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Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist

Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist Background: Cannabinoid receptors and T-type calcium channels are potential targets for treating pain. Here we report on the design, synthesis and analgesic properties of a new mixed cannabinoid/T-type channel ligand, NMP-181. Results: NMP-181 action on CB and CB receptors was characterized in radioligand binding and in vitro GTPγ[ S] 1 2 functional assays, and block of transiently expressed human Cav3.2 T-type channels by NMP-181 was analyzed by patch clamp. The analgesic effects and in vivo mechanism of action of NMP-181 delivered spinally or systemically were analyzed in formalin and CFA mouse models of pain. NMP-181 inhibited peak Ca 3.2 currents with IC values V 50 in the low micromolar range and acted as a CB agonist. Inactivated state dependence further augmented the inhibitory action of NMP-181. NMP-181 produced a dose-dependent antinociceptive effect when administered either spinally or systemically in both phases of the formalin test. Both i.t. and i.p. treatment of mice with NMP-181 reversed the mechanical hyperalgesia induced by CFA injection. NMP-181 showed no antinocieptive effect in Ca 3.2 null mice. The antinociceptive effect of intrathecally delivered NMP-181 in the formalin test was reversed by i.t. treatment of mice with AM-630 (CB antagonist). In contrast, the NMP-181-induced antinociception was not affected by treatment of mice with AM-281 (CB antagonist). Conclusions: Our work shows that both T-type channels as well as CB receptors play a role in the antinociceptive action of NMP-181, and also provides a novel avenue for suppressing chronic pain through novel mixed T-type/cannabinoid receptor ligands. Keywords: T-type Channels, Cannabinoid Receptors, Pain, Patch-Clamp, Mice Background Low-voltage activated (LVA) T-type calcium channels In recent decades, our knowledge of the mechanisms are essential contributors to signalling in electrically underlying pain sensation has improved substantially; excitable cells [2-5] and are well recognized as important however, despite the increased understanding of the regulators of pain transmission [6-8]. T-type channels neurobiology of pain and the discovery of new pain- are highly expressed in primary afferent pain fibers with mediating molecules, only few novel classes of analgesic the highest expression levels in medium sized dorsal compounds have entered the clinic. Therefore, the iden- root ganglion (DRG) neurons [9]. Inhibition of T-type tification of new pharmacophores for analgesics is of channels by intrathecal [7,10] or systemic [11] delivery critical importance. Although the drug discovery sector of synthetic compounds, or through selective subunit frequently focuses on the design of highly specific knockdown via antisense oligonucleotides [7,12-14] channel and receptor modulators, the use of compounds has been shown to produce potent analgesic effects in that interact with more than one molecular target may rodents. Exactly how T-type channels contribute to pain provide opportunities for synergistic actions to increase processing is unclear, but may involve a regulation of analgesic efficacy [1]. the excitability of the primary afferent fiber and/or a contribution to neurotransmission at dorsal horn synap- * Correspondence: zamponi@ucalgary.ca ses [6,15,16]. Cannabinoid receptors on the other hand Department of Physiology and Pharmacology, Hotchkiss Brain Institute, are G‑protein-coupled receptors [17] that are activated University of Calgary, Calgary, Canada by cannabinoid ligands such as the phytocannabinoid Full list of author information is available at the end of the article © 2013 Gadotti et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gadotti et al. Molecular Pain 2013, 9:32 Page 2 of 11 http://www.molecularpain.com/content/9/1/32 9 9 Δ -tetrahydrocannabinol (Δ -THC) and endogenous in our pain model to affect the locomotor activity of cannabinoids such as anandamide and 2-arachidonyl mice on open-field test. glycerol (2-AG) [18]. These ligands bind to the two members of the CB receptor family - CB and CB [19,20]. Results 1 2 Cannanbinoids have shown efficacy in relieving pain in In vitro characterization of putative tricyclic T-type randomized-controlled trials often without serious adverse channel inhibitors effects [21] and also they show therapeutic action in the We previously reported on a novel series of tricyclic treatment of pain associated with diseases such as multiple compounds that were capable of interacting with both sclerosis [22,23]. Recent reports suggest that CB agonism cannabinoid receptors and T-type calcium channels can play a role in the analgesic effects of selective CB [31]. Based on our previous SAR data, we identified a core agonists in the rat CFA model [24]. A very low occupancy pharmacophore and synthesized NMP-181(Figure 1) as a of CB receptors (<10%) by an agonist with a relatively low possible dual CB /T-type channel ligand. We first tested 1 2 intrinsic efficacy can induce neurochemical and behavioral the ability of NMP-181 to inhibit transiently expressed effects resulting in antinociception [25]. Remarkably, T-type channels in tsA-201 cells. A concentration-response many endocannabinoids (such as anandamide) [26-28] curve revealed that the inhibitory effect of NMP-181 on and phytocannabinoids (Δ -tetrahydrocannabinol and Ca 3.2 occurred with an IC of 4.6 μM and a Hill coeffi- V 50 cannabidiol) [29,30] can also block T-type calcium cient of 2.1, indicating cooperativity between multiple channels, resulting in a more pronounced analgesia. This blocking modes (Figure 2A). Figure 2B illustrates the time- then suggests that such mixed cannabinoid receptor course of the effect of NMP-181 on Ca 3.2peakcurrent agonists with low intrinsic efficacy and T-type channel amplitude, revealing a rapid onset of block and only partial antagonists may produce synergistic actions with fewer reversibility. To evaluate whether this compound was side effects that may be exploited for analgesia. able to block other Ca 3 isoforms, 10 μM of NMP-181 In this study, we synthesized and pharmacologically was tested on transiently expressed human Ca 3.1 and characterized a novel compound NMP-181 (Figure 1) that Ca 3.3 channels at a test potential of −20 mV. As seen in exhibits a low intrinsic CB efficacy and potent T-type Figure 2C,D, the degree of inhibition was similar for all channel blocking activity. This compound was character- three Ca 3 isoforms. Application of NMP-181 to Ca 3.2 V V ized in cell models, and was evaluated in various in vivo channels produced a mild but significant hyperpolarizing models for analgesic properties. Our data show that in half-activation potential from −32.7 mV to −38.4 mV NMP-181 interferes with pain transmission through a (n = 5, P < 0.05) (Figure 2E). Many of T-type channel mechanism related to CB receptor activation and Ca 3.2 blockers have state-dependent inhibitory effects, with 2 V channel inhibition but without nonspecific sedative ac- enhanced potency at depolarized holding potentials tions, indicated by the inability of the active dose used [11,31,32]. To determine whether NMP-181 block is simi- larly state dependent, we recorded steady-state inactivation curves before and after application of NMP-181. As shown in Figure 2F, application of 10 μM of NMP-181 shifted the half-inactivation potential of Ca 3.2 channels towards more hyperpolarized potentials from −56.0 mV to −64.1 mV (n = 4, P <0.01). These data implythatatatypical neuronal resting membrane potential additional T-type channel inhibition can occur due to a drug induced reduc- tion in channel availability. Altogether, our data indicate that NMP-181 mediates T-type channel inhibition with affinities in the low micromolar range and possibly lower at more depolarized resting membrane potentials or as a result of frequency-dependent inhibition. Cannabinoid receptor affinity of NMP-181 NMP-181 was also tested for its cannabinoid activities using CB and CB binding assays. NMP-181 displaced 1 2 respectively 65.4% and 60.5% of [ H]CP55,490 in HEK293 cells expressing human CB receptors, and in rat brain ho- mogenates expressing CB receptors at 10 μM. NMP-181 exhibited the best affinity for CB receptors with a K value 2 i Figure 1 Molecular Structure of NMP-181. of 123 nM at human CB receptors and > 2 μMat rat CB 2 1 Gadotti et al. Molecular Pain 2013, 9:32 Page 3 of 11 http://www.molecularpain.com/content/9/1/32 Figure 2 Pharmacological and biophysical properties of NMP-181 block of T-type calcium channels. (A) concentration dependence of NMP-181 inhibition of the Ca 3.2 peak current amplitude. The data were fitted with a Hill equation. The half-maximal inhibitory concentration (IC ) from the fit was 4.6 μM and the Hill coefficient was 2.1. Numbers in parentheses reflect numbers of experiments for each concentration point. (B) representative time course of the development of and recovery from NMP-181 (30 μM) inhibition. (C), NMP-181 (10 μM) did not show selectivity on Ca 3 channels, as illustrated in the histogram and (D) representative traces from NMP-181 inhibition on Ca 3.1, 3.2 and 3.3 currents. V V Statistical significance was determined by one-way ANOVA followed by a Dunnett’s test, when the data were compared to those from Ca 3.2 group. (E) normalized current–voltage relations of Ca 3.2 current before and after application of 10 μM NMP-181. The half-activation potentials were −32.7 ± 2.0 mV and −38.4 ± 2.8 mV before and after application of