AV, Alekseeva SV, Nemova EP, et al. Preclinical safety investigation of GB-115 dipeptide. Eksperimental'naya i klinicheskaya farmakologiya. 2010;73(6):29-32. (In Russ.)]. doi: 10.30906/0869-2092-2010-73-6-29-32.