European Journal of Pharmacology 735 (2014) 38–43
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Chlorpromazine confers neuroprotection against brain ischemia by activating BKCa channel Hua-Juan Li a, Yu-Jiao Zhang a, Li Zhou a, Feng Han b, Ming-Yan Wang a, Mao-Qiang Xue a,n, Zhi Qi a,n,1 a b
Department of Basic Medical Sciences, Medical College of Xiamen University, Xiang’an Nan Lu, Xiamen 361102, China College of Pharmaceutical Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, China
ar t ic l e i nf o
a b s t r a c t
Article history: Received 13 December 2013 Received in revised form 9 April 2014 Accepted 10 April 2014 Available online 19 April 2014
Chlorpromazine (CPZ) is a well-known antipsychotic drug, still widely being used to treat symptoms of schizophrenia, psychotic depression and organic psychoses. We have previously reported that CPZ activates the BKCa (KCa1.1) channel at whole cell level. In the present study, we demonstrated that CPZ increased the single channel open probability of the BKCa channels without changing its single channel amplitude. As BKCa channel is one of the molecular targets of brain ischemia, we explored a possible new use of this old drug on ischemic brain injury. In middle cerebral artery occlusion (MCAO) focal cerebral ischemia, a single intraperitoneal injection of CPZ at several dosages (5 mg/kg, 10 mg/kg and 20 mg/kg) could exert a significant neuroprotective effect on the brain damage in a dose- and time-dependent manner. Furthermore, blockade of BKCa channels abolished the neuroprotective effect of CPZ on MCAO, suggesting that the effect of CPZ is mediated by activation of the BKCa channel. These results demonstrate that CPZ could reduce focal cerebral ischemic damage through activating BKCa channels and merits exploration as a potential therapeutic agent for treating ischemic stroke. & 2014 Elsevier B.V. All rights reserved.
Keywords: Brain ischemia BKCa channels (KCa1.1) Chlorpromazine Middle cerebral artery occlusion
1. Introduction Drugs rarely act specifically to a single target in a disease process and elicit a cure with few side effects. Indeed, recent analyses of drug and drug-target networks show a rich pattern of interactions among drugs and their targets, where drugs acting on a single target are the exception. Side-effects are the unintended consequence of therapeutic treatments, but they can also provide valuable information for repositioning old drugs to bring new therapies (Duran-Frigola and Aloy, 2012; Yang and Agarwal, 2011). The main advantage of drug-repositioning strategies is that, since it starts from approved compounds with well-characterized pharmacology and safety profiles, it should drastically reduce the risk of attrition in clinical phases (Duran-Frigola and Aloy, 2012). Several drugs have been successfully repositioned to new indications. For example, sibutramine and milnacipran were initially developed for depression but now they are used for treating obesity and fibromyalgia syndrome, respectively (Novac, 2013).
n
Corresponding authors. E-mail addresses: [email protected] (M.-Q. Xue), [email protected] (Z. Qi). 1 Tel.: þ86 592 2181330.
http://dx.doi.org/10.1016/j.ejphar.2014.04.017 0014-2999/& 2014 Elsevier B.V. All rights reserved.
Chlorpromazine (CPZ) is a neuroleptic antipsychotic agent with a long history of clinical use (Shen, 1999; McNaughton et al., 2001; Carpenter and Koenig, 2008). Its therapeutic effect is commonly attributed to its ability to potently inhibit dopamine D2 receptors (Carpenter and Koenig, 2008). However, the psychiatric benefit of CPZ is accompanied by numerous side effects, such as antiserotonergic activity (Croll et al., 1997) and post-synaptic α1 adrenoceptor-blockade (Nedergaard and Abrahamsen, 1988). Besides, CPZ has been reported to interfere with a number of receptors and ion channels, such as Ca2 þ channels (McNaughton et al., 2001), K þ channels (Ogata and Tatebayashi, 1993; Nakazawa et al., 1995), nicotinic receptor channels (Benoit and Changeux, 1993), and Cl- channels (Quamme, 1997). Previously, we have shown that CPZ activates the largeconductance, Ca2 þ -activated potassium (BKCa, KCa1.1) channel at the whole cell level (Qi et al., 2005). In the present work, we showed that CPZ increased the single channel open probability of the BKCa channels without change in its single channel amplitude. Activation of BKCa channels has been shown to have neuroprotective effect on brain ischemia (Gribkoff et al., 2001; Chi et al., 2010), because BKCa channel activation would act to limit Ca2 þ entry, reduce excessive release of excitatory neurotransmitter and energy expenditure. Therefore, trying to reposition this old drug for new
H.-J. Li et al. / European Journal of Pharmacology 735 (2014) 38–43
use, we explored the possibility of using CPZ for treating focal cerebral ischemia.
