Effect of MB327 and oximes on rat intestinal smooth muscle function

Chemico-Biological Interactions 204 (2013) 1–5

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Effect of MB327 and oximes on rat intestinal smooth muscle function C. Königer, F. Worek, H. Thiermann, T. Wille ⇑ Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany

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Article history: Received 14 December 2012 Received in revised form 20 March 2013 Accepted 9 April 2013 Available online 18 April 2013 Keywords: Anticholinergic Antimuscarinic Smooth muscle function MB327 Oxime Dose–response-relationship

a b s t r a c t Organophosphorous compounds (OP) are highly toxic compounds. Great efforts have been undertaken in the last decades to develop new reactivators of OP-inhibited acetylcholinesterase. So far, a broad-spectrum oxime bearing efficacy against all OP is still missing and alternative approaches are presently under investigation. Previous experiments demonstrated that the bispyridinium non-oxime MB327 was able to improve OP-impaired muscle force in human, rat and guinea pig respiratory muscles and to increase survival in soman, sarin and tabun poisoned guinea pigs. Recent studies indicate that MB327 exhibits a high affinity to muscarinic acetylcholine receptors but up to now, only scarce information is available on the effects of MB327 in isolated organs. Now, the antimuscarinic effect of MB327 was compared to that of established oximes and atropine in a rat jejunum smooth muscle model. MB327 showed a fully reversible smooth muscle relaxing effect at lower concentrations (EC50  6 lM) than all tested oximes. In fact, MB327 exhibited an antimuscarinic smooth muscle relaxing effect at concentrations which were shown to improve OP-impaired skeletal muscle force. Hence, it may be assumed that the antimuscarinic potency of MB327 may contribute to its therapeutic effect in OP poisoning. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Organophosphorous compounds (OP), particularly nerve agents, are highly life threatening substances and incidences such as the sarin attack of the Aum Shinrikyo sect in 1995 in Tokyo demonstrate that the use of OP is a problem of particular relevance [1]. OP exert their toxic effect primarily via inhibition of the enzyme acetylcholinesterase (AChE), thus leading to increased acetylcholine (ACh) concentrations and finally – if untreated – to death due to central and peripheral respiratory arrest [2]. In general, the therapy consists of a two-way strategy. Oximes, e.g. obidoxime and pralidoxime, are administered in order to restore the blocked AChE function [3,4]. Atropine acts as a direct antagonist at muscarinic receptors and blocks the effect of excessive ACh concentrations at the effector organs [5]. It is administered in rising doses until the cholinergic symptoms stop [6]. Because of the limited efficacy of clinically established pyridinium oximes, e.g. pralidoxime (2-PAM) and obidoxime (Table 1), in poisoning by the nerve agents soman, cyclosarin and tabun [7–9] great efforts were undertaken in the last decades to find more effective ways of treatment. So far, a broad-spectrum oxime bearing efficacy against all nerve agents is still missing. Recently, MB327 (Table 1), a bispyridinium compound without oxime group(s), showed a remarkable therapeutic effect, in combination with hyoscine and physostigmine, in soman, sarin and ta⇑ Corresponding author. Tel.: +49 89 3168 2305; fax: +49 89 3168 2333. E-mail address: [email protected] (T. Wille). 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.04.004

bun poisoned guinea pigs [10,11]. In addition, MB327 improved soman blocked neuromuscular transmission in human intercostal muscle and rat and guinea pig diaphragms preparations in vitro [10,12]. The therapeutic effect of MB327 was attributed to an interaction with nicotinic ion channels [10], however, receptor binding studies with human a7 and Torpedo californica nicotinic receptors showed that MB327 did not interact with the orthosteric binding site [13]. In contrast, MB327 exhibited a moderate affinity towards the human M5 muscarinic receptor [14], comparable to bispyridinium oximes [15]. In addition, previous studies showed a parasympatholytic effect of obidoxime in guinea pig ileum models [16,17]. An antimuscarinic effect of bispyridinium compounds may contribute to their therapeutic effect [18] or may result in adverse reaction. In view of the potential of MB327 to serve as a broad-spectrum antidote against OP poisoning, it was tempting to investigate the functional effects of MB327 in a rat jejunum model and to compare it to various established oximes, i.e. obidoxime, pralidoxime, TMB4, HI-6 and MMB-4 (Table 1). 2. Materials and methods 2.1. Chemicals HI-6 was kindly provided by Dr. Clement (Defence Research Establishment Suffield, Ralston, Alberta, Canada) and MMB-4 by Prof. J. Fusek (Purkyne Military Medical Academy, Hradec Kralove, Czech Republic). Obidoxime was purchased from Merck KG

