Brain Research 868 (2000) 123–127www.elsevier.com/ locate /bres

Short communication

Antinociceptive effect of cilnidipine, a novel N-type calcium channelantagonist

a , c b b*Manabu Murakami , Osamu Nakagawasai , Shigeo Fujii , Masahiro Hosono ,c c c a dSoichi Hozumi , Akihisa Esashi , Ryoo Taniguchi , Tadashi Okamura , Takashi Suzuki ,

d a c cHironobu Sasano , Teruyuki Yanagisawa , Koichi Tan-no , Takeshi Tadano ,e cKenji Kitamura , Kensuke Kisara

aDepartment of Molecular Pharmacology, Tohoku University School of Medicine, Seiryomachi, Aobaku, Sendai 980-8575, JapanbPharmaceuticals Research Laboratories, Fujirebio Inc., Sendai, Japan

cDepartment of Pharmacology, Tohoku Pharmaceutical University, Sendai, JapandDepartment of Pathology, Tohoku University School of Medicine, Sendai, Japan

eDepartment of Pharmacology, Fukuoka Dental College, Fukuoka, Japan

Accepted 14 March 2000

Abstract

We investigated the antinociceptic effects of cilnidipine, a dihydropyridine derivative which acts on both L- and N-type voltage-dependent calcium channels, in mice. Intrathecally injected cilnidipine showed significant analgesic effect in formalin test. Cilnidipinesignificantly suppressed N-type currents in dorsal root ganglion (DRG) cells. Our findings apparently support the idea that cilnidipineattenuates synaptic neurotransmission by inhibiting N-type calcium channels in DRG neurons. 2000 Elsevier Science B.V. All rightsreserved.

Theme: Excitable membranes and synaptic transmission

Topic: Calcium channel physiology, pharmacology, and modulation

Keywords: Calcium channel; Cilnidipine; Nociception

Dihydropyridine (DHP) calcium channel antagonists act The aim of this study was to clarify the antinociceptiveon L-type calcium channels and have been used for the action of cilnidipine in mice. For this purpose, we ex-treatment of cardiovascular disease [4]. Among them, amined the effects of intrathecally injected cilnidipine oncilnidipine (FRC-8653) has a slow-onset, long-lasting the behavioral responses to the nociceptive stimulation byhypotensive effect [12] and unique inhibitory actions on formalin. Furthermore, the expression patterns of a and1B

sympathetic neurotransmission, resulting in inhibition of a subunits, which are pore-forming subunits of N- and1A

norepinephrine release. Because of its inhibitory action on P/Q-type calcium channels, respectively, in the DRGsympathetic neurotransmission [11], cilnidipine has been neurons of the sensory nervous system were examined.used for the treatment of hypertension with suppression of Patch-clamp experiments were done with isolated neuronsreflex tachycardia. Cilnidipine has a marked inhibitory from DRG to evaluate whether cilnidipine inhibits N-typeeffect on both N- and L-type calcium channels in rat dorsal calcium channels.root ganglion (DRG) neurons, which suggests application Experimentally native, 4–5-week-old mice, offspringof this novel DHP derivative to modify neuronal functions from CB57BL6 and 129SV, were used. After intrathecal[6]. injections of cilnidipine in unanesthetized mice at the L5

and L6 intervertebral space, the formalin test was done bysubcutaneously injecting 20 ml of a 2% formaldehyde*Corresponding author. Tel.: 181-22-717-8063; fax: 181-22-717-solution as previously described [18]. Significant differ-8065.

E-mail address: mmura@mail.cc.tohoku.ac.jp (M. Murakami) ences between two groups were determined by Student’s

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02295-2

124 M. Murakami et al. / Brain Research 868 (2000) 123 –127

t-test. Dunnett’s multiple comparison was used for themultiple intergroup test.

Under ether anesthesia, DRG of the mouse were ob-tained, fixed immediately in 4% paraformaldehyde, andsubsequently embedded in paraffin-wax. To prepare cRNAprobe, a and a subunit specific sequences were1B 1A

amplified in RT-PCR from mouse brain RNA. BiotinylatedUTP-labeled cRNA probe, transcribed by T7 or T3 RNApolymerase, was used for in situ hybridization, which wasperformed by a manual capillary action system (Micro-Probe, Fisher Scientific, Pittsburgh, PA, USA) [19].

