Blockade of T-type calcium channels by 6-prenylnaringenin, a hop component, alleviates neuropathic and visceral pain in mice

Accepted Manuscript Blockade of T-type calcium channels by 6-prenylnaringenin, a hop component, alleviates neuropathic and visceral pain in mice Fumiko Sekiguchi, Tomoyo Fujita, Takahiro Deguchi, Sakura Yamaoka, Ken Tomochika, Maho Tsubota, Sumire Ono, Yamato Horaguchi, Maki Ichii, Mio Ichikawa, Yumiko Ueno, Nene Koike, Tadatoshi Tanino, Huy Du Nguyen, Takuya Okada, Hiroyuki Nishikawa, Shigeru Yoshida, Tsuyako Ohkubo, Naoki Toyooka, Kazuya Murata, Hideaki Matsuda, Atsufumi Kawabata PII:

S0028-3908(18)30309-5

DOI:

10.1016/j.neuropharm.2018.06.020

Reference:

NP 7232

To appear in:

Neuropharmacology

Received Date: 7 February 2018 Revised Date:

30 May 2018

Accepted Date: 14 June 2018

Please cite this article as: Sekiguchi, F., Fujita, T., Deguchi, T., Yamaoka, S., Tomochika, K., Tsubota, M., Ono, S., Horaguchi, Y., Ichii, M., Ichikawa, M., Ueno, Y., Koike, N., Tanino, T., Du Nguyen, H., Okada, T., Nishikawa, H., Yoshida, S., Ohkubo, T., Toyooka, N., Murata, K., Matsuda, H., Kawabata, A., Blockade of T-type calcium channels by 6-prenylnaringenin, a hop component, alleviates neuropathic and visceral pain in mice, Neuropharmacology (2018), doi: 10.1016/j.neuropharm.2018.06.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

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NEUROPHARMACOLOGY Research Paper

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Blockade of T-type calcium channels by 6-prenylnaringenin, a hop component, alleviates neuropathic and visceral pain in mice

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Fumiko Sekiguchi a, Tomoyo Fujita a, Takahiro Deguchi b, Sakura Yamaoka a, Ken Tomochika a, Maho Tsubota a, Sumire Ono a, Yamato Horaguchi a, Maki Ichii a, Mio Ichikawa a, Yumiko Ueno a, Nene Koike a, Tadatoshi Tanino c, Huy Du Nguyen d, Takuya Okada d, Hiroyuki Nishikawa a, Shigeru Yoshida e, Tsuyako Ohkubo f, Naoki Toyooka d, g, Kazuya Murata b, Hideaki Matsuda b, †, Atsufumi Kawabata a, * a

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Laboratory of Pharmacology and Pathophysiology, Faculty of Pharmacy, Kindai University, Higashi-Osaka 577-8502, Japan b Division of Natural Drug Resources, Faculty of Pharmacy, Kindai University, Higashi-Osaka 577-8502, Japan c Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, 770-8514, Japan. d Graduate School of Innovative Life Science, University of Toyama, Toyama 930-8555, Japan. e Department of Life Science, Faculty of Science and Engineering, Kindai University, Higashi-Osaka 577-8502, Japan f Division of Basic Medical Sciences and Fundamental Nursing, Faculty of Nursing, Fukuoka Nursing College, Fukuoka 814-0193, Japan g Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555, Japan.

Corresponding author. Address: Laboratory of Pharmacology and Pathophysiology, Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-Osaka 577-8502, Japan. Tel: +81 6 4307 3631; Fax: +81 6 6730 1394 E-mail address: [email protected] (A. Kawabata) Institutional URL: http://www.phar.kindai.ac.jp/byoutai/index.files/English-HP/byoutai-Eng.htm †

Deceased

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ACCEPTED MANUSCRIPT Abstract

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Since Cav3.2 T-type Ca2+ channels (T-channels) expressed in the primary afferents and CNS contribute to intractable pain, we explored T-channel-blocking components in distinct herbal extracts using a whole-cell patch-clamp technique in HEK293 cells stably expressing Cav3.2

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or Cav3.1, and purified and identified sophoraflavanone G (SG) as an active compound from

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SOPHORAE RADIX (SR). Interestingly, hop-derived SG analogues, (2S)-6-prenylnaringenin (6-PNG) and (2S)-8-PNG, but not naringenin, also blocked T-channels; IC50 (µM) of SG, (2S)-6-PNG and (2S)-8-PNG was 0.68-0.75 for Cav3.2 and 0.99-1.41 for Cav3.1. (2S)-6-PNG

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and (2S)-8-PNG, but not SG, exhibited reversible inhibition. The racemic (2R/S)-6-PNG as well as (2S)-6-PNG potently blocked Cav3.2, but exhibited minor effect on high-voltage-activated Ca2+ channels and voltage-gated Na+ channels in differentiated

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NG108-15 cells. In mice, the mechanical allodynia following intraplantar (i.pl.)

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administration of an H2S donor was abolished by oral or i.p. SR extract and by i.pl. SG, (2S)-6-PNG or (2S)-8-PNG, but not naringenin. Intraperitoneal (2R/S)-6-PNG strongly suppressed visceral pain and spinal ERK phosphorylation following intracolonic administration of an H2S donor in mice. (2R/S)-6-PNG, administered i.pl. or i.p., suppressed the neuropathic allodynia induced by partial sciatic nerve ligation or oxaliplatin, an anti-cancer agent, in mice. (2R/S)-6-PNG had little or no effect on open-field behavior, motor

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ACCEPTED MANUSCRIPT performance or cardiovascular function in mice, and on the contractility of isolated rat aorta. (2R/S)-6-PNG, but not SG, was detectable in the brain after their i.p. administration in mice.

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Our data suggest that 6-PNG, a hop component, blocks T-channels, and alleviates neuropathic and visceral pain with little side effects.

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Keywords: T-type calcium channel; 6-prenylnaringenin; sophoraflavanone G; neuropathic

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pain; visceral pain

Abbreviations: HVA-current, high-voltage-activated Ca2+ channel-dependent current;

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Nav-current, voltage-dependent Na+ channel-dependent current; NG, naringenin; OHP, oxaliplatin; PNG, prenylnaringenin; PSNL, partial sciatic nerve ligation; SG, sophoraflavanone G; SR, SOPHORAE RADIX; T-channel, T-type Ca2+ channel; T-current,

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T-channel-dependent current.

