INTRODUCTION:

During the last two-decade, serotonin-3 (5-HT₃) receptor antagonists (RAs) gaining importance because of its role in the treatment of nausea and vomiting induced by endogenous substances produced during cancer chemotherapy and radiotherapy¹. Apart from this, numerous studies suggested that 5-HT₃ RAs can be utilized to treat a variety ofneuropsychiatry disorders^2-6. Cancer patients are more prone to have anxiety during chemotherapy and radiation treatment^7,8. It will be beneficial for the cancer patients if the 5-HT₃ RA suppress nausea and vomiting, and have anxiolytic effect. As a result, we evaluated the anxiolytic potential of our previously reported⁹ molecule of 5-HT₃ RA, 6j {2-[4-(4-nitro-phenyl)-piperazin-1-yl]-[1,8]naphthyridine-3-carbonitrile} along with the computational and molecular modelling studies and compared with the standard 5-HT₃ RA, ondansetron.

MATERIALS AND METHODS:

Computational studies:

Using the Molinspiration cheminformatics¹⁰ and SwissADME¹¹ online tools, a computational analysis^12-14 was conducted on the test drug, 6j, and ondansetron, to predict its drug-likeness, namely Veber's¹⁵,Lipinski's¹⁶ rules, and topological polar surface area (TPSA).ProTox-II¹⁷, an online tool, was used to predict the toxicity profile of the test and standard drugs.

Molecular modelling:

The structure (2D) of the test compound, 6j and ondansetron were drawn (figure 1), converted to 3D structure and the molecule were run for energy minimization using Chem Office tool. For molecular docking analysis the energy minimized ligands was used as an input. The protein target, tropisetron-bound mouse 5-HT3 receptor, (PDB id: 6HIS), were obtained from the RCSB Protein Data Bank. Protein preparation was done using Auto Doc Vina (MGLTools-1.5.6)^18-20. Water molecules and bound ligand were cleared from the protein complex; to the protein polar hydrogen atoms and necessary charges were added. The grid box was created to define the active sites with required attributes (center_x = 133.057286, center_y = 98.941429, center_z = 151.201381; size_x = 24, size_y = 27, size_z = 20).The macromolecule (protein) and the ligand were chosen and saved in required format(.pdbqt). During the docking process, various conformers were created for each ligand along with their binding energy²¹. Biovia Discovery Studio 2020 was used to determine the interactions between the receptor and the ligand by selecting the most favourable conformation with the lowest binding energy²².

Figure 1: Structures of test compound, 6j, and standard 5-HT3 receptor antagonist, ondansetron

Anxiolytic Screening

The animal studies were granted by the Institutional Animal Ethics Committee. Albino mice (20-25 gm) were used in this study and they were maintained as per the CPCSEA guidelines²¹. The day before the experiment, the animals were kept in groups of six in plexiglass cages in the laboratory. The experiment was conducted during the light phase of the cycle (8 am - 12 noon. Diazepam (0.2 mg / kg) was used as a positive control; saline as a negative control; ondansetron and test compound 6j were used at doses of 0.1, 1.0 and 10 mg/kg. The drug was dissolved in saline and injected into the intraperitoneal cavity (i.p.) 1 hr. before the test. The experimental data were analyzed using the one-way analysis of variance (ANOVA) test and Tukey-Kramer multiple comparison test using GraphPad InStat 3 software. Statistical significance was established at p < 0.05. The results of anti-anxiety studies are expressed as mean ± SEM.

Elevated plus maze (EPM)

The device consists of two oppositely open arms (30 x 5 cm) at right angles and two oppositely closed arms of the same size. The latter is surrounded by walls (10 cm) on all sides, but is open; there are no lips on the open arms. The four arms are delimited in the central area (5 x 5 cm). The entire device is raised (30 cm) above the ground. The mouse was placed alone in the center of the maze and its behaviour was observed during the 5-minute test. Time spent in the open arm of the maze (expressed as the percentage of time spent in the open arm), the entries into the open arms (expressed as the percentage of entries in the open arm); the time spent in the center of the device is ignored^22,23.

