Radioligand analysis results on mongrel CD-1mice confirmed the results obtained on human T-cell lymphoblast-like cell line and led to the conclusion that s1 ...
Pharmaceutical Chemistry Journal, Vol. 46, No. 6, September, 2012 (Russian Original Vol. 46, No. 6, June, 2012)
INTERACTION OF 2-MERCAPTOBENZIMIDAZOLE DERIVATIVES WITH SIGMA-1 RECEPTORS E. V. Ryaskina,1 M. V. Voronin,1 and S. B. Seredenin1 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 46, No. 6, pp. 12 – 13, June, 2012. Original article submitted March 28, 2012.
The interaction of afobazole and its main metabolite M-11 {2-[2-(3-oxomorpholin-4-yl)ethylthio]-5ethoxybenzimidazole hydrochloride} with sigma-1 (s1) receptors was studied in vitro in radioligand binding experiments on human T-cell lymphoblast-like cell line. Afobazole exhibited ligand properties towards s1 receptors with IC50 = 7.1 ´ 10 – 6 M. For M-11, the corresponding value was IC50 = 9.6 ´ 10 – 4 M. Experiments in vitro on CD-1 mouse brain homogenate established that afobazole interacted with s1 receptors (IC50 = 1.37 ´ 10 – 5 M) whereas M-11 did not show affinity towards s1 receptors (IC50 > 5 ´ 10 – 4 M). Radioligand analysis results on mongrel CD-1 mice confirmed the results obtained on human T-cell lymphoblast-like cell line and led to the conclusion that s1 receptors did not contribute to the pharmacological effects of M-11. Key words: afobazole, afobazole metabolite, s1 receptor, radioligand binding.
Afobazole {5-ethoxy-2-[2-(morpholino)ethylthio]benzimidazole dihydrochloride} and M-11 {2-[2-(3-oxomorpholin-4-yl)ethylthio]-5-ethoxybenzimidazole hydrochloride} were synthesized at Zakusov Institute of Pharmacology, RAMS. The isolation, purification, detection, and elemental analysis of the compounds were carried out according to standard methods [7, 8]. The range of final concentrations of afobazole and M-11 were 10 – 3 – 10 – 9 M in experiments on Jurkat human T-lymphocyte cell line and 10 – 3 – 10 – 7 M in experiments on CD-1 mongrel mice.
Afobazole, 5-ethoxy-2-[2-(morpholino)ethylthio]benzimidazole dihydrochloride, was introduced to clinical practice as an anxiolytic in 2005 [1]. The neuroprotective activity of afobazole was established in several experimental models [2 – 4]. The compound in a liquid drug form under the trademark Neurofazol was registered in 2010 as a neuroprotective agent (Reg. No. LSR-002102/10). The main metabolite of afobazole, M-11 or 2-[2-(3-oxomorpholin-4-yl)ethylthio]-5ethoxybenzimidazole hydrochloride [5], was isolated in pharmacokinetic experiments. Its pharmacological activity is also interesting. Therefore, it seemed advisable to compare the interaction of these compounds with receptors. Because of the homologous molecular structures of afobazole and M-11 and the contemporary model of the s1 receptor pharmacophore [6], the goal of our work was to study the interaction of afobazole and its main metabolite with s1 receptors in various in vitro models.
EXPERIMENTAL BIOLOGICAL PART Interaction of afobazole and M-11 with s1 receptors in vitro on Jurkat human T-lymphocyte cell line was studied in two independent experiments according to the Seguerre (France) protocol [9]. We used [3H](+)-pentazocine at final concentration 8 nM as the labeled ligand. The incubation time of the labeled and unlabeled ligands with the cell suspension was 120 min at 22°C. Interaction of afobazole and M-11 with s1 receptors in vitro in mongrel CD-1 mice (NPP Laboratory Animal Nursery, FIBKh) was studied in the P2 fraction of brain homogenates in three independent experiments according to Colabufo, et al. with modifications [10]. [3H](+)-Pentazocine
EXPERIMENTAL CHEMICAL PART We used [3H](+)-pentazocine {[Ring-1,3-3H]–, 1 mCi (37 MBq), PerkinElmer} as the labeled s1 receptor ligand. 1
Zakusov Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow, 125315b, Russia.
