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Abstract

Background

NXN-188 is a combined neuronal nitric oxide synthase (nNOS) inhibitor and 5-hydroxytryptamine 1B/1D (5-HT_1B/1D) receptor agonist. Using preclinical models, we evaluated whether these two unique therapeutic principles have a synergistic effect in attenuating stimulated calcitonin gene-related peptide (CGRP) release, a marker of trigeminal activation.

Methods

We examined the effect of NXN-188 on: (1) KCl-, capsaicin- and resiniferatoxin (RTX)-induced immunoreactive CGRP (iCGRP) release from isolated preparation of rat dura mater, trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC); and (2) capsaicin- and electrical stimulation (ES)-induced middle meningeal artery (MMA) dilation in a rat closed-cranial window.

Results

NXN-188 inhibited: (1) KCl-stimulated iCGRP release from dura mater (% decrease mean ± SEM, lowest effective concentration) (35 ± 6%, 30 µM), TG (24 ± 11 %, 10 µM) and TNC (40 ± 8%, 10 µM); (2) capsaicin- and RTX-induced iCGRP release from dura mater; and (3) capsaicin- and ES-induced increase in dural artery diameter (32 ± 5%, 3 mg kg⁻¹ intravenous (i.v.) and 36 ± 1%, 10 mg kg⁻¹ i.v.).

Conclusions

NXN-188 inhibits CGRP release from migraine-relevant cephalic tissues. Its effect is most likely mediated via a combination of nNOS-inhibition and 5-HT_1B/1D receptor agonism in dura mater while the mechanisms of action for inhibition of CGRP release from TG and TNC have to be investigated further.

Keywords

CGRP migraine triptans dura mater trigeminal ganglion trigeminal nucleus caudalis

Introduction

Migraine is a major public health problem which has a great impact on its patients as well as society. Although triptans are the main specific antimigraine agents, there is still a significant proportion of migraineurs who are non-responders to triptans (1), which necessitates the development of new therapeutics for migraine treatment. Nitric oxide (NO) and calcitonin gene-related peptide (CGRP) are key mediators in migraine (2 –4). Infusion of NO donors and CGRP to migraineurs induces migraine-like headaches (5,6). The effectiveness of a non-specific nitric oxide synthase NOS inhibitor against acute migraine attacks (7) and chronic tension-type headaches (8) underscores the role of NO in migraine. The CGRP receptor antagonists olcegepant (9) and telcagepant (10,11) are effective against acute migraine, demonstrating a key role of CGRP in migraine pathogenesis. NOS inhibitors can attenuate neurogenic and CGRP-induced dilation of dural meningeal vessels (12). Furthermore, NOS inhibitors reduce pain-related behaviours in multiple neuropathic pain models (13,14) and in chemically induced pain models (15 –17).

The 5-hydroxytryptamine 1B/1D (5-HT_1B/1D/1F) receptor agonist sumatriptan attenuates the release of CGRP after trigeminal nerve stimulation in cats and rats (18,19). It also inhibits the stimulated CGRP release from cultured trigeminal neurons (20). Sumatriptan and donitriptan inhibit CGRP release followed by the activation of trigeminal vascular system in a rat in vivo model (21). In guinea pig dura mater, KCl and capsaicin cause the release of CGRP, and this release can be attenuated by sumatriptan (22). Sumatriptan also inhibits KCl-induced CGRP release from dura mater of rat isolated skull halves, trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) (23).

NXN-188 is a combined neuronal NOS (nNOS) inhibitor and 5-HT_1B/1D receptor agonist developed by NeurAxon Inc. for the treatment of acute migraine (24). According to the preliminary report, it had a clinically meaningful treatment difference compared to a placebo-treated group in acute migraine attacks (1). In this study, using preclinical models, we evaluated whether these two unique therapeutic principles have a synergistic effect in attenuating stimulated CGRP release, a marker of trigeminal activation. We studied the effect of NXN-188 on stimulated CGRP release in the isolated preparation of rat dura mater, TG and TNC. We also investigated the effect of NXN-188 on capsaicin- and electrical stimulation (ES)-induced dural artery dilation in a rat closed-cranial window model.