RESULTS:

Computational studies

Parameters used in Molinspiration and SwissADME clearly indicate that the test compound, 6j, and ondansetron satisfied Veber¹⁵ and Lipinski¹⁶ rules with zero violations (table 1). Toxicity prediction by ProTox-II¹⁷ showed higher LD₅₀ for 6j (300 mg/kg) than ondansetron (95 mg/kg); both the compounds showed no toxicity except mutagenicity (table 2).

Table 1: Drug-likeness prediction using Molinspiration and SwissADME

S. No.	Properties	6j	Ondansetron
1	MW	360.37	293.36
2	nOHNH	0	0
3	nON	5	2
4	cLogP	1.89	2.54
5	nviol (Lipinski rule)	0	0
6	nrotb	3	2
7	TPSA (Å²)	101.87	39.82
8	nviol (Veber rule)	0	0

MW, molecular weight; nOHNH, number of hydrogen donor; nON, number of hydrogen acceptor; nrotb, number of rotatable bonds; TPSA, topological polar surface area; nviol, number of violations

Molecular Modelling

When we performed docking studies of the test and standard compounds with the 5-HT₃ receptor, the test compound, 6j, showed binding energy -8.5 kcal/mol, whereas ondansetron showed -7.7 kcal/mol. Table 3 shows that the test drug, 6j, and the 5-HT3 RA, ondansetron, have hydrogen bonding and hydrophobic interactions with the 5-HT₃ receptor’s amino acids. The 3D and 2D binding interactions of the test compound, 6j, and ondansetron, with the 5-HT₃ receptor are depicted in figure 2.

Table 2: Toxicity prediction using ProTox-II

Compound	LD₅₀ (mg/kg)	Toxicity Class	Toxicity
Compound	LD₅₀ (mg/kg)	Toxicity Class	Hepatotoxicity	Carcinogenicity	Immunotoxicity	Mutagenicity	Cytotoxicity
6j	300	3	No	No	No	Yes	No
Ondansetron	95	3	No	No	No	Yes	No

Table 3: Binding energy and amino acid interactions of 6j and ondansetron

Ligand	Binding energy (kcal/mol)	Binding interactions
Ligand	Binding energy (kcal/mol)	Hydrogen Bonding	Hydrophobic bonding
6j	-8.5	Trp-63,Arg-169	Ile-44 (π-σ), Trp-63 (π- π, π-Alkyl), Tyr-126 (π-Alkyl)
Ondansetron	-7.7	-	Trp-63 (π-σ), Ile-44 (π-σ, π-Alkyl, Alkyl),Arg-65 (Alkyl, π-Alkyl)

6j	Ondansetron

Figure 2: Binding interactions(3D and 2D) of 6j and ondansetron with 5-HT₃receptor

Anxiolytic evaluation:

The response of drugs on % entries in open arms is given in table 4. ANOVA showed extremely significant difference among the given treatments [F (7, 40) = 7.26, p < 0.0001]. Tukey’s test revealed that diazepam (0.2 mg/kg) (p < 0.50), ondansetron (10 mg/kg) (p < 0.5) and 6j (10 mg/kg) (p < 0.1) significantly increased % entries in open arm, whereas the lower doses (1.0 and 0.1 mg/kg) of ondansetron and 6j significantly did not alter the measure in comparison to saline control.

The response of drugs on % time spent in open arms is given in table 4. ANOVA showed extremely significant difference among the given treatments [F (7, 40) = 5.79, p = 0.0001]. Tukey’s test revealed that diazepam (0.2 mg/kg), ondansetron (10 mg/kg) and 6j (10 mg/kg) significantly (p < 0.05) increased the % time spent in open arms, whereas in the lower doses (1.0 and 0.1 mg/kg) both ondansetron and 6j did not alter the measure significantly in comparison to saline control.