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ACCEPTED MANUSCRIPT 1. Introduction

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Cav3.2 T-type Ca2+ channels (T-channels) are abundantly expressed in the primary sensory neurons, and their excessive activity contributes to the development of various types of

intractable pain including inflammatory, neuropathic and visceral pain (Kawabata et al., 2007;

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Maeda et al., 2009; Marger et al., 2011; Matsunami et al., 2012; Matsunami et al., 2009;

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Nishimura et al., 2009; Orestes et al., 2013; Sekiguchi et al., 2013; Sekiguchi et al., 2016; Takahashi et al., 2010; Tsubota-Matsunami et al., 2012; Zamponi et al., 2015). The upregulation of Cav3.2 in the dorsal root ganglia (DRG) has been reported in animal models

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for neuropathic and inflammatory pain (Garcia-Caballero et al., 2014; Jagodic et al., 2008; Latham et al., 2009; Messinger et al., 2009; Takahashi et al., 2010) and for cystitis-related bladder pain (Matsunami et al., 2012; Ozaki et al., 2018). Cav3.2 expressed in the peripheral

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and central endings of the primary afferents regulates neuronal excitability and spontaneous

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neurotransmitter release, respectively (Todorovic and Jevtovic-Todorovic, 2013; Weiss and Zamponi, 2013; Zamponi et al., 2015). Distinct isoforms of T-channels are also expressed in the brain, particularly thalamus, and regulate pain signals (Todorovic and Jevtovic-Todorovic, 2011). Therefore, blood-brain-barrier (BBB)-permeable T-channel blockers might be more effective as analgesics than peripherally preferential ones. However, T-channel blockers with high BBB permeability may induce sedation (Kraus et al., 2010; Miwa et al., 2011; Sekiguchi

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ACCEPTED MANUSCRIPT et al., 2014; Todorovic and Jevtovic-Todorovic, 2011), which could be a side effect limiting clinical use as analgesics. The phenotype of Cav3.2-null mice, such as elevated anxiety,

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impaired memory and reduced sensitivity to psychostimulants (Gangarossa et al., 2014), should also be taken into consideration. Apart from the preclinical studies employing rodents, clinical studies have shown that ABT-639, a potent T-channel blocker, failed to suppress

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C-fiber activity and did not improve pain scores in prospective, randomized, double-blinded

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controlled trials of subjects with painful diabetes mellitus (Serra et al., 2015; Ziegler et al., 2015). In contrast, a study employing a human experimental pain model (Lee, 2014) has shown the effectiveness of Z944, a more highly BBB-permeable T-channel blocker (Tringham

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et al., 2012). Therefore, ideal T-channel blockers as clinically effective and safe analgesics may have to penetrate into the CNS, to an appropriate extent. In the present study, we explored novel T-channel blocking compounds from distinct herbal extracts, and consequently

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purified and identified an active compound, sophoraflavanone G (SG), from SOPHORAE

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RADIX (SR). Here, we show the pharmacological characteristics of SG and its structural analogues including 6-prenylnaringenin (PNG) and 8-PNG, components of hops (Humulus lupulus L.), as T-channel blocking compounds, and indicate that 6-PNG is one of the most useful and safe T-channel-blockers with appropriate BBB permeability, and suppresses neuropathic and visceral pain in mice.

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ACCEPTED MANUSCRIPT 2. Materials and Methods

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2.1. Purification and identification of sophoraflavanone G

SOPHORAE RADIX (SR), the root of Sophora flavescens, was purchased from Tochimoto

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Tenkaido Co., Ltd. (Osaka, Japan) in June, 2012, air-dried and powdered. The powder was

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subjected to extraction with 50% ethanol, and the extract was evaporated under reduced pressure and lyophilized. The amount of residual ethanol in the lyophilized powder was less than 0.01%. After discovering the T-current-blocking activity of 50% ethanol extract of SR

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(SR-extract), as assessed by a whole-cell patch-clamp technique in Cav3.2-expressing HEK293 cells (see below, and Fig. 1A), we tried to purify and identify the active components employing the chloroform extract of SR (SR-CHCl3-extract) (Fig. 1B). The roots (2 kg) were

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pulverized and subjected to extraction with 1.8 L of chloroform for 2 h with stirring. The

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filtrates of the extract were evaporated under reduced pressure, yielding brown powder, SR-CHCl3-extract, of 33.8 g (resulting extraction yield: 1.7%). The SR-CHCl3-extract (17.6 g) was applied to silicic acid column chromatography [Merck No. 1.07734 silica gel 60, 1.5 kg, 5.0 inner diameter (i.d.) × 44 cm] (Merck, Darmstadt, Germany), and step-wise gradient elution was performed using chloroform and methanol (the ratio: 100/0, 99/1, 9/1, 8/2, 1/1 and 0/100). The eluates were combined according to the results of thin-layer chromatography

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ACCEPTED MANUSCRIPT analysis [Merck No. 1.05735 silica gel 60 F254, n-hexane/acetone=10:1 (v/v), detection; UV and 10% sulfuric acid followed by heating] and evaporated under reduced pressure. The

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obtained six fractions, Fr. b~g, were assessed for T-current-blocking activity, and, consequently, the most active fraction, Fr. d (6.5 g), was subjected to silicic acid column chromatography (500 g, 2.5 i.d. ×20 cm) (Fig. 1B). Step-wise elution was performed using

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chloroform and methanol (the ratio: 20/1, 10/1, 1/1 and 0/100), and the fractions were

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combined and separated into eight fractions, Fr. h~o, according to the thin-layer chromatography data (Fig. 1B). Finally, the most potent fraction, Fr. j, among Fr. h~j (Fig. 1C) was subjected to preparative high-performance liquid chromatography (HPLC) under

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conditions as follows: column, YMC-Pack ODS-AM (250 × 20 mm i.d., YMC Co., Ltd., Kyoto, Japan); mobile phase, 0.1% acetic acid in water and acetonitrile [the ratio: 20/80 (0 min) and 5/95 (30 min)]; flow rate, 10.0 ml/min; detection, UV at 280 nm; retention time, 12

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min. The active compound was finally identified as sophoraflavanone G (SG) (Fig. 1D) by 1H

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and 13C NMR, and MS spectra analysis, on the basis of previous reports (Ryu et al., 1997; Wu et al., 1985).

2.2. Chemicals

Sophoraflavanone G (SG) was isolated and purified from SR-CHCl3-extract, as mentioned

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ACCEPTED MANUSCRIPT above, and (2R/S)-6-prenylnaringenin [(2R/S)-6-PNG] was synthesized in-house, as reported previously (Tischer and Metz, 2007). (2S)-6-PNG, (2S)-8-PNG, (2S)-naringenin,

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(2R/S)-naringenin, verapamil and Na2S were purchased from Sigma-Aldrich (St. Louis, MO). NaHS and oxaliplatin were obtained from Kishida Chemical Co., Ltd. (Osaka, Japan) and Wako Pure Chem. Inc., Ltd. (Osaka, Japan), respectively. SR-extract, fractions of

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SR-CHCl3-extract, SG, (2S)-6-PNG, (2R/S)-6-PNG, (2S)-8-PNG, (2S)-naringenin and

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(2R/S)-naringenin were dissolved in DMSO for in vitro assays. For administration to mice, oxaliplatin was dissolved in 5% glucose solution, and NaHS and Na2S were dissolved in saline or distilled water. SG, (2S)-6-PNG, (2R/S)-6-PNG, (2S)-8-PNG and (2R/S)-naringenin

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were dissolved in 1% DMSO/saline solution for intraplantar (i.pl.) injection and suspended in 0.5% carboxymethyl cellulose sodium salt (CMC-Na) solution (Kishida Chemical Co., Ltd.)

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administration.