Methods

Animals

All experimental procedures were performed in accordance with domestic guidelines and regulations for animal care and treatment. The study protocol was approved by the Danish Animal Experimentation Inspectorate (file: 2009/561-1664). Male Sprague-Dawley rats (260–350 g) were purchased from Taconic Europe (Tornbjergvej 40, Ejby, Denmark). They were housed in a standard light and dark cycle with free access to food and water.

Drugs

NXN-188, NXN-413 and sumatriptan were kind gifts from NeurAxon Inc., Toronto, Ontario, Canada. GR127935 and SB224289 were purchased from Tocris Bioscience, Bristol, United Kingdom. Methiothepin mesylate, capsaicin and resiniferatoxin (RTX) were obtained from Sigma Aldrich, Schnelldorf, Germany. The 10 mM stock solutions of sumatriptan, NXN-188, NXN-413, methiothepin and GR127935 were made in water and further diluted in synthetic interstitial fluid (SIF) (in mM: 108 NaCl, 3.48 KCl, 0.7 MgSO₄, 26 NaHCO₃, 1.37 NaH₂PO₄, 1.5 CaCl₂, 9.6 Na⁺ gluconate, 5.55 glucose and 7.6 sucrose). SB224289 10 mM stock solution was made in dimethyl sulfoxide (DMSO) and further diluted in DMSO. After adding these compounds in 300 µl SIF, pH was measured. The final pH remained stable at 7.4. Capsaicin and RTX were dissolved in 96% ethanol to obtain a 10 mM stock concentration and final dilutions were prepared in SIF.

In vitro immunoreactive CGRP (iCGRP) release studies

The rats were anaesthetized by CO₂ inhalation, decapitated, and the tissues were isolated as described earlier with minor modifications (25). First, the brain stem was isolated and the trigeminal nucleus measuring 6 mm in length, caudally from the obex, was isolated from both sides. Afterwards, the skull was cut mid-sagittally and the brain halves were carefully removed while the cranial dura was left attached to the skull. TGs were dissected 1 mm proximal and distal to the point where the mandibular nerve branches off. The dura attached to the TG was carefully removed. TGs and TNCs were immersed in 10 ml SIF at 37°C for 30 min. Skull halves were transferred to the beaker containing SIF and continuously washed for a minimum of 30 min with 500 ml SIF. The abdominal cavity was opened and testicles were lifted out to expose the vas deferens from both sides. Four equally matched segments of vas deferens were dissected from each side. Sperm were removed from inside the vas deferens by slightly pressing the segments with forceps. Vas deferens segments were washed twice with 10 ml of Mg⁺⁺ free Krebs buffer at the interval of 10 min in plastic disposable beakers.

Sampling and stimulation

After washing for 30 min in carbogen (95% O₂/5% CO₂)-bubbled SIF, the TGs and TNCs were placed in the detached caps of micro-centrifuge tubes in a heating block at 37°C. TGs and TNCs were again washed five times with 300 µl SIF for an interval of 5 min. Both skull halves were placed on clay covered with Vaseline® in a humid chamber above a water bath to maintain temperature at 37°C. The cavities covered by supratentorial dura mater were washed five times with 300 µl SIF. After 10 min incubation with 300 µl SIF, 200 µl samples for measuring the basal iCGRP release were collected from all the tissues, and the samples were mixed with an enzyme immunoassay (EIA) buffer containing protease inhibitors and were stored at –20°C until analysis. No significant difference was observed between the basal iCGRP release from the left and the right side of the tissues, thus, one side served as a control for the other in order to reduce the experimental and biological variations. The release of iCGRP from the skull halves, TG and TNC was induced by 60 mM, 100 mM and 40 mM potassium, respectively. These concentrations of KCl are based on concentration response curves with the respective tissues (23). To maintain equal osmolarity, a proportional amount of Na⁺ was removed from the buffer. Pilot experiments show that 10 min incubation is sufficient for a significant and reproducible release of iCGRP over basal levels.