Table 4: Effect of drugs on % open arm entries and % time in open arm (EPM)

Treatment	% Open arm entries	% Time in open arm
Control	6.11 ± 3.89	0.45 ± 0.29
Diazepam (0.2 mg/kg)	27.5 ± 5.09*	2.34 ± 0.45*
Ondansetron (0.1 mg/kg)	6.69 ± 4.3	0.55 ± 0.35
Ondansetron (1 mg/kg)	7.9 ± 5.46	0.56 ± 0.37
Ondansetron (10 mg/kg)	30.1 ± 3.72*	2.33 ± 0.42*
6j (0.1 mg/kg)	6.41 ± 4.3	0.51 ± 0.32
6j (1 mg/kg)	8.35 ± 5.64	0.64 ± 0.41
6j (10 mg/kg)	34.02 ± 3.57**	2.22 ± 0.39*

Values show mean ± SEM; n = 6. *indicates p < 0.05; **indicates p < 0.01 when compared to vehicle control group

DISCUSSION:

Computational studies using online tools revealed that both 6j and ondansetron have the drug-likeness properties with same level of toxicity profile. The test compound, 6j, showed higher binding affinity (lesser binding energy) than the standard, ondansetron. The EPM is one of the well-used animal models in preclinical anxiety research, in which rodents are naturally afraid of open space and height²². In this animal model both the compounds showed significant anxiolytic property at higher doses i.e., 10 mg/kg, whereas at lower doses they failed to show significant anxiolytic activity. In both the animal models, the test drug, 6j, showed anxiolytic potential at 10 mg/kg compared to vehicle control but it did not show significant anxiolytic activity compared to ondansetron and diazepam at the various doses tested.

ACKNOWLEDGEMENTS:

We sincerely thank Natco Pharma, Hyderabad, India for providing ondansetron as a gift sample and ASTU, Adama for the research facilities.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

REFERENCES:

1. Aapro M. CINV: still troubling patients after all these years. Supportive care in cancer. 2018 Mar 19;26(1):5-9. doi:10.1007/s00520-018-4131-3.

2. Fakhfouri G, Rahimian R, Dyhrfjeld-Johnsen J, Zirak MR, Beaulieu J. 5-HT3 Receptor Antagonists in Neurology and Neuropsychiatry. Pharmacological Reviews. 2019 July 1;71(3):383-412.doi: 10.1124/pr.118.015487

3. Olivier B, van Wijngaarden I, Soudijn W. 5-HT3 receptor antagonists and anxiety; a preclinical and clinical review. European Neuropsychopharmacology. 2000 Mar 8;10(2):77-95. doi: 10.1016/S0924-977X(99)00065-6.

4. Juza R, Vlcek P, Mezeiova E, Musilek K, Soukup O, Korabecny J. Recent advances with 5-HT3 modulators for neuropsychiatric and gastrointestinal disorders. Medicinal Research Reviews. 2020 Mar 1;40(5): 1593-1678. doi: 10.1002/med.21666

5. Jaya Preethi P, Padmini K, Srikanth J, Lohita M, Swetha K. Screening Models for CNS Stimulant Drugs: A Review. Asian J. Pharm. Res. 2013 July-Sept 3(3):151-155.

6. Arun Kumar J. Effect of Prenatal Exposure of Clobazam on Anxiety Parameters in Rat Offspring. Research J. Pharmacology and Pharmacodynamics. 2016 8(2):58-64. doi: 10.5958/2321-5836.2016.00011.2

7. Grassi L, Johansen C, Annunziata MA, Capovilla E, Costantini A, Gritti P, Torta R, Bellani M; Italian Society of Psycho-Oncology Distress Thermometer Study Group. Screening for distress in cancer patients: a multicenter, nationwide study in Italy. Cancer. 2013 May 1;119(9):1714-21. doi: 10.1002/cncr.27902

8. Vijaya kumar S, Sucharitha R, Swathi T, RaoAY., PrakashCSK. Nail Toxicity Induced by Cancer Chemotherapy Patients: Data from the Two Multispecialty Hospital. Research J. Pharm. and Tech. 2015 Jan 8(1): 20-26. doi: 10.5958/0974-360X.2015.00004.9

9. Mahesh R, Venkatesha Perumal R, Vijaya Pandi P. Microwave assisted synthesis of 2-(4-substituted piperazin-1-yl)-1,8- naphthyridine-3-carbonitrile as a new class of serotonin 5-HT3 receptor antagonists. Bioorganic Medicinal Chemistry Letters. 2004 Oct 18;14(20):5179–81. doi: 10.1016/j.bmcl.2004.07.060

10. Molinspiration: Calculation of Molecular Properties and Bioactivity Score. 2021. http://www.molinspiration.com/cgi-bin/properties. Accessed 12 May 2021.