NMP-181, respectively (inset, P < 0.05, paired t test). (F) steady-state inactivation curve obtained from Ca 3.2 channels before and after application of 10 μM NMP-181. The half-inactivation potentials were −56.0 ± 2.8 mV and −64.1 ± 4.0 mV before and after the treatment with NMP-181, respectively (inset, P < 0.01, paired t test). Gadotti et al. Molecular Pain 2013, 9:32 Page 4 of 11 http://www.molecularpain.com/content/9/1/32 Table 1 Radioligand competitive binding assays and GTPγ[ S] functional activity Mean K GTPγ[ S] functional assays Ligand Human CB1 Human CB2 Rat CB1 Human CB2 (nM) (nM) EC (μM) E (%) EC (μM) % activation average at 1 μM 50 max 50 NMP181 2238 ± 515 123 ± 28 NA ND >1 24% ND not done, NA not active at 1 μM. receptors (Table 1). NMP-181 exhibited low intrinsic activ- of mice 30 minutes before formalin injection resulted in a ity at human CB receptors since only 24% of activation significantly (one-way ANOVA) decreased pain response was detected at 1 μMinGTPγ[ S] functional assays. No time in both the first (Figure 3C) and second (Figure 3D) functional activity was detected at lower concentrations. phases (41±3% and 44±8% inhibition, respectively). These data indicate that NMP-181 acts as a preferential Importantly, neither spinal (via i.t.) nor systemic (via i.p.) agonist of CB receptors over the CB subtype. treatment with NMP-181 affected locomotor activity of 2 1 mice assessed via an open-field test (Figure 3E, F). Effect of NMP-181 on formalin-induced nociception Given the agonist activity on CB receptors and the Effect of NMP-181 on CFA-induced persistent concomitant T-type channel antagonist activity, we inflammatory nociception hypothesized that this compound may show efficacy in To ascertain whether NMP-181 modulates pain transmis- models of inflammatory pain. NMP-181 was delivered by sion under chronic inflammatory conditions, we analysed the i.t. route and its effects on both the acute nociceptive the nociceptive response of NMP-181 treated mice after and the slower inflammatory pain phases of a formalin test CFA injection. As shown in Figure 4A, B, mice injected with were evaluated [33]. Mice were treated i.t. with NMP-181 CFA developed mechanical hyperalgesia 3 days after CFA or control solution (PBS + DMSO 5%) 15 minutes prior to injection as indicated by a significant decrease of withdrawal behavioural assessment. One-way ANOVA revealed that i.t. thresholds when compared to pre-CFA baseline levels of the -1 treatment of mice with NMP-181 (1–10 μgi.t. )signifi- control group (P < 0.001). Two-way ANOVA revealed that -1 cantly reduced pain response time in both first (Figure 3A) spinal treatment of mice with NMP-181 (10 μgi.t. )signifi- and second (Figure 3B) phases (55±5% and 66±4% in- cantly attenuated the mechanical hyperalgesia induced by hibition, respectively). Intraperitoneal (i.p.) treatment CFA when compared with the CFA + PBS (control) group, Figure 3 Effect of NMP-181 administered by either i.t. (A,C) or i.p. (B,D) routes on the first and second phases of formalin-induced (A,B) pain and mice crossing in the open-field test (C,D). Each bar or individual point represents the mean responses from 6–8 animals and the error bars indicate the S.E.M. Control values (indicated by “0”) are from animals injected with 5% of DMSO in PBS and the asterisks denote the significance relative to the respective control group. **P<0.01; ***P<0.001. (one-way ANOVA followed by Tukey’s test). Gadotti et al. Molecular Pain 2013, 9:32 Page 5 of 11 http://www.molecularpain.com/content/9/1/32 Figure 4 Effect of NMP-181 delivered via i.t. (A) or i.p. (B) on the mechanical hyperalgesia induced by intraplantar delivery of CFA. Each circle represents the mean responses from 7–8 animals and the error bars indicate the S.E.M. Control values (indicated by black circles) are from animals injected with 5% of DMSO in PBS and the asterisks denote the significance relative to the control group. *P<0.05; **P<0.01 and ### P<0.001 when compared to PBS intraplantar group (two-way ANOVA followed by Tukey’s test). at 20 minutes (P < 0.01) and 40 minutes (P < 0.05) after either through i.t or i.p. routes. Altogether, these data NMP-181 treatment (Figure 4A). When mice were treated indicate that NMP-181 treatment specifically modulates -1 with NMP-181 systemically (1 mg kg ,i.p.),mechanical pain signalling and mediates analgesia when delivered hyperalgesia induced by CFA was significantly attenuated either spinally or systemically to mice. at 30 minutes (P < 0.01) and 60 minutes (P < 0.05) after treatment when compared with the CFA + PBS (control) Analysis of mechanism of action of NMP-181 group (Figure 4B). These data indicate that NMP-181 is To investigate if T-type calcium channels play a role in the a regulator of chronic inflammatory pain when delivered analgesic effect of NMP-181, we performed a formalin test Figure 5 Effect of NMP-181 delivered via i.t. to Ca 3.2 null mice on first (C) and second (D) phases of formalin induced pain. Ca 3.2 null V V mice exhibit decreased pain response time when compared to wild-type animal on first (A) and second (B) phases of formalin induced pain. Each bar represents the mean responses from 6 animals and the error bars indicate the S.E.M. Control values (indicated by “C”) are from animals injected with 5% of DMSO in PBS (one-way ANOVA followed by Tukey’s test). Gadotti et al. Molecular Pain 2013, 9:32 Page 6 of 11 http://www.molecularpain.com/content/9/1/32 in Ca 3.2 null mice that were treated either with vehicle or degrading the endocannabinoid 2-arachidonoylglycerol. -1 with NMP-181 (10 μg i.t. )and as showninFigure 5A, B, Although AM-630 is considered highly selective for CB , Ca 3.2 null mice exhibited a lower mean response time it is remotely possible that AM-630 could somehow have when compared to wild-type mice, in agreement with affected NMP-181 block of T-type channels. However, the previous data from [34]. As indicated in Figure 5C, D fact that NMP-181 was still active in Ca 3.2 KO mice Ca 3.2 null mice appear to be completely insensitive to supports the idea that NMP-181 acts via more than one -1 i.t. treatment with NMP-181 (10 μgi.t. ) as revealed by target, and based on the AM-630 data, we thus conclude one-way ANOVA. At face value, these data suggest that that CB receptors also contribute to the analgesic effect NMP-181 may predominantly inhibit pain signalling via of NMP-181. T-type channel inhibition. In contrast, intrathecal treatment of mice with the CB Intrathecal treatment of mice with the CB antagon- antagonist AM-281 (2 μg/i.t.), in combination with NMP- ist AM-630 (1 μg/i.t.), in combination with NMP-181 181 (10 μg/i.t.), did not modify the antinociception caused (10 μg/i.t.), significantly attenuated the antinociceptive by NMP-181 (10 μg/i.t.) in the formalin test (Figure 6C, D), action of NMP-181 (10 μg/i.t.) (Figure 6A, B) in both excluding an involvement of the CB receptor in the phases of formalin-induced pain as revealed by two-way analgesic action of NMP-181. Importantly the dose of ANOVA ([NMP-181 treatment F(1.65)=5.4, P <0.001 and AM-281 used was able to significantly reverse the anal- NMP-181 × AM-630 + NMP-181 interaction: F(1.75)=4.5, gesic action of URB-597 (10 μg/i.t., a dose that resulted P<0.001] for the first phase and [NMP-181 treatment F in a >50% reduction in response time, data not shown), an (2.87)=5.4, P <0.001 and NMP-181 × AM-630 + NMP-181 inhibitor of the enzyme fatty acid amide hydrolase, which is interaction F(1.67)=5.4, P<0.05] for the second phase). As a the primary degradatory enzyme for the endocannabinoid positive control, we used JZL-184 (3 μg/i.t., a dose which anandamide. Collectively these data show that intrathecal in itself produced a 50% reduction in response time; data delivery of NMP-181 mediates its analgesic actions through not shown), an irreversible inhibitor for monoacylglycerol a combination of Ca 3.2 channel inhibition and CB V 2 lipase, which is the primary enzyme responsible for receptor stimulation. Figure 6 Effect of i.t. treatment with selective CB (A, B) or CB (C, D) receptor antagonists on the antinociceptive action of NMP-181 2 1 on first (A, C) and second (B, D) phases of formalin-induced pain in mice. Each bar represents the mean responses from 6–8 animals and the error bars indicate the S.E.M. Control values (indicated by “C”) are from animals injected with 5% of DMSO in PBS and the asterisks denote the # ### significance relative to the control group. *P<0.05; ***P<0.001 and P<0.05; P<0.001 when compared to NMP-181 alone or positive controls treated groups (two-way ANOVA followed by Tukey’s test). Gadotti et al. Molecular Pain 2013, 9:32 Page 7 of 11 http://www.molecularpain.com/content/9/1/32 Discussion such as neuroplasticity in the nervous system, we also After the identification of the CB [19] and CB [20] assessed the action of NMP-181 in a persistent inflamma- 1 2 receptors for delta-nine-tetrahydrocannabinol (Δ -THC) tory pain model. Indeed, we demonstrated that NMP-181 in mammals, various pharmaceutical strategies have delivered either spinally (via i.t.) or systemically (via i.p.) attempted to explore the potential therapeutic properties of inhibited mechanical hyperalgesia induced by CFA at doses the cannabinoid system while minimizing its problematic that did not seem to be directly associated with nonspecific side effects [35,36]. A significant problem surrounding sedative actions, indicated by the inability of these the medical use of cannabis-related compounds is a con- doses to affect the locomotor activity of mice in an cern regarding their CB -mediated