NXN-188 (F.W. 409.37) is a selective agonist of 5-HT_1B/1D receptor (pKi = 7.02 and 7.49 at human 5-HT_1B and 5-HT_1D receptors, respectively, and is comparable to sumatriptan, pKi = 7.17 and 7.44 at human 5-HT_1B and 5-HT_1D receptors, respectively) (personal communication with NeurAxon Inc., 2007) and nNOS inhibitor (IC₅₀: human nNOS is 0.9 µM and rat nNOS is 2.4 µM). To study the effect of sumatriptan, NXN-188 and NXN-413 (F.W. 570.70), a selective nNOS inhibitor (IC₅₀ human: nNOS 0.1 µM, eNOS 52 µM and iNOS >100 µM) (personal communication with NeurAxon Inc., 2012), the tissues were pre-incubated for 10 minutes with different concentrations of these compounds, and during the final KCl challenge the same concentrations were maintained.

For reversing the effect of NXN-188, three 5-HT receptor antagonists, GR127935 (5-HT_1B/1D), SB224289 (5-HT_1B) and methiothepin (5-HT₁, 5-HT₆, and 5-HT₇), were used. Different concentrations of these antagonists (Table 1) were incubated for 10 min and then samples were collected to see the effect of these antagonists on basal iCGRP release. After that the same skull halves were again incubated with NXN-188 (30 µM) and different concentrations of these antagonists for 10 min. Finally, 60 mM KCl-SIF buffer was incubated for 10 min in the presence of NXN-188 and different concentrations of GR127935 or SB224289 or methiothepin. In control skull halves, only NXN-188 and vehicle of GR127935 (water) or SB224289 (DMSO, 1% final concentration) or methiothepin (water) were added to the tissue prior to the KCl challenge. The same protocol was followed for TGs and TNCs, except a final challenge was performed with 100 mM and 40 mM KCl-SIF buffer, respectively. GR127935 (1 µM) was used for reversing the effect of sumatriptan. The effect of the combination of sumatriptan (30 µM) and NXN-413 (30 µM) was also studied on KCl-stimulated iCGRP release in the skull half preparation.

Table 1.

Effect of 5-hydroxytryptamine 1B/1D (5-HT_1B/1D) antagonists GR127935 and SB224289, and 5-HT₁ antagonist methiothepin on basal immunoreactive calcitonin gene-related peptide (iCGRP) release from skull halves, trigeminal nucleus caudalis (TNCs) and trigeminal ganglions (TGs) preparations of rats. Values are given as percentage change mean ± SEM. The Wilcoxon matched paired test was used for nonparametric analysis. Number of experiments is shown in parentheses.

Drug	Tissue	Concentration (µM)	Percentage change in CGRP release Mean ± SEM (n)
GR127935	Dura	0.1	−16 ± 21 (4)
		1	2 ± 16 (6)
		30	35 ± 14 (4)
		100	37 ± 4^a (6)
	TNC	1	4 ± 21 (10)
	TNC	100	44 ± 11^a (6)
	TG	1	7 ± 20 (6)
	TG	100	59 ± 6^a (6)
SB224289	Dura	3	14 ± 7 (8)
	Dura	10	42 ± 7^b (8)
	TNC	3	4 ± 28 (5)
	TNC	10	33 ± 23 (6)
	TG	3	14 ± 13 (8)
	TG	10	33 ± 9^a (8)
Methiothepin	Dura	100	48 ± 9^a (6)
	TNC	100	59 ± 8^a (6)
	TG	100	62 ± 9^a (6)

p < 0.05 or ^bp < 0.01, compared to basal iCGRP from respective vehicle-treated control half.

Capsaicin 100 nM (26) and RTX 0.1 nM (27,28) were used to release iCGRP from the skull halves. Two concentrations of NXN-188, 30 µM and 100 µM, were used to inhibit the capsaicin- and RTX-induced iCGRP release in dura preparation. The incubation protocol was the same as in the KCl challenge, except after 10 min NXN-188 incubation samples were not collected and finally the capsaicin or RTX challenge was performed for 10 min in the same buffer.

After washing, vas deferens segments were placed in 200 µl carbogen-saturated Krebs buffer in the detached caps of micro-centrifuge tubes in a heating block at 37°C. These segments were again washed five times with 200 µl Krebs buffer with an interval of 5 min followed by 10 min incubation in Krebs buffer, and the samples (100 µl) for basal iCGRP release were collected. NXN-188 (100 µM) was pre-incubated for 10 minutes in tissue from one side, and during the final KCl challenge the same concentration was maintained. Only the KCl challenge was performed in the matched tissues from other side.