2. Materials and methods 2.1. Electrophysiology The single channel current recordings were performed in the inside–out configuration at room temperature. Currents were amplified using EPC-10 patch-clamp amplifier (HEKA), sampled at 2–5 kHz and filtered at 1.5–2.9 kHz via a 4-pole low-pass Bessel filter. A program package Patchmaster 2.1 and TAC 4.1 (HEKA, Germany) were used for data acquisition and analysis. Pipettes had resistances in a range of 2–5 MΩ in the recording solution. Patch electrode and bath solutions were the same (in mM): 140 KCl, 2 MgCl2, 10 EGTA, 10 HEPES (pH 7.3), 320–335 mOsm, CaCl2 adjusted to free Ca2þ of 1 mM on the basis of calculations using a free software CALCON3. All experiments were performed at room temperature (20–25 1C). The value of the single channel open probability (Po) in a patch with multiple channels was calculated by using TAC 4.1 (HEKA, Germany), based on the equation: Po¼(1 Pc1/N), where Pc is the probability when all of the channels are in the closed state and N is the number of channels in the patch. 2.2. Focal ischemia Male Sprague-Dawley rats (weighing 200–250 g, Xiamen University Animal Experiment Center) were used throughout the study. Middle cerebral artery occlusion (MCAO) was induced by using intraluminal filament insertion technique as previously described (Chi et al., 2010). Briefly, the filament thread (4/0 gauge with the tip heat blunted to a diameter of 0.104 mm) was inserted through the left common carotid artery and advanced into the internal carotid artery to occlude the origin of the middle cerebral artery (approximately 18–20 mm). Body temperatures were monitored and controlled at 377 0.5 1C with a homeothermic blanket. To keep the condition as same as possible, only the rats with postoperative neurological score of 2 (circling to the right), a moderate focal neurologic deficit, were selected according to the Longa 5-point scale scoring system (Longa et al., 1989). The shamoperated animals were treated identically, except that the middle cerebral artery was not occluded after the neck incision. Animals were given unrestricted access to food and water. Animals were killed 24 h post-MCAO. Brains were rapidly removed. Brain slices were prepared by sectioning the brain coronally at 2 mm intervals, and stained by immersion in vital dye (2%) 2,3,5-triphenyltetrazolium hydrochloride (TTC). The slices were photographed, and analyzed with Image-J software (NIH freeware) and presented with Photoshop software (Adobe Systems Inc., San Jose, California, USA). Unstained areas were defined as ischemic lesions. An edema index was calculated by dividing the total volume of the hemisphere ipsilateral to MCAO by the total volume of the contralateral hemisphere. The actual infarct volume adjusted for edema was calculated by dividing the infarct volume by the edema index. Relative infarct volume is given as infarcted volume as the percentage of the total brain volume (Reglodi et al., 2000). All manipulations and analyses were performed by individuals blinded to all groups. Animal care and experiments were performed in accordance with procedures approved by the Animal Care and Use Committee of Xiamen University. 2.3. Drug administration CPZ diluted in 0.9% saline was administered intraperitoneally (i.p.) at a concentration of 0.5, 1, 5, 10 and 20 mg/kg. Saline (0.9%)
39
with 2% DMSO was used as a vehicle. Paxilline (Pax), a hydrophobic blocker of BKCa channels, was dissolved in DMSO and applied by i.p. injection. Charybdotoxin (CTX), a hydrophilic blocker of BKCa channels, was dissolved in 0.9% saline and injected into the intracerebroventricle (i.c.v.). I.c.v. injection was performed by stereotaxic technique using a tepper-motorized micro-syringe with cannula inserted stereotactically at 0.8 mm posterior to bregma, 1.5 mm lateral to midline, and 3.8 mm ventral to the dura. Corresponding solutions were injected at a rate of 1 μl/min (final volume ¼ 5 ml/rat). Rats in the control group were given an equal volume of the vehicle. 2.4. Statistical analysis All the data are expressed as mean 7S.E.M. (standard error of mean). Experimental results were compared among groups by ANOVA, followed by Bonferroni's post hoc test. Differences at Po 0.05 were considered to be statistically significant.
3. Results 3.1. Effect of CPZ on BKCa channel at the single channel level We have reported that CPZ could increase the whole cell current of the BKCa channel in a dose-dependent manner (Qi et al., 2005). However, it is not known whether cytosolic factors are involved in the channel activation, whether the increased current is due to increase in single channel open probability or single channel amplitude. To address these questions, we turned to the inside–out patch-clamp configuration. In the inside–out patch configuration, the single channel current traces at different membrane potentials with or without 20 μM CPZ showed that CPZ increased the Po of the channel at every corresponding membrane potential (Fig. 1A and B). Fig. 1C shows the statistical data on the Po of the channel in the control condition and in the presence of 20 μM CPZ. In the control condition, Po was 3.6 71.3%, while it increased to 13.7 70.7% in the presence of 20 μM CPZ. The current–voltage relationship of the single BKCa channel indicated that CPZ increased the single channel open probabilities without changing the single channel amplitude (Fig. 1D), suggesting that the increase in the whole cell current by CPZ was due to increase in the single channel open probabilities but not its single channel conductance. As the excised membrane patch is not affected by the cytosolic factors, the result also indicated that cytosolic factors are not involved in increasing the single channel open probabilities by CPZ. 3.2. Effect of CPZ on brain infarct size after MCAO To determine the effect of CPZ against focal cerebral ischemia induced injury, we tested the protective effect of CPZ on MCAO rats. The brain slices were stained with TTC, which stains healthy tissue red and leaves infarct tissue pale white. At 24 h post-MCAO, the percentage of infarct size was 23.8 71.4% in the vehicle injected MCAO rat (n ¼23). A single injection of CPZ, at dosages of 0.5, 1.0, 5, 10, and 20 mg/kg immediately following MCAO, dosedependently reduced the percentage of the infarct size to 20.1 71.2% (n ¼6), 19.4 71.8% (n ¼11), 14.8 72.6% (n ¼5), 13.9 7 1.3% (n ¼9) and 17.1 72.5% (n ¼5), respectively (Fig. 2), indicating that CPZ could reduce the cerebral infarct induced by MCAO. The same level of dosage has been shown to be effective in neuronal apoptosis induced by ethanol (Wu et al., 2011). As CPZ at a dosage of 10 mg/kg gave the best protective effect on ischemic damage, this dosage is used in the following study. Furthermore,
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H.-J. Li et al. / European Journal of Pharmacology 735 (2014) 38–43
Fig. 1. Effect of CPZ on BKCa channels at the single channel level in the inside–out configuration. Representative single channel current traces of the BKCa channels at membrane potentials ranging from 60 mV to 10 mV in the absence (A) and presence (B) of 20 μM CPZ. Arrows indicat the level that all the channels are in the closed state. (C) Statistical data on the Po of the single BKCa channel at a voltage of 10 mV in the absence and presence of 20 μM CPZ. (D) Statistical data on the single channel current amplitude vs. membrane voltage in the absence and presence of CPZ. Values represented as mean 7S.E.M. of at least three independent experiments.