11. Daina A, Michielin O, Zoete V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific Reports. 2017 Mar 3;7:42717. doi:10.1038/srep42717.

12. Nisha H, Balasankar K, Aswathy. Computational Analysis to Identify the Drug Targets and their Lead Molecules in Pancreatic Cancer. Research J. Pharm. and Tech. 2017 10(6):1708-1716. doi: 10.5958/0974-360X.2017.00302.X

13. Radhika C, Ajitha M. Molecular docking studies and ADMET Predictions of Pyrimidine Coumarin Scaffolds as Potential IDO Inhibitors. Asian J. Research Chem. 2017 10(3):331-340. doi: 10.5958/0974-4150.2017.00056.6

14. Shital NC, Sanjay JK. Computational Studies and Synthesis of few Thiazolidine-2,4-Dion Analogues for Peroxisome Proliferator Activator Receptor- γ Agonist as Useful Antidiabetic Agent. Asian J. Pharm. Res. 2017 7(4):265-268. doi: 10.5958/2231-5691.2017.00042.9

15. Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple KD. Molecular properties that influence the oral bioavailability of drug candidates. Journal of Medicinal Chemistry. 2002 May 11;45(12):2615-23. doi: 10.1021/jm020017n

16. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Review. 1997 Jan 5;23(1-3):4-25. doi:10.1016/S0169-409X(96)00423-1

17. Banerjee P, Eckert AO, Schrey AK, Preissner R. ProTox-II: a webserver for the prediction of toxicity of chemicals. Nucleic Acids Res. 2018 Jul 2;46(W1):W257-W263. doi: 10.1093/nar/gky318

18. Trott O, Olson AJ. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry. 2010 Jan 30;31(2):455-461. doi: 10.1002/jcc.21334

19. Uday MS, Sachin HR. Efficiency of AUTODOCK: Insilico study of Pharmaceutical Drug Molecules. Asian J. Research Chem. 2021 14(1):92-96. doi: 10.5958/0974-4150.2021.00016.X

20. Kannadasan R, Arnold EI, Basha MSS. Docking of HIV-1 with Neem using Autodock in Bioinformatics. Research J. Pharm. and Tech. 2017 10(11):3877-3880. doi: 10.5958/0974-360X.2017.00704.1

21. Otuokere IE, Amaku FJ, Alisa CO. In Silico Geometry Optimization, Excited – State Properties of (2E)-N-Hydroxy-3-[3-(Phenylsulfamoyl) Phenyl] prop-2-Enamide (Belinostat) and its Molecular Docking Studies with Ebola Virus Glycoprotein. Asian J. Pharm. Res. 2015 July-Sept 5(3):131-137. doi: 10.5958/2231-5691.2015.00020.9

22. Shravani S. Pawar, Sachin H. Rohane. Review on Discovery Studio: An important Tool for Molecular Docking. Asian J. Research Chem. 2021 14(1):86-88. doi: 10.5958/0974-4150.2021.00014.6

23. Committee for the purpose of control and supervision of experimental animals. http://cpcsea.nic.in/Content/55_1_GUIDELINES.aspx. Accessed 5 June 2021

24. Walf A, Frye C. The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nature Protocols. 2007 Mar 1;2:322–28. doi:10.1038/nprot.2007.44

25. Hogg S. A review of the validity and variability of the elevated plus-maze as an animal model of anxiety. Pharmacololgy Biochemistry Behavior. 1996 May;54(1):21-30. doi: 10.1016/0091-3057(95)02126-4.

Received on 07.07.2021 Modified on 17.03.2022

Research J. Pharm. and Tech 2023; 16(7):3075-3078.

DOI: 10.52711/0974-360X.2023.00505