psychoactive effects and open-field test. The CFA model of persistent pain pro- abuse potential [37]. The interest in developing compounds duces central sensitization in response to the release of whose mechanism of action involves the CB receptors several pro-inflammatory mediators, which cumulatively without CB involvement, and thus without CB -mediated increase the sensitivity of peripheral and central sensory 1 1 psychotropic side effects, still remains a goal in medical pathways [46]. therapeutics. For this reason, selective CB receptor Our data reveal that the CB receptor, but not the CB 2 2 1 ligands appear as potentially viable compounds for pain receptor, is likely involved in the analgesic effects of NMP- management. CB activation suppresses microglial cell 181, as the effects of NMP-181 were partially reversed by activation [38] and decreases neuroinflammation [39] AM-630, but not AM-281. At the same time, it is interest- which are common underlying mechanisms that lead to ing to note that NMP-181 was completely ineffective in in pathological pain [40] even though involvment of CB Ca 3.2 null mice (Figure 5C, D), although the null mice 1 V receptor cannot be excluded in these effects [24]. In themselves showed only a partial reduction in nocifensive addtion, cannabinoid receptors may couple to other ef- behavior that in itself was smaller than the effect of fectors such as N-type calcium channels that are critical for NMP-181 in wild type animals (compare Figure 5A, B the transmission of pain signals [41-43]. Interestingly, with Figure 3A, B). It is possible that Ca 3.2 null mice either Δ -THC and cannabidiol [30] or the endogenous develop compensatory mechanisms that are insensitive cannabinoid anandamide and its derivatives [26,28,44] to NMP-181. It is also possible that the NMP-181 medi- inhibit T-type channel activity, thus mediating neuronal ated activation of CB receptors might trigger its analgesic excitability via a receptor-independent mechanism. Given effects in part via second messenger mediated inhibition of that blockade of the T-type channel subtype Ca 3.2 results Ca 3.2 channel activity. This then might explain why null V V in antinociceptive, anti-hyperalgesic, and anti-allodynic mice are completely insensitive to NMP-181 even though effects [11], the use of mixed CB /T-type calcium channel they presumably still express CB receptors. Finally, we 2 2 ligand may provide a potential strategy for the development note that the effects of NMP-181 on Ca 3.2 channels of more effective analgesics. Combining different mech- appeared to be state dependent, resulting in analgesia. anisms of action in a single drug could represent many Altogether, our data support a mechanism of which advantages. These may include a gain of potency and dual action of NMP-181 on CB receptors and Ca 3.2 2 V duration of effects, a reduced number of prescribed drugs calcium channels. for a given condition, and perhaps a reduction of side- With regard to Ca 3.2, NMP-181 also mediated a effects, as synergistic action on two targets may require hyperpolarzing shift in half inactivation potential that an overall lower dose. We were thus interested in iden- would be expected to produce additional inhibitory ef- tifying a CB (but not CB ) agonist with T-type channel fects due to reduced channel availability at normal 2 1 blocking ability. Here, we present a new compound of this resting potentials. Such a feature is often associated class, NMP-181, which mediates analgesia in vivo dose- with frequency dependent inhibition [6,32] which is a dependently and through a mechanism involving CB desirable feature in a drug designed to inhibit cellular receptor activation and T-type calcium channel blockade. excitability, such as in epilepsy [47], cardiac arrhyth- Our results demonstrate that NMP-181 administered mias [48] and pain [30,49]. spinally (via i.t.) or systemically (i.p.) inhibits biphasic (neurogenic and inflammatory) pain induced by formalin in mice. This effect was greater during the second inflam- Conclusions matory pain phase of the test, indicating that NMP-181 Altogether, this study shows that NMP-181 exerts a rapid strongly modulates inflammatory pain. In the formalin onset and pronounced antinociceptive effect in mice when test, the neurogenic phase is elicited by direct activation of administered spinally and systemically, at doses that do not nociceptive terminals; whereas a combination of periph- interfere with locomotor activity. The NMP-181 scaffold eral and central mechanisms underlies the inflammatory maythus serveasa newpharmacophorefor the devel- phase [33,45]. As chronic pain differs substantially from opment of new, more potent and longer-lasting mixed acute pain in terms of its persistence and adaptive changes CB /T-type channel ligands. 2 Gadotti et al. Molecular Pain 2013, 9:32 Page 8 of 11 http://www.molecularpain.com/content/9/1/32 Methods in a competitive binding experiment using, respectively, Chemical synthesis membrane fractions prepared from rat brain homogenate NMP-181 was synthesized at the Core Laboratory for expressing CB receptor and HEK293 cells expressing the Neuromolecular Production. Full analytical data and human CB receptor. The competition binding experi- detailed synthesis protocol are available in the supporting ment for CB and CB was performed in 96 well plates 1 2 information (Additional file 1). NMP-181 was prepared in a containing Standard Binding Buffer (50 mM Tris HCl, 1 -1 four-step sequence starting with carbazole which was first mM EDTA, 3 mM MgCl ,5 mg ml fatty acid-free BSA, alkylated and then formylated. Oxidation of the resulting pH 7.4). The radioligand was [ H]CP55940, and the refer- aldhehyde followed by amidification afforded NMP-181. ence compound was CP55940. A solution of the com- -1 NMP-181 base was used for in vitro studies and NMP-181 pound to be tested was prepared as a 1 mg ml stock hydrochloride was used for the in vivo studies. in DMSO and then diluted in Standard Binding Buffer by serial dilution. Radioligand was diluted to five times cDNA constructs the assay concentration in Standard Binding Buffer. The cDNAs encoding human Ca 3.2 and Ca 3.3 were Aliquots (50 μl) of radioligand were dispensed into the V V generously provided by Drs. Arnaud Monteil (CNRS wells of a 96-well plate containing 100 μlofStandard Montpellier) and Terrance Snutch (University of British Binding Buffer. Then, duplicate 50-μl aliquots of the test Columbia), respectively. The isolation of human Ca 3.1 and reference compound dilutions were added. Finally, cDNA in our laboratory was described previously [27]. crude membrane fractions of cells were resuspended in The cDNA encoding the human CB receptor was isolated 3 ml of chilled Standard Binding Buffer and homoge- from a human brain stem cDNA library [50]. Sequencing nized by several passages through a 26 gauge needle, confirmed that it was identical to GenBank Accession then 50 μl were dispensed into each well. The 250-μl X54937. The coding sequence of the human CB receptor reactions were incubated at room temperature for 1.5 was subcloned as a HindIII-XbaI 1.5 kb DNA fragment in hours, and then harvested by rapid filtration onto the expression vector pCDNA3 and in a bicistronic expres- Whatman GF/B glass fiber filters pre-soaked with 0.3% sion vector. The human CB receptor was cloned by PCR polyethyleneimine using a 96-well Brandel harvester. using oligonucleotides based on the sequence published by Four rapid 500-μl washes were performed. Filters were Munro and colleagues [20] with human genomic DNA as placed in 6-ml scintillation tubes and allowed to dry template. Sequencing of the resulting clones identified a overnight. Bound radioactivity was harvested onto 0.3% fragment of 1.1 kb encoding the human cannabinoid 2 polyethyleneamine-treated, 96-well filter mats using a 96- receptor, identical to GenBank Accession X74328. The cod- well Filtermate harvester. The filter mats were dried, then ing sequence of the human CB receptor was inserted into scintillant was melted onto the filters and the radioactivity bicistronic expression plasmids as a BamHI-NheI fragment retained on the filters counted in a Microbeta scintillation and was subcloned as a BamHI-NheI DNA fragment in a counter. Raw data (dpm) representing total radioligand BamHI-XbaI expression vector pCDNA3 (Invitrogen). The binding (i.e., specific + non-specific binding) were plotted sequences of human CB ,usedinthe bindingstudies are as a function of the logarithm of the molar concentration the NCBI Reference Sequence. of the competitor (i.e., test or reference compound). Non- linear regression of the normalized (i.e., percent radioligand Cell culture and transfection resuspendedbinding compared to that observed in the ab- HEK293 cells and CHO cells were used in radioligand sence of test or reference compound) raw data was binding assays while tsA-201 cells were used in elec- performed in Prism 4.0 (GraphPad Software) using the trophysiological studies. Human CB (used in the binding built-in three parameter logistic model describing ligand studies) was cloned into pcDNA5.0FRT and cell lines were competition binding to radioligand-labeled sites: y = made using the FlpIn system from Invitrogen. tsA-201 cell bottom + [(top-bottom)/(1 + 10×-logIC )] where the culture and transient transfection of calcium channels denominator equals the residual radioligand binding mea- were described previously [51]. In brief, Ca 3.1, 3.2 and sured in the presence of 10 μM reference compound (i.e., 3.3 α1 subunits were transfected individually with yellow non-specific binding) and