we found that CPZ has an effect even when injected 1 h postMCAO (Fig. 3). 3.3. Blockade of BKCa channels abolishes the neuroprotective effect of CPZ on MCAO If BKCa channel is the molecular target, then its specific blockers should suppress the neuroprotective effect of CPZ. To determine whether the neuroprotection of CPZ is associated with the BKCa channel activation, CTX (400 nM), a water soluble specific BKCa channel blocker, was given by i.c.v. injection 30 min post-MCAO accompanied by i.p. injection of CPZ (10 mg/kg) at 1 h post-MCAO. In the vehicle-treated group, the percentage of the infarct size was 27.6 þ2.7% at 24 h post-MCAO (n ¼7). In the CPZ-treated group, the
Fig. 2. Effect of CPZ on brain injury after MCAO. (A) Representative images of brain slices obtained at 24 h post-MCAO from sham operation, vehicle-treated MCAO and 0.5–20 mg/kg CPZ-treated MCAO rats. (B) Relative infarct volume in vehicle-treated and 0.5–20 mg/kg CPZ-treated MCAO rats vs. sham operated groups. n: P o 0.05 and nn : P o 0.01.
percentage of the infarct size was 17.6þ1.6% (n¼ 8). In contrast, the percentage of the infarct size was 25.2þ2.4% (n¼11) for the CPZtreated group with i.c.v. injection of CTX (400 nM) (Fig. 4). Therefore, the blockade of BKCa channel by CTX significantly suppressed the neuroprotective effect of CPZ. To further confirm whether BKCa channels are involved, we selected a hydrophobic blocker Paxilline (Pax). In the vehicletreated group, the percentage of the infarct size was 25.6 þ3.6% at 24 h post-MCAO (n ¼6). In the CPZ-treated group, the percentage of the infarct size was 15.6 þ1.7% (n ¼7). In contrast, the percentage of the infarct size was 22.2 þ1.7% (n ¼11) for the CPZ-treated group with i.p. injection of Pax (2.2 mg/kg) (Fig. 5), indicating that Pax could suppress the effect of CPZ. These two blockers have very different properties and different mechanisms of action: One is hydrophobic, another is hydrophilic; one was applied by i.c.v. injection, the other was applied by i.p. injection. However, both blockers could abolish the effect of CPZ, which strongly suggests that CPZ protects ischemia-induced brain injury via activation of BKCa channels and gives further evidence that the BKCa channel is the molecular target of CPZ.
H.-J. Li et al. / European Journal of Pharmacology 735 (2014) 38–43
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Fig. 3. Time-dependent effect of CPZ on the brain infarct size. (A) Representative images of brain slices for vehicle-treated sham operation, vehicle-treated MCAO and CPZ (10 mg/kg)-treated MCAO rats at 0 h and 1 h post-MCAO. (B) Relative infarct size in vehicle-treated and CPZ (10 mg/kg)-treated MCAO rats at 24 h post-MCAO vs. sham operated groups. n: Po 0.05 and nn: Po 0.01.
Fig. 4. Effect of CPZ on the brain infarct size in the presence of a specific hydrophilic BKCa channel blocker CTX. (A) Representative images of brain slices for vehicle-treated MCAO and CPZ (10 mg/kg)-treated MCAO rats at 24 h post-MCAO in the absence and presence of CTX. (B) Relative infarct size in vehicle-treated and CPZ (10 mg/kg)-treated MCAO rats at 24 h post-MCAO in the absence and presence of CTX vs. sham operated groups. nn: Po 0.01.