Enzyme immunoassay (EIA) for immunoreactive CGRP

The samples were processed using commercial EIA kits (SPIbio, Paris, France) to study iCGRP release. From all the tissues, 200 µl of sample was mixed with 50 µl of EIA buffer. While making the CGRP standard, we added 50 µl EIA buffer with different concentrations of CGRP standard. Slope was derived from the CGRP standard curve and was used for analysis of unknown samples. Pilot studies on the samples obtained from TNC showed the amount of iCGRP release was too high and not in the linear range of the standard curve. Therefore, for further analysis we diluted the samples from TNC six times before analysis, and final release was subsequently multiplied by six. The antibody in the iCGRP EIA kit is directed against human-CGRP α/β but it has 100% cross-reactivity with rat and mouse CGRP. The iCGRP detection level was about 2 pg ml⁻¹. The protocol provided with the kit was followed. In short, the samples were incubated in wells at 4°C for 16–20 h. After incubation, the wells were washed and incubated with Ellman's reagent, a colorimetric indicator. The wells were covered with an aluminium sheet and placed in a dark room for 60 min at room temperature. The optical density was measured at 410 nm using a micro-plate photometer (Tecan, Infinite M200, software SW Magellan v.6.3, Männedorf, Switzerland).

Intravital microscopy on a rat closed-cranial window model

Rats were anaesthetized with pentobarbital (Mebumal® 65 mg kg⁻¹ intraperitonial) and depth of anaesthesia was tested by suppression of the hind paw reflex. Anaesthesia was continuously supplemented with pentobarbital (Mebumal® 20 mg kg⁻¹ h⁻¹ intravenous (i.v.)) during the experiment. Body temperature was maintained at 37.0 ± 0.5°C throughout the experiments using an automatic regulated heating plate (Letica HB101, Panlab, Barcelona, Spain). After intubation, the animal was mechanically ventilated by a respirator (Abovent 7025; UGO Basil, Comerio, Italy) with a 30/70% mixture of O₂/N₂O, a stroke volume of 3.5–4.0 ml and a stroke rate of 55–65. Arterial blood samples were collected prior to, during, and at the end of each experiment for analysis of partial pressure of oxygen (PaO₂), carbon dioxide (PaCO₂) and pH (ABL520, Radiometer, Brønshøj, Denmark). All values were kept within normal limits (pH 7.35–7.45, mean arterial blood pressure (MABP) 81.7–127.5 mmHg and PaCO₂ 35.2–42.7 mmHg).The closed-cranial window model experiments were performed as described previously (25). All data, including the changes in diameter of the arteries and MABP, were continuously collected and simultaneously analysed by Perisoft (Version 2.0; Perimed AB, Järfälla, Sweden). As drilling induces dilatation of dural arteries, the rat was left to recover for 45 minutes before proceeding with the pharmacological experiments. After the experiments the animals were sacrificed by an i.v. overdose of 1 M KCl.

Experimental protocols

The middle meningeal artery (MMA) diameter was recorded immediately after drilling and after the recovery period. Before the start of any experiment the baseline diameter of the target artery was measured for at least 5 min to ensure stability. The dilatory effect on the target artery was tested with one single i.v. dose of CGRP 500 ng kg⁻¹ (NeoMPS, Strasbourg, France). To study the dilatory effect of capsaicin on the MMA, a 10 µg kg⁻¹ i.v. bolus dose was given. In order to check the reproducibility of the responses, the same dose of capsaicin was repeated at the interval of 5 min. The protocol used to study the effect of NXN-188, sumatriptan, NXN-413 and the combination of NXN-413 and sumatriptan on capsaicin-induced vasodilatation of MMA is shown in Figure 1A and 1B.

Figure 1.

Flowchart showing different protocols used for closed-cranial window model experiments: (A) Protocol to study the effect of NXN-188 on capsaicin-induced middle meningeal artery (MMA) diameter increase. (B) Protocol to study the effect of sumatriptan, NXN-413 and combination of both on capsaicin-induced MMA diameter increase. (C) Protocol to study the effect of NXN-188 and sumatriptan on electrical stimulation (ES)-induced MMA diameter increase. (D) Protocol to study the effect of GR127935 on NXN-188 and sumatriptan responses on ES-induced MMA diameter increase. The numbers above the black horizontal line represent the sequence of capsaicin or ES challenges. Time interval between two stimuli is shown below horizontal line. Solid arrow shows either capsaicin or ES stimuli; the dashed arrow shows intravenous (i.v.) infusion of different drugs.