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for i.p. administration. SR-extract was suspended in CMC-Na solution for i.p. and oral

2.3. Cell culture

HEK293 cells stably expressing human Cav3.1 or Cav3.2 T-channels (Sekiguchi et al., 2016) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with L-glutamine (Wako Pure Chem. Inc. Ltd.), 10% fetal calf serum (FCS) (Nichirei Biosci. Inc.,

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ACCEPTED MANUSCRIPT Tokyo, Japan), 50 U/ml penicillin, 50 µg/ml streptomycin (Gibco, Carlsbad, CA) and 250 µg/ml G418 (Sigma-Aldrich). NG108-15 cells, mouse neuroblastoma and rat glioma hybrid

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cells, were cultured in high glucose DMEM supplemented with 0.1 mM hypoxanthine, 1 µM aminopterin, 16 µM thymidine, 50 U/ml penicillin, 50 µg/ml streptomycin and 10% FCS. To measure high voltage-activated (HVA) Ca2+ channel-dependent currents (HVA-currents) and

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voltage-dependent Na+ (Nav) channel-dependent currents (Nav-currents), NG108-15 cells

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were differentiated into neuron-like cells by stimulation with dibutyryl cyclic AMP (db-cAMP) at 1 mM for 2 days in the 1% FCS-containing culture medium, considering evidence that the differentiated neuron-like NG108-15 cells abundantly express both

Nagasawa et al., 2009).

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HVA-Ca2+ channels and Nav channels (Chemin et al., 2002; Kawaguchi et al., 2007;

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2.4. Whole-cell patch-clamp recording

The whole-cell patch-clamp recording was performed in human Cav3.1- and Cav3.2-expressing HEK293 (Cav3.1-HEK and Cav3.2-HEK, respectively) cells, and in neuronally differentiated NG108-15 cells, as described previously (Kawabata et al., 2007; Nagasawa et al., 2009). Data were acquired and digitized through Digidata (1332A; Axon Instruments, Foster City, CA) and analyzed by a personal computer using pCLAMP8 software

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ACCEPTED MANUSCRIPT (Axon Instruments). For measurement of T-channel-dependent currents (T-currents), Ba2+ currents were recorded from randomly chosen cells at room temperature 10 min after

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application of SR-extract, fractions of SR-CHCl3-extract, SG, (2S)-6-PNG, (2R/S)-6-PNG or (2S)-8-PNG. In the experiments to determine the reversibility of the inhibitory effects of SG, (2S)-6-PNG, (2S)-8-PNG, (2S)-naringenin or (2R/S)-naringenin on T-channels, T-currents in

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Cav3.2-HEK cells were repeatedly determined at 10-s intervals by repetitive application of

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test pulses at -20 mV for 200 ms from a holding potential at -80 mV. The extracellular solution containing the blockers or vehicle was perfused at a rate of 5 ml/min. The composition of extracellular solution was as follows (mM): 97 N-methyl-D-glucamine

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(NMDG), 10 BaCl2, 10 HEPES, 40 tetraethylammonium (TEA)-Cl and 5.6 glucose (pH 7.4). A patch pipette was filled with an intracellular solution containing (mM): 140 CsCl, 4 MgCl2, 5 EGTA and 10 HEPES (pH 7.2). The resistance of patch electrodes ranged from 3 to 7 MΩ.

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Series-resistance was compensated by 80%, and current recordings were low-pass filtered (<5

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kHz). The cell membrane voltage was held at -80 mV, and whole cell Ba2+ currents were elicited by step pulses of 200 ms duration from -120 to +40 mV with increments of 10 mV. T-currents were determined as the difference between currents of the peak and 150 ms after the beginning of a test pulse at -20 mV. The voltage dependencies of activation and steady-state inactivation were analyzed with single Boltzmann distributions of the following forms:

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ACCEPTED MANUSCRIPT Activation: G/Gmax = 1/{1 + exp[-(V - V1/2)/k]} Inactivation: I/Imax = 1/{1 + exp[(V - V1/2)/k]}.

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In these forms, Imax is the maximal amplitude of currents, Gmax is the maximal conductance, V1/2 is the voltage at which a half of the currents is activated or inactivated, and k represents the voltage dependence (slope) of the distribution. HVA-currents and Nav-currents were

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determined in the differentiated neuron-like NG108-15 cells. HVA-currents were measured as

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persistent Ba2+ currents detected 75 ms after the beginning of a test pulse at +10 mV for 200 ms from a holding potential at -60 mV, using the above-mentioned extracellular and intracellular solutions. Nav-currents were determined as the difference between currents of the

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peak and 15 ms after the beginning of a test pulse at -20 mV for 25 ms from a holding potential at -80 mV, using different extracellular and intracellular solutions, the compositions of which were (mM): extracellular solution, 130 NaCl, 10 HEPES, 0.01 CaCl2, 5 MgCl2, 20

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TEA-Cl and 5.6 glucose (pH 7.4); intracellular solution, 10 NaCl, 130 CsCl, 5 MgCl2, 5

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EGTA and 10 HEPES (pH 7.2). We confirmed that the Nav-currents completely disappeared in the presence of tetrodotoxin at 0.1 µM (data not shown). HVA-currents and Nav-currents were measured before and after application of SG, (2S)-6-PNG or (2R/S)-6-PNG in the same cell.

2.5. Experimental animals

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Male Wistar rats (350-400 g), ddY mice (18-25 g) were purchased from Kiwa Laboratory

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Animals Co. Ltd. (Wakayama, Japan). We also used male C57BL/6J (wild-type) and Cacna1h (Cav3.2 null, background C57BL/6J mice) (23-27 g) purchased from the Jackson Laboratory (Bar Harbor, ME). The animals were housed in a temperature-controlled room (22-24ºC)

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under a 12-h day/night cycle and had free access to food and water. Grouping animals was

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randomized. Blinding was not possible. All experimental protocols were approved by the Committee for the Care and Use of Laboratory Animals at Kindai University, and were in accordance with the guiding principles approved by The Japanese Pharmacological Society

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and the NIH guidelines (Guide for the Care and Use of Laboratory Animals, NIH Publication

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2.6. Assessment of nociceptive threshold and creation of somatic pain models in mice

Mice were placed on a risen wire mesh floor, covered with a clear plastic box (10 × 10 × 10 cm) and acclimated to the experimental environment. Then, the mid-plantar surface of the right hind paw was stimulated with von Frey filaments (0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6 and 1.0 g), and 50% paw withdrawal threshold was determined according to the up-down method (Chaplan et al., 1994).

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ACCEPTED MANUSCRIPT Intraplantar administration of H2S donors, such as NaHS and Na2S, induces Cav3.2-dependent mechanical hyperalgesia and/or allodynia in rats or mice (Maeda et al.,

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2009; Okubo et al., 2012b; Sekiguchi and Kawabata, 2013), which is convenient and useful as animal models to evaluate the efficacy of novel Cav3.2 T-channel blockers as analgesics in vivo (Sekiguchi et al., 2016). Therefore, the NaHS-induced allodynia model in mice was

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employed as the primary nociception assay in the present study. Briefly, the mouse received

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i.pl. administration of NaHS at 0.1 nmol/paw in a volume of 10 µl in the right hindpaw, and the mechanical nociceptive threshold was monitored before and after i.pl. NaHS. SG, (2S)-6-PNG, (2S)-8-PNG or (2R/S)-naringenin was co-administered i.pl. with NaHS. Mice

before i.pl. NaHS.