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H.-J. Li et al. / European Journal of Pharmacology 735 (2014) 38–43
Fig. 5. Effect of CPZ on the brain infarct size in the presence of specific hydrophobic BKCa channel blocker paxiiline (Pax). (A) Representative images of brain slices for vehicletreated MCAO and CPZ (10 mg/kg)-treated MCAO rats at 24 h post-MCAO in the absence and presence of Pax. (B) Relative infarct volume in vehicle-treated and CPZ (10 mg/ kg)-treated MCAO rats at 24 h post-MCAO in the absence and presence of Pax vs. sham operated groups. n: Po 0.05.
4. Discussion CPZ is a widely used antipsychotic drug. It is highly lipophilic and would pass readily through lipid bilayers and the blood brain barrier. Even though the steady state plasma levels of CPZ are in the range of 10–230 ng/ml when patient received 200 mg bid (Cooper, 1978), the concentration of CPZ in the brain might be much higher because CPZ has been shown to be highly bound to plasma proteins (Curry, 1970). It has been reported that brain uptakes 6.04 71.6% of the injected CPZ 15 min after the injection, and the concentration remains constant for 45 min (Comar et al., 1979). The brain to body weight ratio for adult rats is approximately 2%. Therefore, CPZ is substantially concentrated within the brain (Tsuneizumi et al., 1992). In schizophrenic patients, the average serum levels of CPZ in the plasma are in the range of 3.5–21.5 μg/ml (Huang and Ruskin, 1964), which is roughly 1–60 μM. In our study, we demonstrated that a single i.p. injection of CPZ at dosages of 5, 10 and 20 mg/kg, which is several tens of micromole concentration, could exert significant neuroprotective effect on the brain damage. This is in the concentration range that could activate the BKCa channels. These results suggest that CPZ is capable of activating BKCa channels at therapeutic concentrations and in so doing plays a neuroprotective role in brain ischemia. In consistence with our result, CPZ has been shown to be able to exert neuroprotective effect on spinal cord ischemia (Sader et al., 2002), glutamate-induced neurotoxicity (Stone and Pilowsky, 2007), neuronal apoptosis induced by ethanol (Wu et al., 2011) and primary hippocampal neuronal cell death induced by growth medium deprivation (Bastianetto et al., 2006). Based on our results, we infer that patients that frequently take CPZ would have lower possibility of getting ischemic brain injury. An epidemiological
investigation on ischemia occurrence in patients will help to address this issue.
Acknowledgments This work was supported by the National Natural Science Foundation of China (NSFC Grant nos. 31070741, 31270891, and 81000560).
References Bastianetto, S., Danik, M., Mennicken, F., Williams, S., Quirion, R., 2006. Prototypical antipsychotic drugs protect hippocampal neuronal cultures against cell death induced by growth medium deprivation. BMC Neurosci. 7, 28–37. Benoit, P., Changeux, J.P., 1993. Voltage dependencies of the effects of chlorpromazine on the nicotinic receptor channel from mouse muscle cell line So18. Neurosci. Lett. 160, 81–84. Carpenter, W.T., Koenig, J.I., 2008. The evolution of drug development in schizophrenia: past issues and future opportunities. Neuropsychopharmacology 33, 2061–2079. Chi, S., Cai, W., Liu, P., Zhang, Z., Chen, X., Gao, L., Qi, J., Bi, L., Chen, L., Qi, Z., 2010. Baifuzi reduces transient ischemic brain damage through an interaction with the STREX domain of BKCa channels. Cell Death Dis. 1, e13. Comar, D., Zarifian, E., Verhas, M., Soussaline, F., Maziere, M., Berger, G., Loo, H., Cuche, H., Kellershohn, C., Deniker, P., 1979. Brain distribution and kinetics of 11C-chlorpromazine in schizophrenics: positron emission tomography studies. Psychiatry Res. 1, 23–29. Cooper, T.B., 1978. Plasma level monitoring of antipsychotic drugs. Clin. Pharmacokinet. 3, 14–38. Croll, R.P., Baker, M.W., Khabarova, M., Voronezhskaya, E.E., Sakharov, D.A., 1997. Serotonin depletion after prolonged chlorpromazine treatment in a simpler model system. Gen. Pharmacol 29, 91–96. Curry, S.H., 1970. Plasma protein binding of chlorpromazine. J. Pharm. Pharmacol. 22, 193–197.