ES parameters

To investigate whether neurogenically induced vasodilatation was inhibited by NXN-188, a bipolar stimulation electrode (NE-200x, Harvard Instruments, UK) was placed on the surface of the cranial window approximately 200 mm from the investigated arteries. Stimulations at 5 Hz, 1 ms pulse width and of 10 s duration were applied (Grass Stimulator S48, Grass Instruments, USA). Stimulations were done with increasing voltage until maximum dilation was observed. The same voltage was used for the subsequent ES in the same animal (29,30). In order to check the reproducibility of the responses, two consecutive control responses to electrical stimulation were recorded with an interval of 5–10 min. The protocol used to study the effect of NXN-188 and sumatriptan on ES-induced vasodilation of MMA is shown in Figure 1C. The protocol for examining the effects of GR127935 on responses of NXN-188 and sumatriptan is shown in Figure 1D. Response to the ES given immediately before the drug treatment was compared with the response elicited by ES immediately after the drug treatment, except in protocol 2 (Figure 1B), when the fourth ES was compared with the second ES.

Data analysis

The iCGRP release was calculated as percentage change as compared to the control, and presented as % mean ± SEM. A criterion value of p < 0.05 was considered statistically significant. GraphPrism (GraphPad Software Inc., San Diego, USA) was used for statistical analysis. Wilcoxon matched paired test was used for nonparametric analysis of iCGRP release studies. To obtain final iCGRP release from TNC (only stimulated samples), a dilution factor of six was multiplied by the measured iCGRP release. A Kruskal-Wallis test was performed between percentage inhibition by NXN-188, NXN-413 and sumatriptan. For the in vivo studies, the analysis of the test substances was performed as previously described (25). In brief, analysis was based on measurements of two parameters: the changes in the diameter of the artery and changes in MABP. The vessel diameter was measured in arbitrary units and MABP was measured in mmHg. Dilation of the vessels and changes in MABP were calculated as percentage changes from the baseline, which was defined as the peak increases in MMA diameter and peak drop in MABP, which occurred within 1 min of infusion. Responses to the first dose of capsaicin or ES and the responses elicited by capsaicin or ES immediately after the drug treatment were compared using a paired t test. The percentage inhibition was calculated as the percentage difference between the mean maximal inhibitions and mean capsaicin or ES responses.

Results

A total of 138 rats were used in this study. Out of 138 rats, 107 rats were used in the in vitro iCGRP release studies and 31 rats were used in the in vivo closed-cranial window model.

Effect of NXN-188 on iCGRP release from skull halves, TGs and TNCs

The mean basal values of iCGRP release from all skull halves TGs and TNCs were 12.0 ± 1.1 pg ml⁻¹ (n = 54), 35.1 ± 11.5 pg ml⁻¹ (n = 36) and 179.4 ± 17.5 pg ml⁻¹ (n = 34), respectively. No difference was observed in iCGRP release between the two skull halves and TGs and TNCs from the same rat. 60 mM, 100 mM and 40 mM KCl induced a significant increase in iCGRP release from skull halves, TGs and TNCs, respectively (Figure 2). NXN-188 concentrations ranging from 3 µM to 100 µM were used to investigate its effect on KCl-induced iCGRP release in all the tissue preparations. NXN-188 itself did not induce a significant change in baseline iCGRP release. In skull halves, NXN-188 significantly reduced KCl-induced iCGRP release at 30 µM and 100 µM (Figure 3). The 3 µM and 10 µM concentrations of NXN-188 did not have any significant effect on KCl-induced iCGRP release. NXN-188 concentrations beyond 100 µM are not in the pharmacological range and nonspecific responses to other 5-HT receptors, muscarinic and adrenergic receptors are found (personal communication with NeurAxon Inc.). The 100 nM capsaicin and 0.1 nM RTX challenge significantly increased iCGRP release from the skull halves (Figure 2). NXN-188 significantly reduced capsaicin-induced iCGRP release at 30 µM and 100 µM concentrations, and RTX-induced iCGRP release at 30 µM concentration (Figure 3). RTX-induced iCGRP release was not blocked significantly at 100 µM concentration of NXN-188; this may be due to a smaller number of animals used in this protocol (p = 0.06, n = 5). Since the effect of 100 µM concentration was not more than 30 µM concentration of NXN-188, the protocol was not extended to more rats. NXN-188 significantly reduced KCl-induced iCGRP release at 10, 30 and 100 µM concentrations in TG, and at 10 µM and 100 µM concentrations in TNC (Figure 4). We have conducted pilot experiments in three rats with capsaicin-induced iCGRP release in TGs and TNCs. As we did not find an inhibitory effect of NXN-188 on capsaicin-induced CGRP release in TG and TNC (data not shown), we decided not to do experiments with RTX in TGs and TNCs.