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Interaction of 2-Mercaptobenzimidazole Derivatives
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100
Total binding
80 2
60 40
1
20
of [3H](+)-pentazocine, %
Total binding
of [3H](+)-pentazocine, %
100
2
80 60
1
40 20 0
0 –8
–7
–6 –5 –4 –3 log [X], M Fig. 1. Competitive release of [3H](+)-pentazocine by afobazole (1 ) and M-11 (2 ) in experiments in vitro on Jurkat human T-lymphocyte cell line. log[X] is the logarithm of afobazole and M-11 concentration (M).
was used at final concentration 1 nM. The range of afobazole and M-11 concentrations was 10 – 3 – 10 – 7 M. The P2 fraction was incubated with [3H](+)-pentazocine, afobazole, and M-11 in Tris-HCl buffer (500 mL, pH 7.4, 50 mM) at 37°C for 120 min. Radioligand binding was stopped by adding Tris-HCl buffer (10 mL, 50 mM, pH 8.0, 4°C) and subsequent rapid filtration through GF/B glass-wool filters (Whatman) that were treated beforehand with polyethyleneimine (0.05%). The radioactivity of each sample was measured in a Tri-Carb 2900TR liquid scintillation counter (PerkinElmer). Specific binding in each experiment was calculated as the average of three experimental determinations. The IC50 values in experiments in vitro on Jurkat human T-lymphocyte cell line and mongrel CD-1 mice were calculated from Eqs. (1) and (2), respectively, using Graph Pad Prism version 5.02 for Windows and Graph Pad Software (San Diego, CA, USA; www.graphpad.com). Y = D + [(A – D )/(1 + (C/IC50)nH)],
(1)
where Y is the specific binding; A, the maximum specific binding; D, the minimum specific binding; C the afobazole concentration; and nH, the Hill coefficient. Y =D+
( A - D) 1 + 10
(C - LogIC 50 )
,
where Y is the specific binding; A, the maximum specific binding; D, the minimum specific binding; and C, the afobazole concentration.
-7
-6
-5
-4
-3
log[X], M Fig. 2. Competitive release of [3H](+)-pentazocine by afobazole (1 ) and M-11 (2 ) in experiments in vitro on P2 fraction of CD-1 mouse brain homogenate. log[X] is the logarithm of afobazole and M-11 concentrations (M).
RESULTS AND DISCUSSION Competitive release curves (Fig. 1) were obtained and IC50 values were calculated by analyzing afobazole and M-11 binding with human T-lymphocyte culture. Afobazole exhibited experimentally ligand properties for s1 receptors in the micromolar range (IC50 = 7.1 ´ 10 – 6 M). The IC50 value for M-11 under the same experimental conditions (9.6 ´ 10 – 4 M) led to the conclusion that the main metabolite had little pharmacologically significant affinity for s1 receptors. The experimental results provided a basis for further study of the mechanism of interaction of afobazole and M-11 with receptors in model laboratory animals. The release curves (Fig. 2) corresponded to IC50 values close to that obtained in experiments in vitro in human T-lymphocyte cultures, namely 1.37 ´ 10 – 5 M for afobazole and > 5 ´ 10 – 4 M for M-11, in a series of experiments in vitro on CD-1 mouse brain homogenates. The experimental results in the mongrel CD-1 mouse model confirmed that afobazole exhibited ligand properties for s1 receptors in the micromolar concentration range and that the main metabolite of afobazole M-11 had little affinity for these receptors. Recalling the significance of s1 receptors in mechanisms for ensuring the active functional state of the cell membrane and neuronal ion balance [11 – 13], the nature of the interactions of the studied compounds with s1 receptors led to the conclusion that differences in the pharmacological effects of afobazole and M-11 could be explained by an inadequate affinity of M-11 for s1 receptors. The results defined the plan for further pharmacological research in at least two directions. First, a comparison of the pharmacological spectra of
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afobazole and M-11 would help to explain the s1-dependent contribution to the action of afobazole. Second, identification of the pharmacological properties of M-11 would enable a working hypothesis about the significance of interaction with other biotargets to the effects of M-11 to be formulated. REFERENCES 1. G. G. Neznamov, S. A. Syunyakov, D. V. Chumakov, et al., Psikhiatriya Psikhofarmakoter., 4(8), 8 – 13 (2006). 2. I. P. Galaeva, T. L. Garibova, T. A.Voronina, et al., Byull. Eksp. Biol. Med., 140(11), 545 – 548 (2005). 3. T. A. Antipova, D. S. Sapozhnikova, L. Yu. Bakhtina, et al., Eksp. Klin. Farmakol., 72(1), 12 – 14 (2009). 4. J. Cuevas, A. Behensky, W. Deng, et al., J. Pharmacol. Exp. Ther., 339(1), 152 – 160 (2011).
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