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Duran-Frigola, M., Aloy, P., 2012. Recycling side-effects into clinical markers for drug repositioning. Genome Med. 4, 3–6. Gribkoff, V.K., Starrett Jr., J.E., Dworetzky, S.I., Hewawasam, P., Boissard, C.G., Cook, D.A., Frantz, S.W., Heman, K., Hibbard, J.R., Huston, K., Johnson, G., Krishnan, B. S., Kinney, G.G., Lombardo, L.A., Meanwell, N.A., Molinoff, P.B., Myers, R.A., Moon, S.L., Ortiz, A., Pajor, L., Pieschl, R.L., Post-Munson, D.J., Signor, L.J., Srinivas, N., Taber, M.T., Thalody, G., Trojnacki, J.T., Wiener, H., Yeleswaram, K., Yeola, S.W., 2001. Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels. Nat. Med. 7, 471–477. Huang, C.L., Ruskin, B.H., 1964. Determination of serum chlorpromazine metabolities in psychotic patients. J. Nerv. Ment. Dis. 139, 381–386. Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84–91. McNaughton, N.C.L., Green, P.J., Randall, A.D., 2001. Inhibition of human alpha(1E) subunit-mediated Ca2 þ channels by the antipsychotic agent chlorpromazine. Acta Physiol. Scand. 173, 401–408. Nakazawa, K., Ito, K., Koizumi, S., Ohno, Y., Inoue, K., 1995. Characterization of inhibition by haloperidol and chlorpromazine of a voltage-activated K þ current in rat phaeochromocytoma cells. Br. J. Pharmacol. 116, 2603–2610. Nedergaard, O.A., Abrahamsen, J., 1988. Effect of chlorpromazine on sympathetic neuroeffector transmission in the rabbit isolated pulmonary artery and aorta. Br. J. Pharmacol. 93, 23–34. Novac, N., 2013. Challenges and opportunities of drug repositioning. Trends Pharmacol. Sci. 34, 267–272. Ogata, N., Tatebayashi, H., 1993. Differential inhibition of a transient K þ current by chlorpromazine and 4-amino-pyridine in neurones of the rat dorsal root ganglia. Br. J. Pharmacol. 109, 1239–1246.
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Qi, Z., Chi, S., Su, X., Naruse, K., Sokabe, M., 2005. Activation of a mechanosensitive BK channel by membrane stress created with amphipaths. Mol. Membr. Biol. 22, 519–527. Quamme, G.A., 1997. Chlorpromazine activates chloride currents in Xenopus oocytes. Biochim. Biophys. Acta 1324, 18–26. Reglodi, D., Somogyvari-Vigh, A., Vigh, S., Kozicz, T., Arimura, A., 2000. Delayed systemic administration of PACAP38 is neuroprotective in transient middle cerebral artery occlusion in the rat. Stroke 31, 1411–1417. Sader, A.A., Barbieri-Neto, J., Sader, S.L., Mazzetto, S.A., Alves Jr., P., Vanni, J.C., 2002. The protective action of chlorpromazine on the spinal cord of rabbits submitted to ischemia and reperfusion is dose-dependent. J. Cardiovasc. Surg. (Torino) 43, 827–831. Shen, W.W., 1999. A history of antipsychotic drug development. Compr. Psychiatry 40, 407–414. Stone, J.M., Pilowsky, L.S., 2007. Novel targets for drugs in schizophrenia. CNS Neurol. Disord. Drug Targets 6, 265–272. Tsuneizumi, T., Babb, S.M., Cohen, B.M., 1992. Drug distribution between blood and brain as a determinant of antipsychotic drug effects. Biol. Psychiatry 32, 817–824. Wu, J., Song, R., Song, W., Li, Y., Zhang, Q., Chen, Y., Fu, Y., Fang, W., Wang, J., Zhong, Z., Ling, H., Zhang, L., Zhang, F., 2011. Chlorpromazine protects against apoptosis induced by exogenous stimuli in the developing rat brain. PLoS One 6, e21966. Yang, L., Agarwal, P., 2011. Systematic drug repositioning based on clinical sideeffects. PLoS One 6, e28025.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Chlorpromazine confers neuroprotection against brain ischemia by activating BKCa channel Hua-Juan Li a, Yu-Jiao Zhang a, Li Zhou a, Feng Han b, Ming-Yan Wang a, Mao-Qiang Xue a,n, Zhi Qi a,n,1 a b
Department of Basic Medical Sciences, Medical College of Xiamen University, Xiang’an Nan Lu, Xiamen 361102, China College of Pharmaceutical Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, China
ar t ic l e i nf o
a b s t r a c t
Article history: Received 13 December 2013 Received in revised form 9 April 2014 Accepted 10 April 2014 Available online 19 April 2014
Chlorpromazine (CPZ) is a well-known antipsychotic drug, still widely being used to treat symptoms of schizophrenia, psychotic depression and organic psychoses. We have previously reported that CPZ activates the BKCa (KCa1.1) channel at whole cell level. In the present study, we demonstrated that CPZ increased the single channel open probability of the BKCa channels without changing its single channel amplitude. As BKCa channel is one of the molecular targets of brain ischemia, we explored a possible new use of this old drug on ischemic brain injury. In middle cerebral artery occlusion (MCAO) focal cerebral ischemia, a single intraperitoneal injection of CPZ at several dosages (5 mg/kg, 10 mg/kg and 20 mg/kg) could exert a significant neuroprotective effect on the brain damage in a dose- and time-dependent manner. Furthermore, blockade of BKCa channels abolished the neuroprotective effect of CPZ on MCAO, suggesting that the effect of CPZ is mediated by activation of the BKCa channel. These results demonstrate that CPZ could reduce focal cerebral ischemic damage through activating BKCa channels and merits exploration as a potential therapeutic agent for treating ischemic stroke. & 2014 Elsevier B.V. All rights reserved.