the numerator equals the fluorescent protein in tsA-201 cells using the calcium total radioligand binding observed in the absence of phosphate method. competitor. The log IC (i.e., the log of the ligand con- centration that reduces radioligand binding by 50%) is In vitro receptor radioligand CB and CB binding studies thus estimated from the data and used to obtain the K 1 2 i CB and CB radioligand binding data were obtained using by applying the Cheng-Prusoff approximation: K =IC / 1 2 i 50 National Institute of Mental Health (NIMH) Psychoactive (1 + [ligand]/K ) where [ligand] equals the assay radioligand Drug Screening Program (PDSP) resources as de- concentration and K equals the affinity constant of the scribed earlier [31,52-54]. Compounds were screened radioligand for the target receptor. Gadotti et al. Molecular Pain 2013, 9:32 Page 9 of 11 http://www.molecularpain.com/content/9/1/32 GTPγ[ S] functional assays maximum slope conductance, k is a slope factor, and Functional activity was evaluated using GTPγ[ S] assay V is the half activation potential. in CHO cell membrane extracts expressing recombinant human CB or CB receptors as we previously described Animals 1 2 [31,55]. Compounds were solubilized in 100% DMSO at All experiments were conducted following the protocol a concentration of 10 mM within 4 hours of the first approved by the Institutional Animal Care and Use testing session. A pre-dilution for the dose response Committee and all efforts were made to minimize animal curve was performed in 100% DMSO and then diluted suffering according to the policies and recommendations 100 fold in assay buffer at a concentration 2 fold higher of the International Association for the Study of Pain. than the concentration to be tested. Compounds were Adult male C57BL/6J (wild-type) or CACNA1H knockout tested for agonist activities in duplicate with CP55,940 (Ca 3.2 null) mice (20-25 g) were used (total of 265 mice). (Tocris, Bioscience, Ellisville, MI, USA) as reference Animals were housed at a maximum of five per cage agonist. Membranes were mixed with GDP diluted in (30×20 ×15cm) with ad libitum access to food and assay buffer to give 30 μM solution (volume:volume) water. They were kept in controlled temperature of 23 ± 1°C and incubated for at least 15 min on ice. In parallel, on a 12 h light/dark cycle (lights on at 7:00 a.m.). When GTPγ[ S] (GE Healthcare, Catalogue number SJ1308) drugs were delivered by the intraperitoneal (i.p.) route, a were mixed with the beads (PVT-WGA (GE Healthcare, constant volume of 10 ml/kg body weight was injected. -1 RPNQ001), diluted in assay buffer at 50 mg ml When drugs were administered by intrathecal (i.t.) delivery, -1 (0.5 mg 10 μl ) (volume:volume) just before starting the volumes of 10 μl were injected. Intrathecal (i.t) injections reaction. The following reagents were successively added were given to fully conscious mice using the method previ- in the wells of an Optiplate (Perkin Elmer): 50 μlofligand, ously described [56,57]. Animals were manually restrained, 20 μl of the membrane: GDP mix, 10 μl of assay buffer for the dorsal fur of each mouse was shaved, the spinal column agonist testing, and 20 μl of the GTPγ[ S]: beads mix. was arched, and a 30-gauge needle attached in a PE20 The plates were covered with a topseal, agitated on an Polyethylene tube to a 25-μl Hamilton microsyringe orbital shaker for 2 min, and then incubated for 1 hour (Hamilton, Birmingham, UK) was inserted into the at room temperature. Then the plates were centrifuged subarachnoid space between the L and L vertebrae. 4 5 for 10 min at 2000 rpm and counted for 1 min/well with a Correct i.t. positioning of the needle tip was confirmed PerkinElmer TopCount reader. Assay reproducibility was by a characteristic tail-flick response of animal. Appropriate monitored by the use of reference compound CP 55,940. vehicle-treated groups were also assessed simultaneously. For replicate determinations, the maximum variability tol- All drugs were dissolved in DMSO and control animals erated in the test was of ± 20% around the average of the received PBS + DMSO 5%, which was the maximum replicates. Efficacies (E )for CB or CB are expressed as DMSO concentration in solutions delivered to animals. max 1 2 a percentage relative to the efficacy of CP 55,940. Mice with a targeted disruption of the Ca 3.2 gene (Homozygous CACNA1H, also called α -3.2)[58]were purchased from Jackson Laboratories. Different cohorts of Electrophysiology mice were used for each test and each mouse was used only Methods and procedures used in the electrophysiological once. The observer was blind to the experimental condi- studies were described in detail by us previously [31]. tions in the experiment examining the action of NMP-181 Whole-cell currents were recorded from tsA-201 cells 2–4 on formalin, open-field and CFA tests (Figures 3 and 4). days after transfection. NMP compounds were dissolved in DMSO at a 10 mM concentration and diluted into Formalin test external recording solution with a final DMSO concen- The formalin test allows us to evaluate two different types tration no higher than 0.3%. Concentration-response of pain: neurogenic pain (phase 1) is caused by direct acti- studies were analyzed with the Hill equation I/I = control vation of nociceptive nerve terminals, while inflammatory 1/[1 + (IC /[compound]) ], where I is the normalized 50 pain (phase 2) is mediated by a combination of peripheral current at a given concentration of the compound, IC is 50 input and spinal cord sensitization [33,45]. For this test, the concentration of the compound yielding a current that mice were acclimatized in the laboratory for at least is half of the control current, I ,and n is the Hill coeffi- control 60 minutes before experiments. Animals received 20 μl cient. For steady-state inactivation curves, data were fitted of a formalin solution (1.25%) made up in PBS injected (V−Vh)/k using Boltzmann equation I =1/(1+ e ), where V is h intraplantarily (i.pl.) in the ventral surface of the right the half inactivation potential and k is the slope factor. hindpaw. Following i.pl. injections of formalin, the animals Current–voltage (I-V) plots were fitted using the modi- were immediately placed individually into observation −(V−Va)/k fied Boltzmann equation: I =1/(1+ e )× G × chambers and the time spent licking or biting the injected (V - E ), where E is the reversal potential, G is the rev rev paw was recorded and considered as a nociceptive Gadotti et al. Molecular Pain 2013, 9:32 Page 10 of 11 http://www.molecularpain.com/content/9/1/32 response. We observed animals individually from 0–5 receptor type 2; CFA: Complete Freund's adjuvant; DPA: Dynamic plantar aesthesiometer; DRG: Dorsal root ganglion; LVA: Low-voltage activated. min (neurogenic phase) and 15–30 min (inflammatory phase) and the time spent licking or biting the injected paw Competing interests was recorded with a chronometer. The authors declare that they have no competing interests. Author contributions CFA-induced persistent inflammatory pain VMG, HY, RRP and NDB performed experiments and analyzed data. VMG, PD In order to induce persistent inflammatory pain, mice re- and GWZ designed experiments. VMG, PD and GWZ wrote the manuscript. The authors read and approved the final manuscript. ceived 20 μl of Complete Freund's Adjuvant (CFA) injected subcutaneously in the plantar surface of the right hindpaw Acknowledgments (i.pl.) [59]. Control groups received 20 μLofPBS in the This work was supported by an operating grant to GWZ from the Canadian right paw. Animals received NMP-181 either spinally Institutes of Health Research and by National Institutes of Health (NIH) grant -1 -1 P30-NS055022 to PD and RRP. GWZ is a Canada Research Chair and an (1–10 μgi.t. ) or systemically (0.3-3 mg kg , i.p.) 3 days Alberta Innovates-Health Solutions (AI-HS) Scientist. VG and HY were following the CFA injection. Mechanical hyperalgesia was supported by an AI-HS Fellowship, NTB was supported by an AI-HS Summer then measured using the Dynamic Plantar Aesthesiometer Studentship award. Radioligand binding assays were performed by the National Institute of Mental Health’s Psychoactive Drug Screening Program (DPA, Ugo Basile, Varese, Italy). Animals were placed Contract # HHSN-271-2008-00025-C (NIMH/PDSP). NIMH/PDSP is directed by individually in small enclosed testing arenas (20 cm × Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and 18.5 cm × 13 cm, length × width × height) on top of a Project Officer Jamie Driscol at NIMH, Bethesda MD, USA. Marvin was used for drawing, displaying and characterizing chemical structures, substructures wire mesh floor. Mice were allowed to acclimate for a and reactions included in the supporting information, Marvin 5.11.5, 2013, period of 90 minutes. The DPA device was positioned ChemAxon (http://www.chemaxon.com). beneath the animal, so that the filament was directly Author details under the plantar surface of the ipsilateral hind paw. Department of Physiology and Pharmacology, Hotchkiss Brain Institute, Each paw was tested three times per session. 2 University of Calgary, Calgary, Canada. Core Laboratory for Neuromolecular Production, The University of Montana, Missoula, MT, USA. 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Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist

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References (129)

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Springer Journals
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Copyright © 2013 by Gadotti et al.; licensee BioMed Central Ltd.