For electrophysiological experiments, currents wererecorded in a whole-cell configuration from single-cellbodies of DRG neurons isolated from 1- to 5-day-oldWistar rats [6]. Currents were filtered at 3 kHz and storedin a computer. The pipette solution consisted of (in mM)135 CsCl, 5 MgCl , 5 BAPTA (Dojin, Kumamoto, Japan),2

10 Hepes, 5 ATP and 12 glucose (pH 7.0 after titrationwith CsOH). The bathing solution consisted of (in mM)100 Tris, 5 CsCl, 5 BaCl , 1 MgCl , 25 TEA–Cl, 5 Hepes2 2

and 20 glucose (pH 7.4 after titration with Tris–HCl). Allexperiments were performed at room temperature.Capacitative and leak currents were subtracted using theP/4 method.

Pretreatment with intrathecal cilnidipine (300 nmol /kg)10 min prior to subcutaneous formalin significantly re-duced the licking time in the late phase (Fig. 1A).Pretreatment with intrathecal injection with v-conotoxin

Fig. 1. (A) The effect of intrathecal pretreatment with cilnidipine (300GVIA (0.46 nmol /kg), which is isolated from the venomnmol /kg), nicardipine (300 nmol /kg) and v-conotoxin GVIA (0.46

of Conus geogrophus and specifically inhibits N-type nmol /kg) on the behaviour induced by subcutaneous formalin injection.channels [9,16], decreased the licking time in both the Nociceptive behaviour in both early (0–10 min; upper panel) and lateearly and the late phases. Nicardipine (300 nmol /kg), phases (10–30 min; lower panel) is shown as the amount of time spent

licking the injected paw. The data are expressed as the mean6S.E.M.which acts preferentially on L-type channels [1], showedStatistical significance was isolated by the Student’s t-test. (B) The effectno effect on either phase. As shown in Fig. 1B, cilnidipineof cilnidipine on the duration of licking after subcutaneous formalin

(30, 300 and 3000 nmol /kg) showed a significant dose- injections. The data are expressed as the mean6S.E.M. Statisticaldependent reduction in the late phase. The highest dose significance was isolated by Dunnett’s multiple comparison test.(3000 nmol /kg) of cilnidipine reduced the licking time inboth the early and the late phases.

Shaker activity assay was carried out according toOlivera’s method to examine neurological side effects of treatment with 3 mM calciseptine and 2 mM v-agatoxincilnidipine [17]. Eight nmol /kg of v-conotoxin GVIA IVA to block the L- and P-type channels, respectively.

21caused a characteristic shaking tremor after intracereb- Cilnidipine reduced the peak amplitude of the Baroventricular injection (i.c.v.). The onset and duration of currents in a dose-dependent manner (Fig. 2b), while athe tremor was relatively dose dependent. Cilnidipine (300 high dose of nicardipine (10 mM) showed no significantnmol /kg, i.c.v.) never caused shaker tremors (up to 900 s), effect on the v-conotoxin GVIA sensitive current. Cil-while v-conotoxin GVIA (1.72 nmol /kg) caused shaker nidipine (100 nM) caused an approximately 50% reductiontremors with the latent time of 350649 s (the of the N-type current. This dose (100 nM) is similar to themean6S.E.M.). No recognizable effect by cilnidipine was IC of this drug to L-type channels (IC 5100 nM) [6].50 50

observed at up to 1 mmol /kg, during experiment (900 s). We present here the first account of the antinociceptiveThe tissue-specific expression of the N-type (a ) and effect of cilnidipine, a novel DHP derivative, without any1B

P/Q-type (a ) calcium channels was examined by mRNA apparent neurological side effects. Electrophysiological1A

in situ hybridization on mouse DRG sections. A marked examination showed marked inhibitory effect of cilnidipineaccumulation of hybridization signals of a and a was on N-type calcium channels and our results showed high1B 1A

observed (Fig. 2a). expression level of N-type channels in DRG. The results ofTo investigate the effect on N-type calcium channels, the present study demonstrated that acute intrathecal

cilnidipine was examined in DRG neurons after the pre- infusion of cilnidipine or v-conotoxin GVIA results in a

Fig. 2. (a) mRNA in situ hybridization, appearing red as a result of fast red reaction, for a and a subunits mRNA in murine dorsal root ganglions. a and a1B 1A 1B 1A

subunit-specific sequences were amplified in PCR with the primer A1B1 (59-GGG GAT AAG GAA ACC CGA AAT CAC CA-39) and A1B2 (59-CTT GGC CTT CCA GGTTCA TGT TAC CA-39), which correspond to the sequences of the murine a subunit G DKETRNHQ and G NMNLEGQA , and A1A1 (59-CTC CCG AGA ACA1B 319 327 403 411