Keywords: Brain ischemia BKCa channels (KCa1.1) Chlorpromazine Middle cerebral artery occlusion
1. Introduction Drugs rarely act specifically to a single target in a disease process and elicit a cure with few side effects. Indeed, recent analyses of drug and drug-target networks show a rich pattern of interactions among drugs and their targets, where drugs acting on a single target are the exception. Side-effects are the unintended consequence of therapeutic treatments, but they can also provide valuable information for repositioning old drugs to bring new therapies (Duran-Frigola and Aloy, 2012; Yang and Agarwal, 2011). The main advantage of drug-repositioning strategies is that, since it starts from approved compounds with well-characterized pharmacology and safety profiles, it should drastically reduce the risk of attrition in clinical phases (Duran-Frigola and Aloy, 2012). Several drugs have been successfully repositioned to new indications. For example, sibutramine and milnacipran were initially developed for depression but now they are used for treating obesity and fibromyalgia syndrome, respectively (Novac, 2013).
n
Corresponding authors. E-mail addresses: [email protected] (M.-Q. Xue), [email protected] (Z. Qi). 1 Tel.: þ86 592 2181330.
http://dx.doi.org/10.1016/j.ejphar.2014.04.017 0014-2999/& 2014 Elsevier B.V. All rights reserved.
Chlorpromazine (CPZ) is a neuroleptic antipsychotic agent with a long history of clinical use (Shen, 1999; McNaughton et al., 2001; Carpenter and Koenig, 2008). Its therapeutic effect is commonly attributed to its ability to potently inhibit dopamine D2 receptors (Carpenter and Koenig, 2008). However, the psychiatric benefit of CPZ is accompanied by numerous side effects, such as antiserotonergic activity (Croll et al., 1997) and post-synaptic α1 adrenoceptor-blockade (Nedergaard and Abrahamsen, 1988). Besides, CPZ has been reported to interfere with a number of receptors and ion channels, such as Ca2 þ channels (McNaughton et al., 2001), K þ channels (Ogata and Tatebayashi, 1993; Nakazawa et al., 1995), nicotinic receptor channels (Benoit and Changeux, 1993), and Cl- channels (Quamme, 1997). Previously, we have shown that CPZ activates the largeconductance, Ca2 þ -activated potassium (BKCa, KCa1.1) channel at the whole cell level (Qi et al., 2005). In the present work, we showed that CPZ increased the single channel open probability of the BKCa channels without change in its single channel amplitude. Activation of BKCa channels has been shown to have neuroprotective effect on brain ischemia (Gribkoff et al., 2001; Chi et al., 2010), because BKCa channel activation would act to limit Ca2 þ entry, reduce excessive release of excitatory neurotransmitter and energy expenditure. Therefore, trying to reposition this old drug for new
H.-J. Li et al. / European Journal of Pharmacology 735 (2014) 38–43
use, we explored the possibility of using CPZ for treating focal cerebral ischemia.
2. Materials and methods 2.1. Electrophysiology The single channel current recordings were performed in the inside–out configuration at room temperature. Currents were amplified using EPC-10 patch-clamp amplifier (HEKA), sampled at 2–5 kHz and filtered at 1.5–2.9 kHz via a 4-pole low-pass Bessel filter. A program package Patchmaster 2.1 and TAC 4.1 (HEKA, Germany) were used for data acquisition and analysis. Pipettes had resistances in a range of 2–5 MΩ in the recording solution. Patch electrode and bath solutions were the same (in mM): 140 KCl, 2 MgCl2, 10 EGTA, 10 HEPES (pH 7.3), 320–335 mOsm, CaCl2 adjusted to free Ca2þ of 1 mM on the basis of calculations using a free software CALCON3. All experiments were performed at room temperature (20–25 1C). The value of the single channel open probability (Po) in a patch with multiple channels was calculated by using TAC 4.1 (HEKA, Germany), based on the equation: Po¼(1 Pc1/N), where Pc is the probability when all of the channels are in the closed state and N is the number of channels in the patch. 2.2. Focal ischemia Male Sprague-Dawley rats (weighing 200–250 g, Xiamen University Animal Experiment Center) were used throughout the study. Middle cerebral artery occlusion (MCAO) was induced by using intraluminal filament insertion technique as previously described (Chi et al., 2010). Briefly, the filament thread (4/0 gauge with the tip heat blunted to a diameter of 0.104 mm) was inserted through the left common carotid artery and advanced into the internal carotid artery to occlude the origin of the middle cerebral artery (approximately 18–20 mm). Body temperatures were monitored and controlled at 377 0.5 1C with a homeothermic blanket. To keep the condition as same as possible, only the rats with postoperative neurological score of 2 (circling to the right), a moderate focal neurologic deficit, were selected according to the Longa 5-point scale scoring system (Longa et al., 1989). The shamoperated animals were treated identically, except that the middle cerebral artery was not occluded after the neck incision. Animals were given unrestricted access to food and water. Animals were killed 24 h post-MCAO. Brains were rapidly removed. Brain slices were prepared by sectioning the brain coronally at 2 mm intervals, and stained by immersion in vital dye (2%) 2,3,5-triphenyltetrazolium hydrochloride (TTC). The slices were photographed, and analyzed with Image-J software (NIH freeware) and presented with Photoshop software (Adobe Systems Inc., San Jose, California, USA). Unstained areas were defined as ischemic lesions. An edema index was calculated by dividing the total volume of the hemisphere ipsilateral to MCAO by the total volume of the contralateral hemisphere. The actual infarct volume adjusted for edema was calculated by dividing the infarct volume by the edema index. Relative infarct volume is given as infarcted volume as the percentage of the total brain volume (Reglodi et al., 2000). All manipulations and analyses were performed by individuals blinded to all groups. Animal care and experiments were performed in accordance with procedures approved by the Animal Care and Use Committee of Xiamen University. 2.3. Drug administration CPZ diluted in 0.9% saline was administered intraperitoneally (i.p.) at a concentration of 0.5, 1, 5, 10 and 20 mg/kg. Saline (0.9%)
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with 2% DMSO was used as a vehicle. Paxilline (Pax), a hydrophobic blocker of BKCa channels, was dissolved in DMSO and applied by i.p. injection. Charybdotoxin (CTX), a hydrophilic blocker of BKCa channels, was dissolved in 0.9% saline and injected into the intracerebroventricle (i.c.v.). I.c.v. injection was performed by stereotaxic technique using a tepper-motorized micro-syringe with cannula inserted stereotactically at 0.8 mm posterior to bregma, 1.5 mm lateral to midline, and 3.8 mm ventral to the dura. Corresponding solutions were injected at a rate of 1 μl/min (final volume ¼ 5 ml/rat). Rats in the control group were given an equal volume of the vehicle. 2.4. Statistical analysis All the data are expressed as mean 7S.E.M. (standard error of mean). Experimental results were compared among groups by ANOVA, followed by Bonferroni's post hoc test. Differences at Po 0.05 were considered to be statistically significant.