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also received i.p. administration of SG and i.p. or oral administration of SR-extract 20 min

Two distinct neuropathic pain models in mice were used to evaluate the therapeutic

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efficacy of T-channel blockers for treatment of intractable pain. One was neuropathic pain

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caused by partial sciatic nerve ligation (PSNL) in mice (Seltzer et al., 1990). Briefly, under isoflurane anesthesia, the common sciatic nerve at the right hind limb was exposed, and approximately one-second of the nerve was tightly ligated with a silk suture. In sham-operated mice, the right common sciatic nerve was exposed but not ligated. The other neuropathy in mice was produced by a single i.p. administration of oxaliplatin (OHP) at 5 mg/kg, according to the previous reports (Kiguchi et al., 2010; Sakurai et al., 2009; Zhao et al.,

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ACCEPTED MANUSCRIPT 2012). (2R/S)-6-PNG was administered i.pl. or i.p. 8 days after PSNL or OHP treatment in

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mice, to evaluate the therapeutic efficacy.

2.7. Creation and assessment of colonic pain by intracolonic administration of an H2S donor

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in mice

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Mice were acclimated to the experimental environment, as mentioned above, except for the size of a plastic box (23 × 16 × 12 cm), and received intracolonic (i.col.) administration of the H2S donor, Na2S, in a volume of 50 µl, as reported elsewhere (Matsunami et al., 2009;

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Sekiguchi et al., 2016). Visceral pain-like nociceptive behavior was counted for 30 min immediately after i.col. Na2S, and then referred hyperalgesia was evaluated by assessing nociceptive scores, 0, 1 or 2 in responses to stimuli of the lower abdomen with von Frey

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filaments with distinct strengths, 0.008, 0.02, 0.16 and 1.0 g, according to the previous report

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(Matsunami et al., 2009). Briefly, each mouse was stimulated with each filament 10 times, and the total score could be up to 20 after 10-time stimuli with each filament for each mouse. The data are presented as the total score. (2R/S)-6-PNG was administered i.p. 15 min before i.col. administration of Na2S.

2.8. Immunohistochemical detection of phosphorylated ERK in the spinal dorsal horn

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ACCEPTED MANUSCRIPT following intracolonic stimulation with an H2S donor in mice

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Acute somatic and visceral pain causes prompt phosphorylation of ERK in the spinal cord, which is useful as a rapid marker for nociceptor excitation (Ji et al., 1999; Matsunami et al., 2009). According to the previously reported method (Matsunami et al., 2009), the mouse was

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anesthetized with i.p. administration of urethane at 1.5 g/kg, and received intracolonic

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administration of Na2S. Exactly 5 min after intracolonic Na2S injection, the mouse was perfused transcardially with 1% paraformaldehyde and subsequently with 4% paraformaldehyde for fixation. The excised spinal cord segments at the thoraco-lumbar levels

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(T13-L1) and lumbo-sacral levels (L5-S1), to which the splanchnic and pelvic nerves project, respectively, were postfixed and serially sectioned. Phosphorylated ERK was immunostained with a rabbit polyclonal antibody against human phopho-p44/42 MAP kinase

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(Thr202/Tyr204) (Cell Signaling, Beverly, MA) and a biotinylated goat antibody against

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rabbit IgG, using a peroxidase-conjugated avidin-biotin complex (Vectastain ABC kit, Vector Laboratories, Burlingame, CA). (2R/S)-6-PNG was administered i.p. 15 min before intracolonic Na2S.

2.9. Isometric tension recording in rat aortic ring preparations

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ACCEPTED MANUSCRIPT Male Wistar rats were sacrificed by decapitation under urethane (1.5 g/kg, i.p.) anesthesia, and the ring segments of the thoracic aorta (1 mm in length) were prepared in an ice-cooled

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Krebs-Henseleit solution (composition in mM: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 KH2PO4, 10 glucose). The endothelium was removed by gently rubbing the inner surface with a thin rubber band. The ring preparations were mounted between two

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triangle wire hooks, and the isometric tension was measured with a force-displacement

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transducer in an organ bath containing the Krebs-Henseleit solution maintained at 37ºC and bubbled with 95% O2/5% CO2 (pH 7.4). Possible T-channel blockers were cumulatively

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applied to the preparation precontracted with 50 mM KCl.

2.10. Measurement of blood pressure and heart rate in mice

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The blood pressure and heart rate of mice were monitored by the tail-cuff method (BP-98A,

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Softron Co., Ltd., Tokyo, Japan), in a warming holder maintained at 37ºC, before and after i.p. administration of (2R/S)-6-PNG.

2.11. Assessment of open-field behavior and rota-rod performance

The open-field apparatus had 40-cm high black wall with 6 pairs of beam/sensor and 60-cm

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ACCEPTED MANUSCRIPT diameter circular meshed floor. Mice were acclimated to the apparatus for 30 min for 4 consecutive days, and received i.p. administration of (2R/S)-6-PNG immediately after the

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final acclimatization period. Each mouse was then placed on the center of the apparatus, and the animal’s behavior was observed for 30-min in total. The measurements included 5

parameters: locomotor activity (the number of mouse’s crossing the beam/sensor), time spent

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grooming, the number of penetration into the 30-cm diameter center circle and time spent in

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the same center area (center-field penetration), and the number of rearing and defecation. The results in the early and later 15-min periods are analyzed separately. The motor performance was evaluated by the rota-rod test for 300 s, using Rotor Load (Natsume Seisakusho Co. Ltd.,

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Tokyo, Japan).

2.12. Assay of concentrations of (2R/S)-6-PNG and SG in the plasma and brain after the

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systemic administration in mice

Plasma and brain tissue levels of (2R/S)-6-PNG and SG were determined to evaluate their penetration into the CNS after the systemic administration. HPLC analysis was performed using a system equipped with SPD-10A, a UV detector, LC10A pump and CR-4A chromatopac integrator (Shimadzu, Kyoto, Japan). The citrated blood was collected from mice under urethane (1.5 g/kg, i.p.) anesthesia, followed by excision of the brain, 5, 10 and 30

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ACCEPTED MANUSCRIPT min after i.p. administration of (2R/S)-6-PNG or SG. Concentrations of (2R/S)-6-PNG and SG in the plasma and brain were determined as reported previously (Possemiers et al., 2005),

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with some modifications. Briefly, 45 µL of plasma was mixed with 5 µL DMSO and 100 µL acetonitrile containing 25 µg/ml flurbiprofen as an internal standard. After centrifugation, the supernatant was loaded onto an HPLC column (COSMOSIL 5C8-AR-II column, particle size

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5 µm, 4.6 × 150 mm, Nacalai Tesque Co., Kyoto, Japan). The brain was homogenized with a

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Teflon homogenizer in three volumes (v/w) of ice-cold phosphate-buffered saline, and an aliquot (990 µL) of the homogenate was mixed with 5 µL DMSO, 100 µL acetonitrile containing 25 µg/ml flurbiprofen and 3 ml ethyl acetate. After centrifugation, the organic

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layer was collected and evaporated, and the residue was reconstituted in acetonitrile-0.01% formic acid (50:50, v/v), a mobile phase solution for HPLC, and loaded onto the HPLC column. (2R/S)-6-PNG and SG were eluted with the mobile phase at a flow rate of 1.0 ml/min

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and detected by absorbance at 280 nm.

2.13. Statistics

Data are represented as the mean ± SEM. Statistical significance for parametric data was analyzed by the Student’s t-test for two-group data and an analysis of variance followed by the Tukey’s test for multiple comparisons. For non-parametric analyses, the Kruskal-Wallis H

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ACCEPTED MANUSCRIPT test followed by a least significant difference-type test was used. significance was set at a

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level of P < 0.05.