Figure 2.

Representative figure showing basal and stimulated immunoreactive calcitonin gene-related peptide (iCGRP) release from skull halves (dura mater), trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC). Values are given as mean ± SEM. The Wilcoxon matched paired test was used for nonparametric analysis; ***p < 0.001, compared with respective basal iCGRP release (n = 12). ^###p < 0.001, compared with basal iCGRP, this group is not paired with basal iCGRP release. Mann Whitney test was used for nonparametric analysis (n = 11).

Figure 3.

Percentage decrease in 60 mM KCl-, 100 nM capsaicin- and 0.1 nM resiniferatoxin (RTX)-induced immunoreactive calcitonin gene-related peptide (iCGRP) release by increasing concentration of NXN-188 in hemi-sected skull preparation of rat. Values are given as percentage decrease in iCGRP release mean ± SEM. The Wilcoxon matched paired test was used for nonparametric analysis; *p < 0.05 as compared with KCl- or capsaicin- or RTX induced iCGRP release (n = 5–6).

Figure 4.

Percentage decrease in KCl-induced immunoreactive calcitonin gene-related peptide (iCGRP) release by increasing concentrations of NXN-188 in trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) preparations of rat. Values are given as percentage decrease in iCGRP release mean ± SEM. The Wilcoxon matched paired test was used for nonparametric analysis; *p < 0.05 as compared with KCl-induced iCGRP release (n = 6; except four animals were used for NXN-188 3 µM in TNC).

Effect of different 5-HT₁ receptor antagonists on NXN-188-mediated inhibition of KCl-stimulated iCGRP release from skull halves, TGs and TNCs

The lower concentration of GR127935 (0.1 µM and 1 µM), a 5-HT_1B/1D receptor antagonist, did not have any effect on basal iCGRP release but high concentration of GR127935 significantly increased iCGRP release from skull halves, TGs and TNCs (Table 1). The effect of NXN-188 (30 µM) on KCl-stimulated iCGRP release was not reversed by GR127935 (0.1 and 1 µM). On the contrary, GR127935 (1 µM) seemed to potentiate the effect of NXN-188 in the three tissues (Table 2). However, more experiments should be conducted for significance. In the TNC preparation, NXN-188 gave similar inhibition at 10 µM and 100 µM concentrations, thus we assumed NXN-188 produced similar decrease in iCGRP release at 30 µM concentration.

Table 2.

The effect of 5-hydroxytryptamine 1B/1D (5-HT _1B/1D) antagonists, GR127935 and SB224289 on NXN-188-induced inhibition of KCl-stimulated immunoreactive calcitonin gene-related peptide (iCGRP) release in the skull halves, trigeminal ganglions (TGs) and trigeminal nucleus caudalis (TNCs) preparations of rats. Values are given as CGRP release pg ml^–1 and percentage change mean ± SEM. The Wilcoxon matched paired test was used for nonparametric analysis. Number of experiments is shown in parentheses.