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C. Königer et al. / Chemico-Biological Interactions 204 (2013) 1–5

Table 1 Chemical structure and smooth muscle relaxation of investigated compounds. R1 = substituent at the first pyridinium ring, R2 = substituent at the second pyridinium ring, a, b = position of the substituent, Y = linker-chain, EC50 = concentration in lM which shows half-maximal smooth muscle relaxation ± SD; ⁄ = contains only one pyridinium ring.

Compound

R1

a

Y

R2

b

EC50 (means ± SD) (lM)

MB327 TMB-4 Obidoxime Pralidoxime⁄ MMB-4 HI-6 Atropine

C–(CH3)3 CHNOH CHNOH CHNOH CHNOH CONH2 –

4 4 4 2 4 4 –

(CH2)3 (CH2)3 CH2–O–CH2 – CH2 CH2–O–CH2 –

C–(CH3)3 CHNOH CHNOH – CHNOH CHNOH –

4 4 4 – 4 2 –

6.21 ± 1.77 41.07 ± 14.23 90.47 ± 26.67 249.00 ± 57.90 264.50 ± 24.08 711.50 ± 112.50 0.015 ± 0.006

(Darmstadt, Germany). Atropine, MgCl2, carbachol, pralidoxime (2PAM) and TMB-4 were supplied by Sigma–Aldrich Chemie GmbH (Taufkirchen, Germany) and NaCl, CaCl2, NaHCO3, NaH2PO4, Glucose and KCl were delivered by Carl Roth GmbH + Co. KG (Karlsruhe, Germany). MB327 (>98%; Table 1) was synthesized at DSTL (Porton Down, UK).

tubes with a 5 l containing vacuum flask (Radnoti) to allow rapid buffer changes. Rats were anesthetized with carbon dioxide and killed by cervical dislocation. Then, the aboral 6 cm of the jejunum were excised and cut into five segments of 1 cm length. The segments were mounted vertically in the organ bath. At the lower side they were fixed to a glass retainer, the upper side was mounted to a force transducer which was connected to an amplifier (both Experimetria). Each of the five mounted jejunum segments from a single animal was put into one of the 15 ml single organ baths, containing Tyrode solution aerated with 95% O2 and 5% CO2, and was allowed to equilibrate for 45 min. During the equilibrationphase the buffer was changed every 15 min to maintain stable motility and muscle force. At the beginning of each experiment the basal activity of the individual segments was recorded for 5 min and from the last 2 min the absolute integral of the basal activity (AUCbasal activity) was calculated. Afterwards, 20 ll of a 1.5 mM carbachol solution were administered, resulting in an organ bath concentration of 2 lM. Immediately after addition of carbachol the activity at maximum stimulation was recorded for 5 min. Again, the last 2 min of this interval were used to calculate the integral of maximum smooth muscle stimulation (AUCcarbachol). Thereafter the jejunum segments were washed two times followed by a 10 min resting interval (for original registration see Fig. 1). In order to evaluate the effect of the test compounds the jejunum segments were stimulated with 2 lM carbachol. After 2 min the compounds were added as a 20 ll aliquot. The alteration of the smooth muscle force was recorded for further 3 min. The last 2 min of this interval were used for the calculation of the AUCsubstance integral. Afterwards, washing was performed followed by a 10 min resting phase. The test procedure was carried out

2.2. Animals Male Wistar rats (Charles River, Sulzfeld, Germany), weighing 250–300 g, were hold in small groups of six animals with an 12/ 12 h light–dark cycle with standard laboratory diet (Altromin, Lage, Germany) and water ad libitum. The animals were given a 10 days resting phase in order to accommodate to the new environment and food. At the point of examination the animals showed no signs of infections or intestinal disorders. The study protocol was approved by the local authorities. 2.3. Methods The model consisted of five water-jacketed single organ baths (Experimetria Ltd, Budapest, Hungary) and a water-jacketed buffer reservoir (Radnoti, Monrovia, CA, USA). The temperature of the water bath was adjusted to 37 °C. The Tyrode buffer-containing reservoir was connected with each of the five organ baths with a silicone tube. For the preparation and the organ bath Tyrode buffer with the following composition was used: NaCl 137 mM, CaCl2 1.8 mM, NaHCO3 22 mM, Glucose 5.5 mM, KCl 2.68 mM, MgCl2 1.05 mM, NaH2PO4 0.42 mM, aerated with 95% O2 and 5% CO2 (5 ml/min) at 37 °C. The organ baths were connected via silicone

d1

d2

c

c

d3

c

d4

c

c

F [mN]