3. Results

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active component from SOPHORAE RADIX (SR)

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3.1. Purification and identification of sophoraflavanone G (SG) as a T-channel-blocking

In Cav3.2-HEK cells, SR-extract, a 50% ethanol extract of SR, inhibited T-currents in a

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concentration-dependent manner in a range of 5-50 µg/ml (Fig. 1A). We then tried to purify and identify the active components from SR, using SR-CHCl3 extract. On the basis of the T-current inhibitory activity in Cav3.2-HEK cells, SR-CHCl3 extract was subjected to 2-step

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separations (Fig. 1B), and fraction j (Fr. j) among Frs. h-j separated from Fr. d exhibited the

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most potent activity in a range of 0.3 to 1 µg/ml (Fig. 1B, C). From Fr. j, SG (Fig. 1D) was purified and finally identified as an active compound. SG strongly inhibited T-currents in both Cav3.1-HEK and Cav3.2-HEK cells, but exhibited only minor inhibitory effect on HVA-currents in differentiated NG108-15 cells that express L-, N-, P/Q- and R-types of Ca2+ channels (Chemin et al., 2002; Nagasawa et al., 2009) (Fig. 1E, F, G).

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ACCEPTED MANUSCRIPT 3.2. Comparison of T-channel-blocking activity of commercially available SG analogues,

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6-PNG, 8-PNG and naringenin

We next evaluated T-channel-blocking activity of hop-derived SG analogues, (2S)-6-PNG (Fig. 2A) and (2S)-8-PNG (Fig. 2B), and of (2R/S)-naringenin (Fig. 2C) or (2S)-naringenin

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having a basic skeleton structure of SG, 6-PNG and 8-PNG in Cav3.1-HEK and Cav3.2-HEK

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cells. (2S)-6-PNG and (2S)-8-PNG suppressed T-currents in a range of 0.1-10 µM, and the proportion (Cav3.2/Cav3.1) of the inhibition potency of (2S)-6-PNG and (2S)-8-PNG on T-channels was 1.53 and 1.37, respectively, on the basis of IC50 values (Fig. 2D, E, F, G). On

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the other hand, neither (2R/S)-naringenin nor (2S)-naringenin even at 30 µM affected T-currents (Fig. 2H, I). SG, (2S)-6-PNG or (2S)-8-PNG did not change voltage-dependent activation and steady-state inactivation curves in Cav3.1-HEK cells (Fig. 2J and

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Supplementary Table S1). In Cav3.2-HEK cells, on the other hand, all three chemicals tended

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to shift the activation curve toward positive potentials, and (2S)-6-PNG and (2S)-8-PNG, but not SG, tended to shift the steady-state inactivation curve toward negative potentials (Fig. 2K and Supplementary Table S1). Interestingly, in Cav3.2-HEK cells, the inhibitory effect of (2S)-6-PNG and (2S)-8-PNG on T-currents was reversible, whereas that of SG was irreversible (Fig. 3A-D). Given the well-known estrogenic activity of 8-PNG (Keiler et al., 2013), we focused on 6-PNG as a

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ACCEPTED MANUSCRIPT reversible T-channel blocker for treatment of pain, and compared the effects of (2S)-6-PNG and (2R/S)-6-PNG on T-currents in Cav3.2-HEK cells, and HVA-currents and Nav-currents in

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neuron-like differentiated NG108-15 cells, known to express distinct HVA Ca2+ channels and Nav-channels, especially Nav1.7 (Chemin et al., 2002; Kawaguchi et al., 2007; Nagasawa et al., 2009). Both (2S)-6-PNG and (2R/S)-6-PNG potently inhibited Cav3.2-currents with

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similar IC50 values (Fig. 3E, F), while they exhibited relatively weaker inhibitory effects on

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HVA-currents and Nav-currents (Fig. 3G-J). On the basis of IC50 values (Fig. 3E-J), the proportion (Cav3.2/HVA) of the inhibition potency of (2S)-6-PNG and (2R/S)-6-PNG on Cav3.2 and HVA-currents was 2.16 and 5.20, respectively, and that (Cav3.2/Nav) on Cav3.2

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and Nav-currents was 1.79 and 3.54. Thus, apparent selectivity of (2R/S)-6-PNG to Cav3.2 was higher than (2S)-6-PNG.

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3.3. Effects of SR-extract, SG, (2S)-6-PNG, (2S)-8-PNG and (2R/S)-naringenin on mechanical

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allodynia induced by i.pl. NaHS, an H2S donor, in mice

Intraplantar administration of NaHS, a donor of H2S, at 0.1 nmol/paw maximally decreased mechanical nociceptive threshold at 15 min in mice (Fig. 4), an effect known to be dependent on both Cav3.2 and TRPA1 channels (Okubo et al., 2012b; Sekiguchi et al., 2016). The i.pl. NaHS-induced mechanical allodynia was significantly prevented by oral or i.p.

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ACCEPTED MANUSCRIPT preadministration of SR-extract at 200-400 mg/kg (Fig. 4A, B). SG, (2S)-6-PNG and (2S)-8-PNG, but not (2R/S)-naringenin, at 1 or 10 pmol/paw, when co-administered with i.pl.

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NaHS, also abolished the NaHS-induced allodynia in mice (Fig. 4C-F).

3.4. Effects of (2R/S)-6-PNG on visceral nociception induced by i.col. administration of a

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donor of H2S in mice

Cav3.2 is involved in the colonic pain induced by luminal H2S or zinc chelators in mice (Matsunami et al., 2011; Matsunami et al., 2009; Sekiguchi et al., 2016; Tsubota-Matsunami

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et al., 2012) and by repeated i.col. administration of butyrate in rats (Marger et al., 2011). Thus, we evaluated the effect of systemic administration of (2R/S)-6-PNG, which can be easily synthesized, on luminal H2S-induced colonic pain in mice. As did i.col. NaHS

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(Matsunami et al., 2009; Sekiguchi et al., 2016; Tsubota-Matsunami et al., 2012), i.col. Na2S,

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another H2S donor, at 5 nmol/mouse produced visceral pain-like nociceptive behavior (Fig. 5A) followed by referred hyperalgesia in the lower abdomen (Fig. 5B). (2R/S)-6-PNG, preadministered i.p. at 10-30 mg/kg, significantly reduced the Na2S-induced nociceptive behavior and/or referred hyperalgesia (Fig. 5A, B). In anesthetized mice, as did i.col. NaHS, i.col. Na2S at 5 nmol/mouse caused prompt phosphorylation of ERK in the superficial layers of the spinal dorsal horn at T13-L1 and L5-S1 levels to which the splanchnic and pelvic

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ACCEPTED MANUSCRIPT nerves project, an effect reduced by i.p. preadministration of (2R/S)-6-PNG at 30 mg/kg (Fig. 5C). In the spinal cord at T13-L1 and L5-S1, the number of phosphorylated ERK-positive

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cells significantly increased following i.col. Na2S in laminae I-II, V-VI and X to which the primary afferent neurons project, and the Na2S-induced increase in the phosphorylated

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ERK-positive cell number was prevented by i.p. (2R/S)-6-PNG at 30 mg/kg (Fig. 5D).