Subject
Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology; Neurosciences; Neurology
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1744-8069
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1744-8069
DOI
10.1186/1744-8069-9-32
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23815854
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Abstract

Background: Cannabinoid receptors and T-type calcium channels are potential targets for treating pain. Here we report on the design, synthesis and analgesic properties of a new mixed cannabinoid/T-type channel ligand, NMP-181. Results: NMP-181 action on CB and CB receptors was characterized in radioligand binding and in vitro GTPγ[ S] 1 2 functional assays, and block of transiently expressed human Cav3.2 T-type channels by NMP-181 was analyzed by patch clamp. The analgesic effects and in vivo mechanism of action of NMP-181 delivered spinally or systemically were analyzed in formalin and CFA mouse models of pain. NMP-181 inhibited peak Ca 3.2 currents with IC values V 50 in the low micromolar range and acted as a CB agonist. Inactivated state dependence further augmented the inhibitory action of NMP-181. NMP-181 produced a dose-dependent antinociceptive effect when administered either spinally or systemically in both phases of the formalin test. Both i.t. and i.p. treatment of mice with NMP-181 reversed the mechanical hyperalgesia induced by CFA injection. NMP-181 showed no antinocieptive effect in Ca 3.2 null mice. The antinociceptive effect of intrathecally delivered NMP-181 in the formalin test was reversed by i.t. treatment of mice with AM-630 (CB antagonist). In contrast, the NMP-181-induced antinociception was not affected by treatment of mice with AM-281 (CB antagonist). Conclusions: Our work shows that both T-type channels as well as CB receptors play a role in the antinociceptive action of NMP-181, and also provides a novel avenue for suppressing chronic pain through novel mixed T-type/cannabinoid receptor ligands. Keywords: T-type Channels, Cannabinoid Receptors, Pain, Patch-Clamp, Mice Background Low-voltage activated (LVA) T-type calcium channels In recent decades, our knowledge of the mechanisms are essential contributors to signalling in electrically underlying pain sensation has improved substantially; excitable cells [2-5] and are well recognized as important however, despite the increased understanding of the regulators of pain transmission [6-8]. T-type channels neurobiology of pain and the discovery of new pain- are highly expressed in primary afferent pain fibers with mediating molecules, only few novel classes of analgesic the highest expression levels in medium sized dorsal compounds have entered the clinic. Therefore, the iden- root ganglion (DRG) neurons [9]. Inhibition of T-type tification of new pharmacophores for analgesics is of channels by intrathecal [7,10] or systemic [11] delivery critical importance. Although the drug discovery sector of synthetic compounds, or through selective subunit frequently focuses on the design of highly specific knockdown via antisense oligonucleotides [7,12-14] channel and receptor modulators, the use of compounds has been shown to produce potent analgesic effects in that interact with more than one molecular target may rodents. Exactly how T-type channels contribute to pain provide opportunities for synergistic actions to increase processing is unclear, but may involve a regulation of analgesic efficacy [1]. the excitability of the primary afferent fiber and/or a contribution to neurotransmission at dorsal horn synap- * Correspondence: zamponi@ucalgary.ca ses [6,15,16]. Cannabinoid receptors on the other hand Department of Physiology and Pharmacology, Hotchkiss Brain Institute, are G‑protein-coupled receptors [17] that are activated University of Calgary, Calgary, Canada by cannabinoid ligands such as the phytocannabinoid Full list of author information is available at the end of the article © 2013 Gadotti et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gadotti et al. Molecular Pain 2013, 9:32 Page 2 of 11 http://www.molecularpain.com/content/9/1/32 9 9 Δ -tetrahydrocannabinol (Δ -THC) and endogenous in our pain model to affect the locomotor activity of cannabinoids such as anandamide and 2-arachidonyl mice on open-field test. glycerol (2-AG) [18]. These ligands bind to the two members of the CB receptor family - CB and CB [19,20]. Results 1 2 Cannanbinoids have shown efficacy in relieving pain in In vitro characterization of putative tricyclic T-type randomized-controlled trials often without serious adverse channel inhibitors effects [21] and also they show therapeutic action in the We previously reported on a novel series of tricyclic treatment of pain associated with diseases such as multiple compounds that were capable of interacting with both sclerosis [22,23]. Recent reports suggest that CB agonism cannabinoid receptors and T-type calcium channels can play a role in the analgesic effects of selective CB [31]. Based on our previous SAR data, we identified a core agonists in the rat CFA model [24]. A very low occupancy pharmacophore and synthesized NMP-181(Figure 1) as a of CB receptors (<10%) by an agonist with a relatively low possible dual CB /T-type channel ligand. We first tested 1 2 intrinsic efficacy can induce neurochemical and behavioral the ability of NMP-181 to inhibit transiently expressed effects resulting in antinociception [25]. Remarkably, T-type channels in tsA-201 cells. A concentration-response many endocannabinoids (such as anandamide) [26-28] curve revealed that the inhibitory effect of NMP-181 on and phytocannabinoids (Δ -tetrahydrocannabinol and Ca 3.2 occurred with an IC of 4.6 μM and a Hill coeffi- V 50 cannabidiol) [29,30] can also block T-type calcium cient of 2.1, indicating cooperativity between multiple channels, resulting in a more pronounced analgesia. This blocking modes (Figure 2A). Figure 2B illustrates the time- then suggests that such mixed cannabinoid receptor course of the effect of NMP-181 on Ca 3.2peakcurrent agonists with low intrinsic efficacy and T-type channel amplitude, revealing a rapid onset of block and only partial antagonists may produce synergistic actions with fewer reversibility. To evaluate whether this compound was side effects that may be exploited for analgesia. able to block other Ca 3 isoforms, 10 μM of NMP-181 In this study, we synthesized and pharmacologically was tested on transiently expressed human Ca 3.1 and characterized a novel compound NMP-181 (Figure 1) that Ca 3.3 channels at a test potential of −20 mV. As seen in exhibits a low intrinsic CB efficacy and potent T-type Figure 2C,D, the degree of inhibition was similar for all channel blocking activity. This compound was character- three Ca 3 isoforms. Application of NMP-181 to Ca 3.2 V V ized in cell models, and was evaluated in various in vivo channels produced a mild but significant hyperpolarizing models for analgesic properties. Our data show that in half-activation potential from −32.7 mV to −38.4 mV NMP-181 interferes with pain transmission through a (n = 5, P < 0.05) (Figure 2E). Many of T-type channel mechanism related to CB receptor activation and Ca 3.2 blockers have state-dependent inhibitory effects, with 2 V channel inhibition but without nonspecific sedative ac- enhanced potency at depolarized holding potentials tions, indicated by the inability of the active dose used [11,31,32]. To determine whether NMP-181 block is simi- larly state dependent, we recorded steady-state inactivation curves before and after application of NMP-181. As shown in Figure 2F, application of 10 μM of NMP-181 shifted the half-inactivation potential of Ca 3.2 channels towards more hyperpolarized potentials from −56.0 mV to −64.1 mV (n = 4, P <0.01). These data implythatatatypical neuronal resting membrane potential additional T-type channel inhibition can occur due to a drug induced reduc- tion in channel availability. Altogether, our data indicate that NMP-181 mediates T-type channel inhibition with affinities in the low micromolar range and possibly lower at more depolarized resting membrane potentials or as a result of frequency-dependent inhibition. Cannabinoid receptor affinity of NMP-181 NMP-181 was also tested for its cannabinoid activities using CB and CB binding assays. NMP-181 displaced 1 2 respectively 65.4% and 60.5% of [ H]CP55,490 in HEK293 cells expressing human CB receptors, and in rat brain ho- mogenates expressing CB receptors at 10 μM. NMP-181 exhibited the best affinity for CB receptors with a K value 2 i Figure 1 Molecular Structure of NMP-181. of 123 nM at human CB receptors and > 2 μMat rat CB 2 1 Gadotti et al. Molecular Pain 2013, 9:32 Page 3 of 11 http://www.molecularpain.com/content/9/1/32 Figure 2 Pharmacological and biophysical properties of NMP-181 block of T-type calcium channels. (A) concentration dependence of NMP-181 inhibition of the Ca 3.2 peak current amplitude. The data were fitted with a Hill equation. The half-maximal inhibitory concentration (IC ) from the fit was 4.6 μM and the Hill coefficient was 2.1. Numbers in parentheses reflect numbers of experiments for each concentration point. (B) representative time course of the development of and recovery from NMP-181 (30 μM) inhibition. (C), NMP-181 (10 μM) did not show selectivity on Ca 3 channels, as illustrated in the histogram and (D) representative traces from NMP-181 inhibition on Ca 3.1, 3.2 and 3.3 currents. V V Statistical significance was determined by one-way ANOVA followed by a Dunnett’s test, when the data were compared to those from Ca 3.2 