Background & Aims
Recent studies had shown that pharmacological actions of benzodiazepines (BZD) are determined by GABAA receptor (GABAARs) ?-subunits [1, 2]. Binding to the ?1 subunits cause sedation and anticonvulsive effects, anxiolytic effects are modulated by the ?2- and for the ?5-containing GABAARs. ?2- and ?3 GABAARs subtypes also associated with myorelaxation, motor coordination is induced by modulation of ?1 or ?3 subtypes. Moreover, the antihyperalgesic effect of BZD depends mainly on ?2-containing GABAARs in the spinal cord [3, 4].
Propoxazepam as the promising analgesic agent, similar to gabapentinoid drugs, used for the treatment of neuropathic pain [5], and has an anticonvulsant effect [6-8]. Our data suggest [6-8] GABAergic and glycinergic systems involving in analgesic and anticonvulsant properties of Propoxazepam. The aim of this study was the revealing of molecular mechanism of Propoxazepam analgesic action by estimation of the selectivity binding to different isoforms of GABAA-Rs.
Methods
Electrophysiological experiments were performed on automated SP384PE Patch Clamp (Syncropatch SP384PE) system on GABA h132, h232, h332, h432 and h532 ionotropic GABAARs expressed in HEK293 cells.
GABAARs agonist, GABA, (5 µM) was used as a positive control agonist. Diazepam, the positive allosteric modulator (PAM), as positive control PAM on h132 GABAARs was used at concentrations 0.001-3 µM (with step 0.5 lg). Propoxazepam, obtained according to [9], was tested at final concentrations 0.001-3 nM (with step 0.5 lg) 0.1, 0.3, 1, 3, 10, 30, 100, 300 nM. All measures were conducted at 4 replicate wells/concentration.
From the concentration-response curve the Emin and Emax (bottom and top plateau), C50 and Hill coefficient were calculated. Statistical analysis was be performed, using an unpaired, 2-tailed, Student’s t-test at p<0.05.
Results
The reference PAM, diazepam (reference substance for only ?1 subunint-containing receptor, since the demonstration of its high affinity to this isoform [10]), showed a robust PAM effect with stimulation of the h1?22 GABAARs in the presence of 5 µM GABA (EC50PAM=0.04 µM and Emax ~200%. Propoxazepam did not produce a significant, concentration dependent inhibition of the GABAA receptors and showed robust PAM effect at all GABAA receptors and demonstrated notable difference in GABA isoforms selectivity with exception of the h4?22 (determined ED50 were 0.018, 0.038, 0.098 and 0.02 µM for ?1, ?2, ?3 and ?5 subunits correspondingly; > 0.3 µM for 4). There is also the difference in Emax values (assuming the 100 % at zero concentration – 179.6; 280.1; 455.1; 129.1 and 199.8 % for 1-5 isoforms).
Conclusions
On the results of propoxazepam binding to GABAA h*?22 it is shown that the ? subunit plays a significant role in the receptor’s affinity for Propoxazepam. The rank order of EC50 decreasing are ?1 = ?5>?2>?3>?4 and for Emax ?3>?2>?5>?1>?4. The magnitude of Emax values allows to suggest that functional impact of these two isoforms can be much more intensive, despite the certain formal difference in affinity indicators (EC50). The results indicate that propoxazepam causes the maximum effect only on receptors containing ?2 and ?3 subunits.
Propoxazepam is a potent ligand of the GABAARs allosteric BDZ site, which exhibits functional selectivity for receptors containing a2, a3, or ?5 over those containing ?1. Thus, propoxazepam has the potential to provide analgesia with less sedation than non-selective BDZ.
References
1. Sieghart W. Unraveling the function of GABA(A) receptor subtypes. Trends Pharmacol Sci. 2000 Nov;21(11):411-413. doi: 10.1016/s0165-6147(00)01564-9.
2. Engin E, Liu J, Rudolph U. ?2-containing GABA(A) receptors: a target for the development of novel treatment strategies for CNS disorders. Pharmacol Ther. 2012;136(2):142-152. doi:10.1016/j.pharmthera.2012.08.006
3. Rudolph U, Möhler H. GABAA receptor subtypes: Therapeutic potential in Down syndrome, affective disorders, schizophrenia, and autism. Annu Rev Pharmacol Toxicol. 2014;54:483-507. doi: 10.1146/annurev-pharmtox-011613-135947.
4. Ralvenius WT, Acuña MA, Benke D, Matthey A, Daali Y, Rudolph U, Desmeules J, Zeilhofer HU, Besson M. The clobazam metabolite N-desmethyl clobazam is an ?2 preferring benzodiazepine with an improved therapeutic window for antihyperalgesia. Neuropharmacology. 2016 Oct;109:366-375. doi: 10.1016/j.neuropharm.2016.07.004.
5. Chincholkar M. Gabapentinoids: pharmacokinetics, pharmacodynamics and considerations for clinical practice. Br J Pain. 2020;14(2):104-114. doi:10.1177/2049463720912496.
6. Golovenko N.Ya., Larionov V.B., Reder A.S., Valivodz I.P. An ?ffector analysis of the interaction of propoxazepam with antagonists of GABA and glycine receptors. Neurochemical Journal. 2017. 11(4): 302–308. doi: 10.1134/S1819712417040043.
7. Golovenko M., Reder A., Andronati S., Larionov V. Evidence for the involvement of the GABA-ergic pathway in the anticonvulsant and antinociception activity of Propoxazepam in mice and rats. J. Pre-Clin. Clin. Res. 2019. 13(3): 99–105. doi: 10.26444/jpccr/110430.
8. Golovenko N.Ya., Larionov V.B., Andronati S.A., Valivodz I.P., Yurpalova T.A. ?harmacodynami?s of interaction between Propoxazepam and a GABA-benzodiazepine receptor-ionophor complex. Neurophysiology. 2018. 50(1): 2–10. doi: 10.1007/s11062-018-9711-9.
9. Andronati S., Semenishyna E., Pavlovsky V., Simonov Y., Makan S., Boyko I., Burenkova N., Gdaniec M., Cardinael P., Bouillon J. Synthesis, structure and affinity of novel 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones for CNS central and peripheral benzodiazepine receptors. European Journal of Medicinal Chemistry. 2009. 45(4): 1346–1351. doi: 10.1016/j.ejmech.2009.12.027.
10. Möhler H. Molecular regulation of cognitive functions and developmental plasticity: impact of GABAA receptors. J Neurochem. 2007 Jul;102(1):1-12. doi: 10.1111/j.1471-4159.2007.04454.x.
Presenting Author
Vitalii Larionov
Poster Authors
Topics
- Mechanisms: Biological-Molecular and Cell Biology