GCC TTA TC-39) and A1A2 (59-GTC TGC CTC CTC TTC CTC TTT CTT C-39) which correspond to the sequences of the murine a subunit T PENSLIVT and1A 403 411

E KKEEEEAD , respectively. Hybridization products were visualized as red by fast red salt. As a negative control, hybridizations were performed with adjacent sections463 471

with the corresponding sense probes. Magnification, 3100; hematoxylin was used as nuclear stain. Scale bar is the same for both panels (50 mm). (b) Effects of cilnidipine(1–100 nM) and nicardipine (10 mM) on the N-type current in rat dorsal root ganglion neurons pretreated with 3 mM calciseptine and 2 mM v-agatoxin IVA. The current wasevoked by step depolarization to 0 mV from a holding potential of 260 mV (30 ms duration, 0.18 Hz). It was confirmed that the current was almost abolished by the applicationof 3 mM v-conotoxin GVIA (not shown). Each drug was added to the superfusate. To evaluate the effects of cilnidipine and nicardipine, the reduction of the current amplitudeinduced by 3 mM v-conotoxin GVIA was normalized as 1.0, and the percent reduction relating to the v-conotoxin GVIA (3 mM)-sensitive currents was examined. Inset showstypical current traces before and after the application of cilnidipine doses (10 and 100 nM). Each column and vertical bar represents the mean and S.E.M. from three to sevendifferent neurons, respectively.

126 M. Murakami et al. / Brain Research 868 (2000) 123 –127

Fig. 2. (continued)

significant decrease in the licking time of the late phase of onstrated that cilnidipine has a potent antinociceptivethe formalin test. effect in mice, and inhibits N-type calcium channels in the

Four different calcium channels (L-, N-, P-, R-types) rat DRG neurons. Considering the high expression level ofexist in DRG [10]. It is widely accepted that N- and P-type the a (N-type) subunit in DRG, cilnidipine exerts its1B

21calcium channels are highly localized at the pre-synaptic antinociceptive action by inhibiting Ca influx, probablyterminals, playing important roles in the neurotransmitter through N-type channels and suppressing neurotransmitterrelease, and that L-type channels are in the soma [2,3,20]. release.

Autoradiographic studies have shown that N-type chan- Although cilnidipine and v-conotoxin GVIA are N-typenels are highly localized in the superficial laminae I and II channel antagonists, our results show some difference inof the spinal cord where the small myelinated (laminae I) their pharmacological effects. A much higher dose ofand unmyelinated (laminae II) nociceptive afferents termi- cilnidipine was needed to elicit an antinociceptive effect,nate [7,13]. Our results in the in situ hybridization experi- and cilnidipine preferentially acted in the late phase. Onlyment showed high expression levels of a and a the highest dose of cilnidipine (3000 nmol /kg) acted in the1B 1A

subunits in murine DRG as reported in chick [5], sug- early phase. On the other hand, v-conotoxin GVIA showedgesting the important roles of N- and P-type channels in marked effects in both the early and late phases in thethe signal transduction in nociceptive afferent fibers of formalin test. These differences between the two drugsmice. may be due to the irreversible effect of v-conotoxin GVIA,

It has been shown that presynaptic blockade of N-type or to differences in the binding sites. It also might be duechannels by v-conotoxin GVIA inhibits glutamatergic to the different use-dependency. Considering the strongsynaptic transmission between DRG neurons and spinal shaker tremor induced by the toxin, cilnidipine might becord neurons [8]. Intrathecal administration of v-conotoxin more advantageous for clinical use, since injection (i.c.v.)GVIA and v-agatoxin-IVA, but not L-type calcium chan- of cilnidipine showed no shaker tremor or other neurologi-nel antagonists, inhibits the dorsal horn neurons and has cal symptoms.antinociceptive effects [2,14,15]. Our present study dem- In the electrophysiological experiments, cilnidipine, but

M. Murakami et al. / Brain Research 868 (2000) 123 –127 127

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Acknowledgements [12] K. Ikeda, M. Hosono, H. Iida, H. Ohnishi, Antihypertensive andcardiovascular profiles of a newly synthesized dihydropyridinederivative 2-methoxyethyl (E)-3-phenyl-2-propen-1-y (6)-1,4-We thank Mrs. Tetsuko Sueta and Mrs. Agnieszkadihydro-2,6-dimethyl-4-(3-nitrophenyl) pyridine-3,5-dicarboxylateMurakami for technical assistance, We also thank Dr.(FRC-8653), Pharmacometrics 44 (1992) 433–442.

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