group. (E) normalized current–voltage relations of Ca 3.2 current before and after application of 10 μM NMP-181. The half-activation potentials were −32.7 ± 2.0 mV and −38.4 ± 2.8 mV before and after application of NMP-181, respectively (inset, P < 0.05, paired t test). (F) steady-state inactivation curve obtained from Ca 3.2 channels before and after application of 10 μM NMP-181. The half-inactivation potentials were −56.0 ± 2.8 mV and −64.1 ± 4.0 mV before and after the treatment with NMP-181, respectively (inset, P < 0.01, paired t test). Gadotti et al. Molecular Pain 2013, 9:32 Page 4 of 11 http://www.molecularpain.com/content/9/1/32 Table 1 Radioligand competitive binding assays and GTPγ[ S] functional activity Mean K GTPγ[ S] functional assays Ligand Human CB1 Human CB2 Rat CB1 Human CB2 (nM) (nM) EC (μM) E (%) EC (μM) % activation average at 1 μM 50 max 50 NMP181 2238 ± 515 123 ± 28 NA ND >1 24% ND not done, NA not active at 1 μM. receptors (Table 1). NMP-181 exhibited low intrinsic activ- of mice 30 minutes before formalin injection resulted in a ity at human CB receptors since only 24% of activation significantly (one-way ANOVA) decreased pain response was detected at 1 μMinGTPγ[ S] functional assays. No time in both the first (Figure 3C) and second (Figure 3D) functional activity was detected at lower concentrations. phases (41±3% and 44±8% inhibition, respectively). These data indicate that NMP-181 acts as a preferential Importantly, neither spinal (via i.t.) nor systemic (via i.p.) agonist of CB receptors over the CB subtype. treatment with NMP-181 affected locomotor activity of 2 1 mice assessed via an open-field test (Figure 3E, F). Effect of NMP-181 on formalin-induced nociception Given the agonist activity on CB receptors and the Effect of NMP-181 on CFA-induced persistent concomitant T-type channel antagonist activity, we inflammatory nociception hypothesized that this compound may show efficacy in To ascertain whether NMP-181 modulates pain transmis- models of inflammatory pain. NMP-181 was delivered by sion under chronic inflammatory conditions, we analysed the i.t. route and its effects on both the acute nociceptive the nociceptive response of NMP-181 treated mice after and the slower inflammatory pain phases of a formalin test CFA injection. As shown in Figure 4A, B, mice injected with were evaluated [33]. Mice were treated i.t. with NMP-181 CFA developed mechanical hyperalgesia 3 days after CFA or control solution (PBS + DMSO 5%) 15 minutes prior to injection as indicated by a significant decrease of withdrawal behavioural assessment. One-way ANOVA revealed that i.t. thresholds when compared to pre-CFA baseline levels of the -1 treatment of mice with NMP-181 (1–10 μgi.t. )signifi- control group (P < 0.001). Two-way ANOVA revealed that -1 cantly reduced pain response time in both first (Figure 3A) spinal treatment of mice with NMP-181 (10 μgi.t. )signifi- and second (Figure 3B) phases (55±5% and 66±4% in- cantly attenuated the mechanical hyperalgesia induced by hibition, respectively). Intraperitoneal (i.p.) treatment CFA when compared with the CFA + PBS (control) group, Figure 3 Effect of NMP-181 administered by either i.t. (A,C) or i.p. (B,D) routes on the first and second phases of formalin-induced (A,B) pain and mice crossing in the open-field test (C,D). Each bar or individual point represents the mean responses from 6–8 animals and the error bars indicate the S.E.M. Control values (indicated by “0”) are from animals injected with 5% of DMSO in PBS and the asterisks denote the significance relative to the respective control group. **P<0.01; ***P<0.001. (one-way ANOVA followed by Tukey’s test). Gadotti et al. Molecular Pain 2013, 9:32 Page 5 of 11 http://www.molecularpain.com/content/9/1/32 Figure 4 Effect of NMP-181 delivered via i.t. (A) or i.p. (B) on the mechanical hyperalgesia induced by intraplantar delivery of CFA. Each circle represents the mean responses from 7–8 animals and the error bars indicate the S.E.M. Control values (indicated by black circles) are from animals injected with 5% of DMSO in PBS and the asterisks denote the significance relative to the control group. *P<0.05; **P<0.01 and ### P<0.001 when compared to PBS intraplantar group (two-way ANOVA followed by Tukey’s test). at 20 minutes (P < 0.01) and 40 minutes (P < 0.05) after either through i.t or i.p. routes. Altogether, these data NMP-181 treatment (Figure 4A). When mice were treated indicate that NMP-181 treatment specifically modulates -1 with NMP-181 systemically (1 mg kg ,i.p.),mechanical pain signalling and mediates analgesia when delivered hyperalgesia induced by CFA was significantly attenuated either spinally or systemically to mice. at 30 minutes (P < 0.01) and 60 minutes (P < 0.05) after treatment when compared with the CFA + PBS (control) Analysis of mechanism of action of NMP-181 group (Figure 4B). These data indicate that NMP-181 is To investigate if T-type calcium channels play a role in the a regulator of chronic inflammatory pain when delivered analgesic effect of NMP-181, we performed a formalin test Figure 5 Effect of NMP-181 delivered via i.t. to Ca 3.2 null mice on first (C) and second (D) phases of formalin induced pain. Ca 3.2 null V V mice exhibit decreased pain response time when compared to wild-type animal on first (A) and second (B) phases of formalin induced pain. Each bar represents the mean responses from 6 animals and the error bars indicate the S.E.M. Control values (indicated by “C”) are from animals injected with 5% of DMSO in PBS (one-way ANOVA followed by Tukey’s test). Gadotti et al. Molecular Pain 2013, 9:32 Page 6 of 11 http://www.molecularpain.com/content/9/1/32 in Ca 3.2 null mice that were treated either with vehicle or degrading the endocannabinoid 2-arachidonoylglycerol. -1 with NMP-181 (10 μg i.t. )and as showninFigure 5A, B, Although AM-630 is considered highly selective for CB , Ca 3.2 null mice exhibited a lower mean response time it is remotely possible that AM-630 could somehow have when compared to wild-type mice, in agreement with affected NMP-181 block of T-type channels. However, the previous data from [34]. As indicated in Figure 5C, D fact that NMP-181 was still active in Ca 3.2 KO mice Ca 3.2 null mice appear to be completely insensitive to supports the idea that NMP-181 acts via more than one -1 i.t. treatment with NMP-181 (10 μgi.t. ) as revealed by target, and based on the AM-630 data, we thus conclude one-way ANOVA. At face value, these data suggest that that CB receptors also contribute to the analgesic effect NMP-181 may predominantly inhibit pain signalling via of NMP-181. T-type channel inhibition. In contrast, intrathecal treatment of mice with the CB Intrathecal treatment of mice with the CB antagon- antagonist AM-281 (2 μg/i.t.), in combination with NMP- ist AM-630 (1 μg/i.t.), in combination with NMP-181 181 (10 μg/i.t.), did not modify the antinociception caused (10 μg/i.t.), significantly attenuated the antinociceptive by NMP-181 (10 μg/i.t.) in the formalin test (Figure 6C, D), action of NMP-181 (10 μg/i.t.) (Figure 6A, B) in both excluding an involvement of the CB receptor in the phases of formalin-induced pain as revealed by two-way analgesic action of NMP-181. Importantly the dose of ANOVA ([NMP-181 treatment F(1.65)=5.4, P <0.001 and AM-281 used was able to significantly reverse the anal- NMP-181 × AM-630 + NMP-181 interaction: F(1.75)=4.5, gesic action of URB-597 (10 μg/i.t., a dose that resulted P<0.001] for the first phase and [NMP-181 treatment F in a >50% reduction in response time, data not shown), an (2.87)=5.4, P <0.001 and NMP-181 × AM-630 + NMP-181 inhibitor of the enzyme fatty acid amide hydrolase, which is interaction F(1.67)=5.4, P<0.05] for the second phase). As a the primary degradatory enzyme for the endocannabinoid positive control, we used JZL-184 (3 μg/i.t., a dose which anandamide. Collectively these data show that intrathecal in itself produced a 50% reduction in response time; data delivery of NMP-181 mediates its analgesic actions through not shown), an irreversible inhibitor for monoacylglycerol a combination of Ca 3.2 channel inhibition and CB V 2 lipase, which is the primary enzyme responsible for receptor stimulation. Figure 6 Effect of i.t. treatment with selective CB (A, B) or CB (C, D) receptor antagonists on the antinociceptive action of NMP-181 2 1 on first (A, C) and second (B, D) phases of formalin-induced pain in mice. Each bar represents the mean responses from 6–8 animals and the error bars indicate the S.E.M. Control values (indicated by “C”) are from animals injected with 5% of DMSO in PBS and the asterisks denote the # ### significance relative to the control group. *P<0.05; ***P<0.001 and P<0.05; P<0.001 when compared to NMP-181 alone or positive controls treated groups (two-way ANOVA followed by Tukey’s test). Gadotti et al. Molecular Pain 2013, 9:32 Page 7 of 11 http://www.molecularpain.com/content/9/1/32 Discussion such as neuroplasticity in the nervous system, we also After the identification of the CB [19] and CB [20] assessed the action of NMP-181 in a persistent inflamma- 1 2 receptors for delta-nine-tetrahydrocannabinol (Δ -THC) tory pain model. Indeed, we demonstrated that NMP-181 in mammals, various pharmaceutical strategies have delivered either spinally (via i.t.) or systemically (via i.p.) attempted to explore the potential therapeutic properties of inhibited mechanical hyperalgesia induced by CFA at doses the cannabinoid system while minimizing its problematic that did not seem to be directly associated with nonspecific side effects [35,36]. A significant problem surrounding