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3.5. Effects of (2R/S)-6-PNG on neuropathic pain induced by sciatic nerve injury or chemotherapy in mice

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Two distinct neuropathic pain models induced by PSNL and by treatment with OHP, an anti-cancer agent, in mice were used to evaluate the effects of i.pl. and i.p. administration of (2R/S)-6-PNG. Intraplantar administration of (2R/S)-6-PNG at 0.01-1 and 0.1-10 nmol/paw

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restored the mechanical allodynia induced by PSNL (Fig. 6A) and by i.p. administration of

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OHP (Fig. 6B), respectively, in a dose-dependent manner. Intraperitoneal administration of (2R/S)-6-PNG also significantly reversed the PSNL-induced allodynia in a range of 20-30 mg/kg (Fig. 6C), and OHP-induced allodynia in a range of 10-20 mg/kg (Fig. 6D). In wild-type C57BL/6J mice, i.p. administration of (2R/S)-6-PNG or SG at 30 mg/kg more clearly restored the PSNL-induced mechanical allodynia (Fig. 7A). Cav3.2-knockout mice, when subjected to PSNL, developed mechanical allodynia (Fig. 7B) that is similar to the one

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ACCEPTED MANUSCRIPT observed in wild-type animals (Fig. 7A), which could be due to compensatory processes, as suggested elsewhere (Gadotti et al., 2015). However, the anti-allodynic activity of

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(2R/S)-6-PNG or SG at 30 mg/kg completely disappeared in the Cav3.2-null mice (Fig. 7B).

3.6. Evaluation of central and cardiovascular side effects of (2R/S)-6-PNG and its analogues

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in mice

Intraperitoneal administration of (2R/S)-6-PNG at 30 mg/kg did not cause any significant changes in the open-field behavior or in rota-rod performance in mice (Fig. 8A, B). In the

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endothelium-removed rat aortic ring preparations precontracted with 50 mM KCl, known to abundantly express L-type Ca2+ channels, SG, (2S)-6-PNG and (2R/S)-6-PNG up to 30 µM caused no or only slight relaxation, although (2S)-8-PNG at 10-30 µM greatly induced

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relaxation, as did verapamil, an L-channel blocker, at 0.01-1 µM (Fig. 8C). Finally, we

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confirmed that i.p. administration of (2R/S)-6-PNG at 20 mg/kg only slightly affected mean blood pressure (Fig. 8D) and had no effect on heart rate in conscious mice (Fig. 8E).

3.7. Concentrations of (2R/S)-6-PNG and SG in the plasma and brain after the systemic administration in mice

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ACCEPTED MANUSCRIPT (2R/S)-6-PNG or SG at 30 mg/kg was administered i.p. to the mice, and their concentrations in the plasma and brain were monitored. After the i.p. administration, the levels of

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(2R/S)-6-PNG peaked at 10 min in the plasma and at 30 min or later in the brain, and the maximal concentrations were 1.036 µg/ml (approximately 3.04 µM) in the plasma and 0.798 µg/g (equivalent to 2.34 µM) in the brain (Table 1), which were consistent to its effective

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concentrations for blockade of Cav3.2 in vitro (see Fig. 3F). Surprisingly, SG was not

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detectable in the brain 5-30 min after i.p. administration, although its plasma concentrations reached a maximum, 1.579 µg/ml (approximately 3.72 µM), 10 min after the administration

4. Discussion

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(Table 1).

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In the present study, we purified and identified SG as a T-channel-blocking component from

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SR, and then demonstrated its structural analogues, (2S)-6-PNG and (2S)-8-PNG, hop components, also blocked T-channels, particularly Cav3.2. The pharmacological analysis of each compound indicates that the T-channel-blocking actions of (2R/S)-6-PNG or (2S)-6-PNG are relatively selective and reversible (see Fig. 3), and that (2R/S)-6-PNG is highly permeable to BBB (see Table 1) and suppresses the somatic allodynia and colonic pain/referred hyperalgesia caused by H2S donors, respectively, such as NaHS and Na2S, and the

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ACCEPTED MANUSCRIPT neuropathic pain induced by PSNL or OHP treatment (see Figs. 4-6), without producing remarkable sedation or hemodynamic changes (see Fig. 8). SG is considered an irreversible

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T-channel blocker (see Fig. 3A) and does not appear to penetrate into the CNS (see Table 1). Therefore, the antinociceptive activity of SG is attributable exclusively to peripheral

mechanisms because of the lack of its penetration into the CNS (see Table 1), which might be

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disadvantageous in its clinical application, because highly BBB-permeable T-channel blockers

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could be more effective in humans (Serra et al., 2015; Tringham et al., 2012; Ziegler et al., 2015). (2S)-8-PNG is considered a reversible T-channel blocker equipotent to (2S)-6-PNG (see Fig. 2), but causes vasorelaxation at 10 µM or higher concentrations, which is in contrast

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to the lack of such effect of (2S)-6-PNG, (2R/S)-6-PNG and SG (see Fig. 8C). (2S)-8-PNG is well-known as a potent hop-derived estrogenic compound (Keiler et al., 2013), and the substitution of the prenyl group at C(8) with alkyl chains of varying lengths and branching

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patterns greatly alters the agonistic activity on estrogen receptors (Roelens et al., 2006). In

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contrast, (2S)-6-PNG does not have such estrogenic activity (Overk et al., 2005). Taken together, we propose that (2S)-6-PNG, a hop component, as well as (2R/S)-6-PNG, which can be easily synthesized, serves as an analgesic, or is useful as one of the best seeds for the development of novel T-channel blocking analgesics. Our findings that (2S)-naringenin or (2R/S)-naringenin having the basic skeleton structure of SG, 6-PNG and 8-PNG, did not show any inhibitory effect on T-currents (see Fig.

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ACCEPTED MANUSCRIPT 2H, I) suggest the importance of alkyl chains at C(6) or C(8) for T-channel blockade. It is very interesting that the T-channel-blocking effects of (2S)-8-PNG, but not SG, were reversible or

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washable (see Fig. 3A, C), although both compounds have alkyl chains at C(8) (see Figs. 1D and 2B). The structural characteristics responsible for the irreversibility of T-channel blockade by SG is still open to question. The comparisons of the racemic mixture and (S)-enantiomer of

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6-PNG provide interesting evidence that (2R/S)-6-PNG and (2S)-6-PNG are equipotent in

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blocking Cav3.2, while the former is less potent than the latter in inhibiting HVA-currents or Nav-currents (see Fig. 3E-J). Therefore, it is likely that (2R)-6-PNG, if available, might be more selective to Cav3.2 than (2S)-6-PNG, which has yet to be ascertained. Nonetheless, the

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blockade of HVA-Ca2+ channels and/or Nav-channels by (2R/S)-6-PNG might contribute to its analgesic activity, particularly in neuropathic pain models, considering the preclinical and clinical evidence that gabapentinoids acting on the α2δ subunit of HVA-Ca2+ channels and

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Nav-channel blockers are effective for treatment of neuropathic pain (Bouhassira and Attal,

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2016; Cardoso and Lewis, 2017).