3. Results 3.1. Effect of CPZ on BKCa channel at the single channel level We have reported that CPZ could increase the whole cell current of the BKCa channel in a dose-dependent manner (Qi et al., 2005). However, it is not known whether cytosolic factors are involved in the channel activation, whether the increased current is due to increase in single channel open probability or single channel amplitude. To address these questions, we turned to the inside–out patch-clamp configuration. In the inside–out patch configuration, the single channel current traces at different membrane potentials with or without 20 μM CPZ showed that CPZ increased the Po of the channel at every corresponding membrane potential (Fig. 1A and B). Fig. 1C shows the statistical data on the Po of the channel in the control condition and in the presence of 20 μM CPZ. In the control condition, Po was 3.6 71.3%, while it increased to 13.7 70.7% in the presence of 20 μM CPZ. The current–voltage relationship of the single BKCa channel indicated that CPZ increased the single channel open probabilities without changing the single channel amplitude (Fig. 1D), suggesting that the increase in the whole cell current by CPZ was due to increase in the single channel open probabilities but not its single channel conductance. As the excised membrane patch is not affected by the cytosolic factors, the result also indicated that cytosolic factors are not involved in increasing the single channel open probabilities by CPZ. 3.2. Effect of CPZ on brain infarct size after MCAO To determine the effect of CPZ against focal cerebral ischemia induced injury, we tested the protective effect of CPZ on MCAO rats. The brain slices were stained with TTC, which stains healthy tissue red and leaves infarct tissue pale white. At 24 h post-MCAO, the percentage of infarct size was 23.8 71.4% in the vehicle injected MCAO rat (n ¼23). A single injection of CPZ, at dosages of 0.5, 1.0, 5, 10, and 20 mg/kg immediately following MCAO, dosedependently reduced the percentage of the infarct size to 20.1 71.2% (n ¼6), 19.4 71.8% (n ¼11), 14.8 72.6% (n ¼5), 13.9 7 1.3% (n ¼9) and 17.1 72.5% (n ¼5), respectively (Fig. 2), indicating that CPZ could reduce the cerebral infarct induced by MCAO. The same level of dosage has been shown to be effective in neuronal apoptosis induced by ethanol (Wu et al., 2011). As CPZ at a dosage of 10 mg/kg gave the best protective effect on ischemic damage, this dosage is used in the following study. Furthermore,
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Fig. 1. Effect of CPZ on BKCa channels at the single channel level in the inside–out configuration. Representative single channel current traces of the BKCa channels at membrane potentials ranging from 60 mV to 10 mV in the absence (A) and presence (B) of 20 μM CPZ. Arrows indicat the level that all the channels are in the closed state. (C) Statistical data on the Po of the single BKCa channel at a voltage of 10 mV in the absence and presence of 20 μM CPZ. (D) Statistical data on the single channel current amplitude vs. membrane voltage in the absence and presence of CPZ. Values represented as mean 7S.E.M. of at least three independent experiments.
we found that CPZ has an effect even when injected 1 h postMCAO (Fig. 3). 3.3. Blockade of BKCa channels abolishes the neuroprotective effect of CPZ on MCAO If BKCa channel is the molecular target, then its specific blockers should suppress the neuroprotective effect of CPZ. To determine whether the neuroprotection of CPZ is associated with the BKCa channel activation, CTX (400 nM), a water soluble specific BKCa channel blocker, was given by i.c.v. injection 30 min post-MCAO accompanied by i.p. injection of CPZ (10 mg/kg) at 1 h post-MCAO. In the vehicle-treated group, the percentage of the infarct size was 27.6 þ2.7% at 24 h post-MCAO (n ¼7). In the CPZ-treated group, the
Fig. 2. Effect of CPZ on brain injury after MCAO. (A) Representative images of brain slices obtained at 24 h post-MCAO from sham operation, vehicle-treated MCAO and 0.5–20 mg/kg CPZ-treated MCAO rats. (B) Relative infarct volume in vehicle-treated and 0.5–20 mg/kg CPZ-treated MCAO rats vs. sham operated groups. n: P o 0.05 and nn : P o 0.01.