sedative actions, indicated by the inability of these the medical use of cannabis-related compounds is a con- doses to affect the locomotor activity of mice in an cern regarding their CB -mediated psychoactive effects and open-field test. The CFA model of persistent pain pro- abuse potential [37]. The interest in developing compounds duces central sensitization in response to the release of whose mechanism of action involves the CB receptors several pro-inflammatory mediators, which cumulatively without CB involvement, and thus without CB -mediated increase the sensitivity of peripheral and central sensory 1 1 psychotropic side effects, still remains a goal in medical pathways [46]. therapeutics. For this reason, selective CB receptor Our data reveal that the CB receptor, but not the CB 2 2 1 ligands appear as potentially viable compounds for pain receptor, is likely involved in the analgesic effects of NMP- management. CB activation suppresses microglial cell 181, as the effects of NMP-181 were partially reversed by activation [38] and decreases neuroinflammation [39] AM-630, but not AM-281. At the same time, it is interest- which are common underlying mechanisms that lead to ing to note that NMP-181 was completely ineffective in in pathological pain [40] even though involvment of CB Ca 3.2 null mice (Figure 5C, D), although the null mice 1 V receptor cannot be excluded in these effects [24]. In themselves showed only a partial reduction in nocifensive addtion, cannabinoid receptors may couple to other ef- behavior that in itself was smaller than the effect of fectors such as N-type calcium channels that are critical for NMP-181 in wild type animals (compare Figure 5A, B the transmission of pain signals [41-43]. Interestingly, with Figure 3A, B). It is possible that Ca 3.2 null mice either Δ -THC and cannabidiol [30] or the endogenous develop compensatory mechanisms that are insensitive cannabinoid anandamide and its derivatives [26,28,44] to NMP-181. It is also possible that the NMP-181 medi- inhibit T-type channel activity, thus mediating neuronal ated activation of CB receptors might trigger its analgesic excitability via a receptor-independent mechanism. Given effects in part via second messenger mediated inhibition of that blockade of the T-type channel subtype Ca 3.2 results Ca 3.2 channel activity. This then might explain why null V V in antinociceptive, anti-hyperalgesic, and anti-allodynic mice are completely insensitive to NMP-181 even though effects [11], the use of mixed CB /T-type calcium channel they presumably still express CB receptors. Finally, we 2 2 ligand may provide a potential strategy for the development note that the effects of NMP-181 on Ca 3.2 channels of more effective analgesics. Combining different mech- appeared to be state dependent, resulting in analgesia. anisms of action in a single drug could represent many Altogether, our data support a mechanism of which advantages. These may include a gain of potency and dual action of NMP-181 on CB receptors and Ca 3.2 2 V duration of effects, a reduced number of prescribed drugs calcium channels. for a given condition, and perhaps a reduction of side- With regard to Ca 3.2, NMP-181 also mediated a effects, as synergistic action on two targets may require hyperpolarzing shift in half inactivation potential that an overall lower dose. We were thus interested in iden- would be expected to produce additional inhibitory ef- tifying a CB (but not CB ) agonist with T-type channel fects due to reduced channel availability at normal 2 1 blocking ability. Here, we present a new compound of this resting potentials. Such a feature is often associated class, NMP-181, which mediates analgesia in vivo dose- with frequency dependent inhibition [6,32] which is a dependently and through a mechanism involving CB desirable feature in a drug designed to inhibit cellular receptor activation and T-type calcium channel blockade. excitability, such as in epilepsy [47], cardiac arrhyth- Our results demonstrate that NMP-181 administered mias [48] and pain [30,49]. spinally (via i.t.) or systemically (i.p.) inhibits biphasic (neurogenic and inflammatory) pain induced by formalin in mice. This effect was greater during the second inflam- Conclusions matory pain phase of the test, indicating that NMP-181 Altogether, this study shows that NMP-181 exerts a rapid strongly modulates inflammatory pain. In the formalin onset and pronounced antinociceptive effect in mice when test, the neurogenic phase is elicited by direct activation of administered spinally and systemically, at doses that do not nociceptive terminals; whereas a combination of periph- interfere with locomotor activity. The NMP-181 scaffold eral and central mechanisms underlies the inflammatory maythus serveasa newpharmacophorefor the devel- phase [33,45]. As chronic pain differs substantially from opment of new, more potent and longer-lasting mixed acute pain in terms of its persistence and adaptive changes CB /T-type channel ligands. 2 Gadotti et al. Molecular Pain 2013, 9:32 Page 8 of 11 http://www.molecularpain.com/content/9/1/32 Methods in a competitive binding experiment using, respectively, Chemical synthesis membrane fractions prepared from rat brain homogenate NMP-181 was synthesized at the Core Laboratory for expressing CB receptor and HEK293 cells expressing the Neuromolecular Production. Full analytical data and human CB receptor. The competition binding experi- detailed synthesis protocol are available in the supporting ment for CB and CB was performed in 96 well plates 1 2 information (Additional file 1). NMP-181 was prepared in a containing Standard Binding Buffer (50 mM Tris HCl, 1 -1 four-step sequence starting with carbazole which was first mM EDTA, 3 mM MgCl ,5 mg ml fatty acid-free BSA, alkylated and then formylated. Oxidation of the resulting pH 7.4). The radioligand was [ H]CP55940, and the refer- aldhehyde followed by amidification afforded NMP-181. ence compound was CP55940. A solution of the com- -1 NMP-181 base was used for in vitro studies and NMP-181 pound to be tested was prepared as a 1 mg ml stock hydrochloride was used for the in vivo studies. in DMSO and then diluted in Standard Binding Buffer by serial dilution. Radioligand was diluted to five times cDNA constructs the assay concentration in Standard Binding Buffer. The cDNAs encoding human Ca 3.2 and Ca 3.3 were Aliquots (50 μl) of radioligand were dispensed into the V V generously provided by Drs. Arnaud Monteil (CNRS wells of a 96-well plate containing 100 μlofStandard Montpellier) and Terrance Snutch (University of British Binding Buffer. Then, duplicate 50-μl aliquots of the test Columbia), respectively. The isolation of human Ca 3.1 and reference compound dilutions were added. Finally, cDNA in our laboratory was described previously [27]. crude membrane fractions of cells were resuspended in The cDNA encoding the human CB receptor was isolated 3 ml of chilled Standard Binding Buffer and homoge- from a human brain stem cDNA library [50]. Sequencing nized by several passages through a 26 gauge needle, confirmed that it was identical to GenBank Accession then 50 μl were dispensed into each well. The 250-μl X54937. The coding sequence of the human CB receptor reactions were incubated at room temperature for 1.5 was subcloned as a HindIII-XbaI 1.5 kb DNA fragment in hours, and then harvested by rapid filtration onto the expression vector pCDNA3 and in a bicistronic expres- Whatman GF/B glass fiber filters pre-soaked with 0.3% sion vector. The human CB receptor was cloned by PCR polyethyleneimine using a 96-well Brandel harvester. using oligonucleotides based on the sequence published by Four rapid 500-μl washes were performed. Filters were Munro and colleagues [20] with human genomic DNA as placed in 6-ml scintillation tubes and allowed to dry template. Sequencing of the resulting clones identified a overnight. Bound radioactivity was harvested onto 0.3% fragment of 1.1 kb encoding the human cannabinoid 2 polyethyleneamine-treated, 96-well filter mats using a 96- receptor, identical to GenBank Accession X74328. The cod- well Filtermate harvester. The filter mats were dried, then ing sequence of the human CB receptor was inserted into scintillant was melted onto the filters and the radioactivity bicistronic expression plasmids as a BamHI-NheI fragment retained on the filters counted in a Microbeta scintillation and was subcloned as a BamHI-NheI DNA fragment in a counter. Raw data (dpm) representing total radioligand BamHI-XbaI expression vector pCDNA3 (Invitrogen). The binding (i.e., specific + non-specific binding) were plotted sequences of human CB ,usedinthe bindingstudies are as a function of the logarithm of the molar concentration the NCBI Reference Sequence. of the competitor (i.e., test or reference compound). Non- linear regression of the normalized (i.e., percent radioligand Cell culture and transfection resuspendedbinding compared to that observed in the ab- HEK293 cells and CHO cells were used in radioligand sence of test or reference compound) raw data was binding assays while tsA-201 cells were used in elec- performed in Prism 4.0 (GraphPad Software) using the trophysiological studies. Human CB (used in the binding built-in three parameter logistic model describing ligand studies) was cloned into pcDNA5.0FRT and cell lines were competition binding to radioligand-labeled sites: y = made using the FlpIn system from Invitrogen. tsA-201 cell bottom + [(top-bottom)/(1 + 10×-logIC )] where the culture and transient transfection of calcium channels denominator equals the residual radioligand