2

w 3

w

w

w

w t [min]

Fig. 1. Original registration of a rat jejunum segment pre-constricted with carbachol and smooth muscle relaxation by MB327. After equilibration segments were stimulated with 2 lM carbachol (c) and basal activity was recorded for 2 min. 2 min after carbachol addition, non-cumulative doses of MB327 (d16 = 1-lM) were added and smooth muscle activity was recorded for 2 min. This was followed by washing (w) and a 10 min resting phase.

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simultaneously for all five organ baths and was repeated 6 times with six different substance concentrations. Solutions of the tested drugs were prepared daily with distilled water. The effect of the test compounds on smooth muscle activity was calculated according to: D AUC(substance) = (AUCsubstance)  (AUCbasal activity) and D AUC(carbachol) = (AUCcarbachol)  (AUCbasal activity) Finally, the isometric force was calculated as % of maximal carbachol stimulation by the equation: % isometric force = D AUC(substance)/D AUC(carbachol) ⁄100 SPEL Advanced Isosys software v3.25 (Experimetria Ltd, Budapest, Hungary) was used for data recording and analysis. 2.4. Statistical analysis The EC50 was calculated from semi-logarithmic plots of the test compound concentration versus the % isometric force using a sigmoidal dose–response model. Data are expressed as means ± standard deviation (SD). Gaussian distribution was analysed by Kolmogorov–Smirnov test. A p < 0.05 was considered as statistically significant.

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instead of tert-butyl groups (Table 1), was the most potent oxime tested followed by the closely related bispyridinium oxime obidoxime (Table 1). 2-PAM, MMB-4 and especially HI-6 showed a substantially lower smooth muscle relaxing effect (Table 1 and Fig. 2). Comparison of the EC50 values of the tested pyridinium compounds revealed a 7-fold (TMB-4) to 115-fold (HI-6) difference compared to MB327. This leads to the following order of potency: MB327  TMB-4 > obidoxime > pralidoxime > MMB-4 > HI-6. For comparison, the smooth muscle relaxation by the muscarinic antagonist atropine was investigated. The calculated EC50 of this OP antidote was 15 nM. Hence, atropine was more than 400fold (MB327) and up to 5000-fold (HI-6) more potent compared to the tested pyridinium compounds. To reveal effects of the combination of atropine and MB327, 25 nM atropine (resembling a therapeutic plasma concentration [5]) and MB327 were added to the specimens simultaneously. The combination of atropine (25 nM) and MB327 (1 lM) showed a similar effect as 25 nM atropine (log10 = 7.60) alone (Fig. 2). Subsequent increase of the MB327 concentration up to 45 lM in combination with atropine (25 nM) resulted in an even more pronounced antimuscarinic effect. 4. Discussion

3. Results 3.1. Method evaluation The basal activity of the smooth muscle preparations (AUC of 2 min) was determined at five different time points (30, 60, 90, 120 and 150 min) and showed a standard deviation within a range of less than 18%. Recording and calculating the smooth muscle activity over 2 min after stimulation, at 45, 60, 75, 90, 105, 120, 135 and 150 min, revealed a slight decrease of the stimulated smooth muscle activity (mean: 80 ± 8% of control at 150 min). The recorded data revealed Gaussian distribution according to the modified Kolmogorov–Smirnov test. 3.2. Effect on intestinal smooth muscle activity All tested compounds exhibited a concentration-dependent smooth muscle relaxing effect which followed a sigmoidal dose– response relationship (Fig. 2). After washing this effect was reversible for all investigated substances. Among the pyridinium compounds tested, MB327 showed to be most potent (EC50 = 6 lM; Table 1). The structural analogue TMB-4, bearing two oxime