percentage of the infarct size was 17.6þ1.6% (n¼ 8). In contrast, the percentage of the infarct size was 25.2þ2.4% (n¼11) for the CPZtreated group with i.c.v. injection of CTX (400 nM) (Fig. 4). Therefore, the blockade of BKCa channel by CTX significantly suppressed the neuroprotective effect of CPZ. To further confirm whether BKCa channels are involved, we selected a hydrophobic blocker Paxilline (Pax). In the vehicletreated group, the percentage of the infarct size was 25.6 þ3.6% at 24 h post-MCAO (n ¼6). In the CPZ-treated group, the percentage of the infarct size was 15.6 þ1.7% (n ¼7). In contrast, the percentage of the infarct size was 22.2 þ1.7% (n ¼11) for the CPZ-treated group with i.p. injection of Pax (2.2 mg/kg) (Fig. 5), indicating that Pax could suppress the effect of CPZ. These two blockers have very different properties and different mechanisms of action: One is hydrophobic, another is hydrophilic; one was applied by i.c.v. injection, the other was applied by i.p. injection. However, both blockers could abolish the effect of CPZ, which strongly suggests that CPZ protects ischemia-induced brain injury via activation of BKCa channels and gives further evidence that the BKCa channel is the molecular target of CPZ.
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Fig. 3. Time-dependent effect of CPZ on the brain infarct size. (A) Representative images of brain slices for vehicle-treated sham operation, vehicle-treated MCAO and CPZ (10 mg/kg)-treated MCAO rats at 0 h and 1 h post-MCAO. (B) Relative infarct size in vehicle-treated and CPZ (10 mg/kg)-treated MCAO rats at 24 h post-MCAO vs. sham operated groups. n: Po 0.05 and nn: Po 0.01.
Fig. 4. Effect of CPZ on the brain infarct size in the presence of a specific hydrophilic BKCa channel blocker CTX. (A) Representative images of brain slices for vehicle-treated MCAO and CPZ (10 mg/kg)-treated MCAO rats at 24 h post-MCAO in the absence and presence of CTX. (B) Relative infarct size in vehicle-treated and CPZ (10 mg/kg)-treated MCAO rats at 24 h post-MCAO in the absence and presence of CTX vs. sham operated groups. nn: Po 0.01.
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Fig. 5. Effect of CPZ on the brain infarct size in the presence of specific hydrophobic BKCa channel blocker paxiiline (Pax). (A) Representative images of brain slices for vehicletreated MCAO and CPZ (10 mg/kg)-treated MCAO rats at 24 h post-MCAO in the absence and presence of Pax. (B) Relative infarct volume in vehicle-treated and CPZ (10 mg/ kg)-treated MCAO rats at 24 h post-MCAO in the absence and presence of Pax vs. sham operated groups. n: Po 0.05.
4. Discussion CPZ is a widely used antipsychotic drug. It is highly lipophilic and would pass readily through lipid bilayers and the blood brain barrier. Even though the steady state plasma levels of CPZ are in the range of 10–230 ng/ml when patient received 200 mg bid (Cooper, 1978), the concentration of CPZ in the brain might be much higher because CPZ has been shown to be highly bound to plasma proteins (Curry, 1970). It has been reported that brain uptakes 6.04 71.6% of the injected CPZ 15 min after the injection, and the concentration remains constant for 45 min (Comar et al., 1979). The brain to body weight ratio for adult rats is approximately 2%. Therefore, CPZ is substantially concentrated within the brain (Tsuneizumi et al., 1992). In schizophrenic patients, the average serum levels of CPZ in the plasma are in the range of 3.5–21.5 μg/ml (Huang and Ruskin, 1964), which is roughly 1–60 μM. In our study, we demonstrated that a single i.p. injection of CPZ at dosages of 5, 10 and 20 mg/kg, which is several tens of micromole concentration, could exert significant neuroprotective effect on the brain damage. This is in the concentration range that could activate the BKCa channels. These results suggest that CPZ is capable of activating BKCa channels at therapeutic concentrations and in so doing plays a neuroprotective role in brain ischemia. In consistence with our result, CPZ has been shown to be able to exert neuroprotective effect on spinal cord ischemia (Sader et al., 2002), glutamate-induced neurotoxicity (Stone and Pilowsky, 2007), neuronal apoptosis induced by ethanol (Wu et al., 2011) and primary hippocampal neuronal cell death induced by growth medium deprivation (Bastianetto et al., 2006). Based on our results, we infer that patients that frequently take CPZ would have lower possibility of getting ischemic brain injury. An epidemiological
investigation on ischemia occurrence in patients will help to address this issue.
Acknowledgments This work was supported by the National Natural Science Foundation of China (NSFC Grant nos. 31070741, 31270891, and 81000560).
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