The present results that i.pl. administration of SG, (2S)-6-PNG or (2S)-8-PNG prevented the mechanical allodynia induced by i.pl. NaHS, an H2S donor (Fig. 4C-F), and restored the neuropathic pain caused by PSNL or OHP treatment (Fig. 6A, C), are in agreement with the previous reports demonstrating the effectiveness of i.pl. administration of other T-channel blockers including NNC 55-0396, RQ-00311651 and mibefradil in rats or mice with

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ACCEPTED MANUSCRIPT neuropathic pain induced by L5 spinal nerve cutting or repeated treatment with paclitaxel, an anti-cancer agent (Okubo et al., 2012a; Sekiguchi et al., 2016; Takahashi et al., 2010). These

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findings support the current understanding that Cav3.2 expressed in the peripheral axons or endings of the primary afferents appears to plays a role in the regulation of neuronal

excitability, particularly in the peripheral neuropathy (Todorovic and Jevtovic-Todorovic,

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2013). On the other hand, Cav3.2 expressed in the central terminals of the sensory neurons is

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responsible for spontaneous neurotransmitter release without action potentials in the spinal dorsal horn, and participates in the pathogenesis of neuropathic pain (Todorovic and Jevtovic-Todorovic, 2013; Weiss and Zamponi, 2013). There is also evidence that T-channels

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also regulate pain sensation at supraspinal levels (Todorovic and Jevtovic-Todorovic, 2011). The concentrations of (2R/S)-6-PNG, after the i.p. administration at 30 mg/kg, reached 1.03 µg/ml (= 3.04 µM) at 10 min in the plasma, and 0.663 and 0.798 µg/g (equivalent to 1.95 and

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2.34 µM) at 10 and 30 min in the brain (see Table 1). These concentrations in the plasma and

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CNS were high enough to block Cav3.2, considering the IC50 value, 0.917 µM (see Fig. 3F). Therefore, the effects of i.p. administration of (2R/S)-6-PNG on the neuropathic pain (see Fig. 6B, D) and colonic pain/referred hyperalgesia (see Fig. 5) may be outcome of the blockade of both peripheral and central T-channels. Our data from the experiments with Cav3.2-knockout mice (see Fig. 7) clearly suggest that the suppressive effects of (2R/S)-6-PNG and SG on the PSNL-induced neuropathic pain are the outcome of Cav3.2 blockade, whereas genetic

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ACCEPTED MANUSCRIPT deletion of Cav3.2 is compensated by molecules other than T-channels in the development of PSNL-induced neuropathy, as suggested elsewhere (Gadotti et al., 2015). It is noteworthy that

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i.p. administration of 6-PNG at 30 mg/kg does not affect open-field behavior or rota-rod performance, because NNC 55-0396, the highly BBB-permeable T-channel blocker,

administered i.p. at 20 mg/kg caused remarkable disturbance in the same behavioral assay

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systems (Sekiguchi et al., 2016).

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Recently, a number of novel T-channel blockers have been developed and evaluated for anti-hyperalgesic or anti-allodynic activity in distinct pain models (Snutch and Zamponi, 2017). The IC50 values, 0.6-1 µM, for T-current inhibition and anti-hyperalgesic/anti-allodynic

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doses, 10-30 mg/kg (i.p.), of (2R/S)-6-PNG or (2S)-6-PNG are similar to those of many of T-channel blockers including NNC 55-0396 and RQ-0311651 (Matsunami et al., 2012; Okubo et al., 2011; Sekiguchi et al., 2016). TTA-A2 is one of the most potent T-channel blockers

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with state-dependency and use-dependency; the IC50 value for T-channel inhibition is less

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than 0.1 µM, and the anti-hyperalgesic/anti-allodynic dose is around 1 mg/kg (i.p.) (Francois et al., 2013; Kraus et al., 2010). However, it is noteworthy that TTA-A2 easily penetrates into the CNS and causes suppression of active wake and promotion of slow-wave sleep (Kraus et al., 2010). Considering the lack of effect of 6-PNG on locomotion and motor performance, 6-PNG is thus considered as an analgesic with a well-balanced BBB permeability.

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ACCEPTED MANUSCRIPT 5. Conclusions

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In conclusion, (2S)-6-PNG, a hop component, and (2R/S)-6-PNG are considered potent T-channel blockers, and alleviate T-channel-dependent somatic and visceral pain in mice

without behavioral and cardiovascular side effects. We thus propose that (2R/S)-6-PNG and/or

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(2S)-6-PNG serve as medicines for treatment of neuropathic pain and also for clinical control

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of visceral pain in patients with colonic diseases such as irritable bowel syndrome.

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ACCEPTED MANUSCRIPT Declaration of interest

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The authors have no conflicts of interest to declare.

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Funding

This work was supported in part by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (2014-2018) (S1411037) and the “Antiaging” Project for

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Private Universities.

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require activation of both Cav3.2 and TRPA1 channels in mice. Br. J. Pharmacol. 166,

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ACCEPTED MANUSCRIPT Figure Legends

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Fig. 1. Purification, identification and characterization of sophoraflavanone G (SG) as a novel T-type Ca2+ channel blocker from SOPHORAE RADIX (SR). (A) Inhibitory effect of the 50% ethanol extract of SR (SR extract) on T-currents in Cav3.2-HEK cells. (B) Protocol

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diagram for fractionation of 100% chloroform extract of SR (SR-CHCl3 extract) by a

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silica-gel column chromatography. Each fraction (Fr.) was tested for T-current-blocking activity in Cav3.2-HEK cells. (C) Concentration-dependent T-current-blocking activity of Frs. h, i and j in Cav3.2-HEK cells. (D) Structure of SG purified from SR. (E-G)

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Concentration-dependent inhibitory effects of SG on T-currents in Cav3.1-HEK (E) or Cav3.2-HEK (F) cells and on HVA-currents in neuron-like differentiated NG108-15 cells (G). V, vehicle. Data show the mean ± SEM for 5-9 (A), 6 - 10 (C), 6-27 (E), 5-18 (F) and 6-10

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(G) experiments.* P < 0.05, ** P < 0.01 vs. vehicle.

Fig. 2. T-type Ca2+ channel blocking activity of hop-derived SG analogues, (2S)-6-prenylnaringenin (PNG) (A) and (2S)-8-PNG (B), and of (2R/S)-naringenin (NG) (C) or (2S)-NG having a basic skeleton structure of SG, 6-PNG and 8-PNG. Concentration-related effects of each compound on T-currents were evaluated in Cav3.1-HEK cells (D, F, H) and Cav3.2-HEK cells (E, G, I). Activation curves and steady-state inactivation curves in the

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ACCEPTED MANUSCRIPT absence and presence of SG, (2S)-6-PNG or (2S)-8-PNG were compared in Cav3.1-HEK cells (J) or Cav3.2- HEK cells (K). V, vehicle. Data show the mean ± SEM for 7-19 (D), 5-6 (E),

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8-11 (F), 6-13 (G), 4-7 (H), 5-13 (I), 5-19 (J) or 13-33 (K) experiments. * P < 0.05, ** P < 0.01 vs. vehicle.

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Fig. 3. Assessment of the reversibility of Cav3.2 blockade by SG, (2S)-6-PNG and

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(2S)-8-PNG, and comparison of the Cav3.2-blocking selectivity of (2S)-6-PNG and (2R/S)-6-PNG.