The results of the present study demonstrated a concentrationdependent smooth muscle relaxation by the prospective OP antidote MB327. The calculated EC50 of 6.21 lM correlates with the affinity of human M5 muscarinic receptors (Ki = 3.3 lM) [14]. The effect of the MB327 on smooth muscle activity was fully reversible (Fig. 1). These findings together with previous receptor binding experiments [14] suggest that the effect of MB327 on smooth muscles is predominantly mediated by muscarinic receptors [19–22]. The non-oxime bispyridinium MB327 was 7- to 115-fold more potent compared to the tested mono- and bispyridinium oxime reactivators. These results are in line with previous studies showing that the MB327 analogue SAD-128, bearing an ether bridge instead of a trimethylene linker between the two pyridinium rings, had a substantially higher antimuscarinic potency compared to bispyridinium oximes [15,23–25]. These results show that a tertbutyl group instead of an oxime group is a decisive structural element for the antimuscarinic potential of bispyridinium compounds. This assumption is supported by the fact that, depending on the experimental protocol, the potency of SAD-128 was 3- to 7-fold higher compared to obidoxime [15,23–25] which is comparable to the data of MB327 and TMB-4. However, one has to take

Fig. 2. Smooth muscle relaxation of rat jejunum by MB327 and related bispyridinium compounds. Dose response curves of MB327 and oximes compared to atropine. Data are given as % isometric force (means ± SD) after jejunum segments were pretreated with 2 lM carbachol (n P 10 segments).

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into account, that MB327 and SAD-128 although bearing a higher antimuscarinic potency as their structural related oximes, are not able to reactivate inhibited acetylcholinesterase. Previous studies described an antimuscarinic effect of pralidoxime, obidoxime, TMB-4, MMB-4 and HI-6 in brain and intestinal smooth muscle models [15,17,18,23–28]. For this antimuscarinic effect much higher concentrations are necessary than clinically achieved plasma concentrations for reactivation in clinical toxicology [3] and can thus be interpreted as irrelevant side effect of oximes. However, due to different experimental protocols and species the results are hardly comparable. The present work demonstrates differences in the smooth muscle relaxing effect of oximes under identical conditions. Hereby, small structural differences had an in part remarkable effect on the calculated EC50 values (Table 1). Replacement of a trimethylene linker (TMB-4) by an ether bridge (obidoxime) resulted in a more than 2-fold higher EC50 while MMB-4, bearing a methyl linker, had a more than 6-fold higher EC50 compared to TMB-4. Modification of the position of the oxime group as well as replacement of an oxime group by an aminocarbonyl group (HI-6) caused an 8-fold higher EC50 compared to obidoxime. Due to unavailable pharmacokinetic and tissue distribution data of MB327 the effective concentration of this compound in target organs is unknown. Thus, a potential contribution of the antimuscarinic potential of MB327 to its overall therapeutic effect in OP poisoning is difficult to assess. If one assumes a pharmacokinetic profile comparable to obidoxime (due to the lack of pharmacokinetic data of MB327 in the literature), a similar plasma concentration of 10–20 lM [3] would already be approximately twice as high as the EC50 and might result in a pronounced antimuscarinic effect. Standard treatment of OP poisoning includes the administration of the muscarinic antagonist atropine and an oxime [29,30]. Hereby, atropine is considered as basic therapy being essential in counteracting the toxic OP effects at central and peripheral muscarinic receptors. Based on the experimental data with the combination of atropine and MB327, the antimuscarinic effect of the combination was higher than that of 25 nM atropine alone (Fig. 2). However, the present data do not allow to determine the exact mechanism and dynamic interaction of the antimuscarinic impact of both compounds. In conclusion, this study presents for the first time data on the effect of MB327 on smooth muscle activity. This bispyridinium non-oxime exhibits a remarkable smooth muscle relaxing effect which is probably mediated by an antagonistic effect at muscarinic receptors. In comparison to clinically used oximes MB327 had a 7- to 115-fold higher potency. At present, the relevance of the antimuscarinic properties of MB327 for its overall therapeutic effect in OP poisoning is difficult to assess. Additional studies are needed to evaluate the potential of MB327 as a broad-spectrum OP antidote and to investigate in detail its mechanisms of action in vivo.

5. Conflict of interest statement The authors declare that there are no conflicts of interest. The design, performance, data interpretation and manuscript writing was under the control of the authors and has not been influenced by the German Ministry of Defence.

Acknowledgements This study was funded by the German Ministry of Defence. The authors are grateful to Dr. C.M. Timperley, DSTL (Porton Down, UK) for donating MB327 and to Mrs. Brosig and Mrs. Binder for their excellent expert technical assistance.

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