binding mea- were described previously [51]. In brief, Ca 3.1, 3.2 and sured in the presence of 10 μM reference compound (i.e., 3.3 α1 subunits were transfected individually with yellow non-specific binding) and the numerator equals the fluorescent protein in tsA-201 cells using the calcium total radioligand binding observed in the absence of phosphate method. competitor. The log IC (i.e., the log of the ligand con- centration that reduces radioligand binding by 50%) is In vitro receptor radioligand CB and CB binding studies thus estimated from the data and used to obtain the K 1 2 i CB and CB radioligand binding data were obtained using by applying the Cheng-Prusoff approximation: K =IC / 1 2 i 50 National Institute of Mental Health (NIMH) Psychoactive (1 + [ligand]/K ) where [ligand] equals the assay radioligand Drug Screening Program (PDSP) resources as de- concentration and K equals the affinity constant of the scribed earlier [31,52-54]. Compounds were screened radioligand for the target receptor. Gadotti et al. Molecular Pain 2013, 9:32 Page 9 of 11 http://www.molecularpain.com/content/9/1/32 GTPγ[ S] functional assays maximum slope conductance, k is a slope factor, and Functional activity was evaluated using GTPγ[ S] assay V is the half activation potential. in CHO cell membrane extracts expressing recombinant human CB or CB receptors as we previously described Animals 1 2 [31,55]. Compounds were solubilized in 100% DMSO at All experiments were conducted following the protocol a concentration of 10 mM within 4 hours of the first approved by the Institutional Animal Care and Use testing session. A pre-dilution for the dose response Committee and all efforts were made to minimize animal curve was performed in 100% DMSO and then diluted suffering according to the policies and recommendations 100 fold in assay buffer at a concentration 2 fold higher of the International Association for the Study of Pain. than the concentration to be tested. Compounds were Adult male C57BL/6J (wild-type) or CACNA1H knockout tested for agonist activities in duplicate with CP55,940 (Ca 3.2 null) mice (20-25 g) were used (total of 265 mice). (Tocris, Bioscience, Ellisville, MI, USA) as reference Animals were housed at a maximum of five per cage agonist. Membranes were mixed with GDP diluted in (30×20 ×15cm) with ad libitum access to food and assay buffer to give 30 μM solution (volume:volume) water. They were kept in controlled temperature of 23 ± 1°C and incubated for at least 15 min on ice. In parallel, on a 12 h light/dark cycle (lights on at 7:00 a.m.). When GTPγ[ S] (GE Healthcare, Catalogue number SJ1308) drugs were delivered by the intraperitoneal (i.p.) route, a were mixed with the beads (PVT-WGA (GE Healthcare, constant volume of 10 ml/kg body weight was injected. -1 RPNQ001), diluted in assay buffer at 50 mg ml When drugs were administered by intrathecal (i.t.) delivery, -1 (0.5 mg 10 μl ) (volume:volume) just before starting the volumes of 10 μl were injected. Intrathecal (i.t) injections reaction. The following reagents were successively added were given to fully conscious mice using the method previ- in the wells of an Optiplate (Perkin Elmer): 50 μlofligand, ously described [56,57]. Animals were manually restrained, 20 μl of the membrane: GDP mix, 10 μl of assay buffer for the dorsal fur of each mouse was shaved, the spinal column agonist testing, and 20 μl of the GTPγ[ S]: beads mix. was arched, and a 30-gauge needle attached in a PE20 The plates were covered with a topseal, agitated on an Polyethylene tube to a 25-μl Hamilton microsyringe orbital shaker for 2 min, and then incubated for 1 hour (Hamilton, Birmingham, UK) was inserted into the at room temperature. Then the plates were centrifuged subarachnoid space between the L and L vertebrae. 4 5 for 10 min at 2000 rpm and counted for 1 min/well with a Correct i.t. positioning of the needle tip was confirmed PerkinElmer TopCount reader. Assay reproducibility was by a characteristic tail-flick response of animal. Appropriate monitored by the use of reference compound CP 55,940. vehicle-treated groups were also assessed simultaneously. For replicate determinations, the maximum variability tol- All drugs were dissolved in DMSO and control animals erated in the test was of ± 20% around the average of the received PBS + DMSO 5%, which was the maximum replicates. Efficacies (E )for CB or CB are expressed as DMSO concentration in solutions delivered to animals. max 1 2 a percentage relative to the efficacy of CP 55,940. Mice with a targeted disruption of the Ca 3.2 gene (Homozygous CACNA1H, also called α -3.2)[58]were purchased from Jackson Laboratories. Different cohorts of Electrophysiology mice were used for each test and each mouse was used only Methods and procedures used in the electrophysiological once. The observer was blind to the experimental condi- studies were described in detail by us previously [31]. tions in the experiment examining the action of NMP-181 Whole-cell currents were recorded from tsA-201 cells 2–4 on formalin, open-field and CFA tests (Figures 3 and 4). days after transfection. NMP compounds were dissolved in DMSO at a 10 mM concentration and diluted into Formalin test external recording solution with a final DMSO concen- The formalin test allows us to evaluate two different types tration no higher than 0.3%. Concentration-response of pain: neurogenic pain (phase 1) is caused by direct acti- studies were analyzed with the Hill equation I/I = control vation of nociceptive nerve terminals, while inflammatory 1/[1 + (IC /[compound]) ], where I is the normalized 50 pain (phase 2) is mediated by a combination of peripheral current at a given concentration of the compound, IC is 50 input and spinal cord sensitization [33,45]. For this test, the concentration of the compound yielding a current that mice were acclimatized in the laboratory for at least is half of the control current, I ,and n is the Hill coeffi- control 60 minutes before experiments. Animals received 20 μl cient. For steady-state inactivation curves, data were fitted of a formalin solution (1.25%) made up in PBS injected (V−Vh)/k using Boltzmann equation I =1/(1+ e ), where V is h intraplantarily (i.pl.) in the ventral surface of the right the half inactivation potential and k is the slope factor. hindpaw. Following i.pl. injections of formalin, the animals Current–voltage (I-V) plots were fitted using the modi- were immediately placed individually into observation −(V−Va)/k fied Boltzmann equation: I =1/(1+ e )× G × chambers and the time spent licking or biting the injected (V - E ), where E is the reversal potential, G is the rev rev paw was recorded and considered as a nociceptive Gadotti et al. Molecular Pain 2013, 9:32 Page 10 of 11 http://www.molecularpain.com/content/9/1/32 response. We observed animals individually from 0–5 receptor type 2; CFA: Complete Freund's adjuvant; DPA: Dynamic plantar aesthesiometer; DRG: Dorsal root ganglion; LVA: Low-voltage activated. min (neurogenic phase) and 15–30 min (inflammatory phase) and the time spent licking or biting the injected paw Competing interests was recorded with a chronometer. The authors declare that they have no competing interests. Author contributions CFA-induced persistent inflammatory pain VMG, HY, RRP and NDB performed experiments and analyzed data. VMG, PD In order to induce persistent inflammatory pain, mice re- and GWZ designed experiments. VMG, PD and GWZ wrote the manuscript. The authors read and approved the final manuscript. ceived 20 μl of Complete Freund's Adjuvant (CFA) injected subcutaneously in the plantar surface of the right hindpaw Acknowledgments (i.pl.) [59]. Control groups received 20 μLofPBS in the This work was supported by an operating grant to GWZ from the Canadian right paw. Animals received NMP-181 either spinally Institutes of Health Research and by National Institutes of Health (NIH) grant -1 -1 P30-NS055022 to PD and RRP. GWZ is a Canada Research Chair and an (1–10 μgi.t. ) or systemically (0.3-3 mg kg , i.p.) 3 days Alberta Innovates-Health Solutions (AI-HS) Scientist. VG and HY were following the CFA injection. Mechanical hyperalgesia was supported by an AI-HS Fellowship, NTB was supported by an AI-HS Summer then measured using the Dynamic Plantar Aesthesiometer Studentship award. Radioligand binding assays were performed by the National Institute of Mental Health’s Psychoactive Drug Screening Program (DPA, Ugo Basile, Varese, Italy). Animals were placed Contract # HHSN-271-2008-00025-C (NIMH/PDSP). NIMH/PDSP is directed by individually in small enclosed testing arenas (20 cm × Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and 18.5 cm × 13 cm, length × width × height) on top of a Project Officer Jamie Driscol at NIMH, Bethesda MD, USA. Marvin was used for drawing, displaying and characterizing chemical structures, substructures wire mesh floor. Mice were allowed to acclimate for a and reactions included in the supporting information, Marvin 5.11.5, 2013, period of 90 minutes. The DPA device was positioned ChemAxon (http://www.chemaxon.com). beneath the animal, so that the filament was directly Author details under the plantar surface of the ipsilateral hind paw. Department of Physiology and Pharmacology, Hotchkiss Brain Institute, Each paw was tested three times per session. 2 University of Calgary, Calgary, Canada. Core Laboratory for Neuromolecular Production, The University of Montana, Missoula, MT, USA. 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Molecular PainSpringer Journals

Published: Jul 1, 2013

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