(A-D) In Cav3.2-HEK cells, the extracellular solution was perfused at a rate of 5 ml/min, and

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T-currents were repeatedly measured every 10 s. Perfusion solution containing SG (A), (2S)-6-PNG (B), (2S)-8-PNG (C) or vehicle (0.1% DMSO) (D) was applied for 5-6 min, shown as black bars. (E-J) Effects of (2S)-6-PNG (E, G and I) and (2R/S)-6-PNG (F, H and J)

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on T-currents in Cav3.2-HEK cells (E and F), and on HVA-currents (G and H) and

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Nav-currents (I and J) in neuronally differentiated NG108-15 cells. V, vehicle. Data show the mean ± SEM for 7-8 (E), 19-20 (F), 4-6 (G), 4-5 (H), 5-10 (I) or 5-10 (J) experiments. * P < 0.05, ** P < 0.01 vs. vehicle.

Fig. 4. Effects of SR-extract (50% ethanol extract of SR), SG, (2S)-6-PNG, (2S)-8-PNG and (2R/S)-naringenin (NG) on the somatic mechanical allodynia induced by intraplantar (i.pl.)

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ACCEPTED MANUSCRIPT injection of NaHS, an H2S donor, in mice. (A and B) SR-extract was administered p.o. or i.p. 20 min before i.pl. NaHS. (C-F) SG, (2S)-6-PNG, (2S)-8-PNG and (2R/S)-NG were

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co-administered i.pl. with NaHS. Nociceptive thresholds 15 min after i.pl. injection of NaHS or vehicle are shown. V, vehicle. Data show the mean ± SEM for 4-8 (A, B), 7-9 (C) or 4-9

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(D-F) mice. ** P < 0.01 vs. vehicle + vehicle; † P < 0.05, †† P < 0.01 vs. vehicle + NaHS.

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Fig. 5. Effect of (2R/S)-6-PNG on the visceral nociception caused by intracolonic (i.col.) administration of Na2S, an H2S donor, in mice. (A, B) (2R/S)-6-PNG at 10 or 30 mg/kg was administered i.p. 15 min before i.col. Na2S at 5 nmol/mouse in conscious mice. Visceral

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pain-like nociceptive behavior was observed and counted for 30 min starting immediately after i.col. Na2S (A), and subsequently, referred hyperalgesia was evaluated by von Frey test (B). (C, D) (2R/S)-6-PNG at 30 mg/kg was administered i.p. 15 min before i.col. Na2S at 5

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nmol/mouse in anesthetized mice. The mice were transcardially perfused exactly 5 min after

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i.col. Na2S, for fixation of the spinal cord. (C) Typical microphotographs of immunostaining of phosphorylated ERK (p-ERK) in the spinal dorsal horn at T13-L1 and L5-S1 levels. Scale bars indicate 100 µm, and arrows in sky blue show p-ERK-positive cells. (D) The number of p-ERK-positive cells in laminae I-II, III-IV, V-VI, VII-IX and X of the bilateral spinal cord. V, vehicle. Data show the mean with S.E.M. for 5-7 mice (A, B) or 25 slices from 5 mice (D). **P<0.01 vs. vehicle + vehicle; ††P<0.01vs. vehicle + Na2S.

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Fig. 6. Effects of intraplantar (i.pl) or i.p. administration of (2R/S)-6-PNG on the neuropathic

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mechanical allodynia induced by PSNL or treatment with oxaliplatin (OHP) in mice. (2R/S)-6-PNG was administered i.pl. (A, B) or i.p. (C, D) to mice 8 days after PSNL (PSNL mice) or sham operation (sham mice) (A, C) and after a single i.p. administration of OHP at 5

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mg/kg (OHP-mice) or vehicle (Control-mice) (B, D). V, vehicle. Data show the mean ± SEM

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for 7-10 (A), 5-8 (B), 5-12 (C) or 5 (D) mice. * P < 0.05, ** P < 0.01 vs. vehicle in Sham-mice (A, C) or Control-mice (B, D); † P < 0.05, †† P < 0.01 vs. vehicle in PSNL-mice

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(A, C) or OHP-mice (B, D).

Fig. 7. Disappearance of the suppressing effect of SG or (2R/S)-6-PNG on the neuropathic mechanical allodynia induced by PSNL in Cav3.2-knockout mice. SG or (2R/S)-6-PNG at 30

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mg/kg was administered i.p. to wild-type C57BL/6J (WT) mice (A) or Cav3.2-knockout

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(Cav3.2-KO) mice (B) 8 days after PSNL. V, vehicle. Data show the mean ± SEM for 5-6 mice. * P < 0.05, ** P < 0.01 vs. vehicle in Sham-WT-mice (A) or Sham-Cav3.2-KO-mice (B); † P < 0.05, †† P < 0.01 vs. vehicle in PSNL-WT-mice (A).

Fig. 8. Effects of i.p. administration of (2R/S)-6- PNG on the behavior, motor performance, aortic smooth muscle contractility and blood pressure in mice. (A, B) Open-field behavior (A)

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ACCEPTED MANUSCRIPT and rota-rod performance (B) were assessed 30 min after i.p. (2R/S)-6-PNG at 30 mg/kg. (C) Concentration-response curves for relaxation induced by SG, (2S)-6-PNG, (2R/S)-6-PNG and

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(2S)-8-PNG, T-channel-blocking compounds, and by verapamil, an L-type Ca2+ channel blocker, in endothelium-removed ring preparations of rat aorta precontracted by high-K+ solution. Each compound was cumulatively applied to the preparatons. IC50 values for

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verapamil and (2S)-8-PNG were 0.141 and 8.571 µM, respectively, and those for other

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chemicals were higher than 30 µM. (D and E) Mean blood pressure (MBP) and heart rate (HR) were measured by the tail-cuff method after i.p. administration of (2R/S)-6-PNG at 20 mg/kg in conscious mice. Data show the mean ± SEM for 4-5 mice (A, B, D, E) or

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preparations (C). * P < 0.05 vs. vehicle.

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ACCEPTED MANUSCRIPT Table 1. Time-related concentrations of (2R/S)-6- PNG and SG in the plasma and brain after their i.p. administration in mice.

5

0.540 ± 0.340

0.223 ± 0.129

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10

1.036 ± 0.098

0.663 ± 0.062

0.64

30

0.579 ± 0.096

5

1.104 ± 0.400

0.000 ± 0.000

0.00

10

1.579 ± 0.129

0.000 ± 0.000

0.00

30

0.644 ± 0.209

0.000 ± 0.000

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Brain (µg/g)

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SG

Plasma (µg/mL)

0.798 ± 0.107

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(2R/S)-6-PNG

Ratio (Brain/Plasma)

Time (min)

1.38

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Blood collection and brain excision were conducted 5, 10 or 30 min after i.p. administration of (2R/S)-6-PNG or SG at 30 mg/kg. Data show the mean ± SEM for 4 mice. To obtain the

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ACCEPTED MANUSCRIPT Highlights

• Cav3.2 T-type Ca2+ channels contribute to intractable pain.

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• We identified sophoraflavanone G from SOPHORAE RADIX as a T-channel blocker.

• The analogues, 6- and 8-prenylnaringenin, derived from hops, also blocked T-channels.

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• 6-Prenylnaringenin had the most preferable characteristics as a T-channel blocker.

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• 6-Prenylnaringenin alleviated neuropathic and visceral pain with little side effects.