CGRP release (pg ml⁻¹)			Percentage change in CGRP release
Dura mater
KCl (60 mM) + NXN-188 (30 µM)	KCl (60 mM) + NXN-188 (30 µM) + GR127935 (1 µM)	KCl (60 mM) + NXN-188 (30 µM) + SB224289 (3 µM)	GR127935	SB224289
158 ± 2 (4)	133 ± 9 (4)		−21 ± 7 (4)
154 ± 12 (8)		176 ± 19 (8)		8 ± 8 (8)
TG
CGRP release (pg ml⁻¹)			Percentage change in CGRP release
KCl (100 mM) + NXN-188 (30 µM)	KCl (100 mM) + NXN-188 (30 µM) + GR127935 (1 µM)	KCl (100 mM) + NXN-188 (30 µM) + SB224289 (3 µM)	GR127935	SB224289
254 ± 97 (4)	184 ± 36 (4)		−43 ± 49 (4)
153 ± 12 (8)		176 ± 19 (8)		10 ± 8 (8)
TNC
CGRP release (pg ml⁻¹)			Percentage change in CGRP release
KCl (40 mM) + NXN-188 (30 µM)	KCl (40 mM) + NXN-188 (30 µM) + GR127935 (1 µM)	KCl (40 mM) + NXN-188 (30 µM) + SB224289 (3 µM)	GR127935	SB224289
669 ± 146 (4)	488 ± 67 (4)		−34 ± 17 (4)
882 ± 142 (7)		999 ± 180 (7)		−4 ± 32 (7)

The 3 µM concentration of SB224289, a 5-HT_1B receptor antagonist, did not have any effect on basal iCGRP release. The effect of NXN-188 (30 µM) was not reversed by 3 µM concentration of SB224289 (Table 2). SB224289 at 10 µM concentration significantly increased basal iCGRP release from skull halves and TGs (Table 1). Methiothepin, a 5-HT₁, 5-HT₆, and 5-HT₇ receptor antagonist, showed significant increase in basal iCGRP release by ∼50% from all three tissues at 100 µM concentration (Table 1). The high concentrations of these three antagonists with an effect on basal iCGRP release were not used to reverse the effect of NXN-188.

Effect of NXN-188 on KCl-stimulated iCGRP release from vas deferens segments

The 60 mM KCl challenge induced a significant (p = 0.008) increase in iCGRP release (13 ± 2 pg ml⁻¹) as compared to the basal iCGRP release (2 ± 0.5 pg ml⁻¹) in vas deferens segments (n = 8 segments, n = 2 rats). NXN-188 itself did not induce a significant change in baseline iCGRP release. NXN-188 did not change 60 mM KCl-induced iCGRP release from vas deferens segments (−17 ± 21 %, p = 0.42, 100 µM).

Effect of NXN-413 and sumatriptan on KCl-stimulated iCGRP release from skull halves

NXN-413 and sumatriptan also inhibited the stimulated iCGRP release from skull halves individually. The combination of NXN-413 and sumatriptan inhibited simulated iCGRP release more than that achieved by each compound alone. NXN-188, NXN-413 and sumatriptan inhibited KCl-induced iCGRP release to the same extent if measured in percentage change. The effect of sumatriptan was reversed by GR127935 (Table 3). NXN-413 did not change the amount of KCl-induced iCGRP release from TG (−30 ± 26 %, p = 0.56, 30 µM) and TNC (−28 ± 25 %, p = 0.56, 30 µM).

Table 3.

Effect of NXN-413, sumatriptan, and combination of these compounds on 60 mM KCl-induced calcitonin gene-related peptide (CGRP) release from skull halves of rat. Combination of NXN-413 and sumatriptan inhibited simulated iCGRP release more than achieved by each compound itself. 5-hydroxytryptamine 1B/1D (5-HT _1B/1D) antagonist GR127935 reversed the effect of sumatriptan. Values are given as CGRP release pg ml⁻¹ and percentage change mean ± SEM. The Wilcoxon matched paired test was used for nonparametric analysis. Number of experiments is shown in parentheses.

CGRP release (pg ml⁻¹)						Percentage change in CGRP release
KCl (60 mM)	KCl (60 mM) + sumatriptan (30 µM)	KCl (60 mM) + sumatriptan (30 µM) + GR127935 (1 µM)	KCl (60 mM) + NXN-413 (30 µM)	KCl (60 mM) + NXN-413 (30 µM) + sumatriptan (30 µM)	KCl (60 mM) + NXN-188 (30 µM)	Percentage change in CGRP release
202 ± 39 (6)	159 ± 31^a (6)					20 ± 5 (6)
	84 ± 10 (6)	130 ± 31^a (6)				26 ± 9 (6)
157 ± 26 (6)			117 ± 10^a (6)			25 ± 3 (6)
	109 ± 22 (6)			75 ± 11^a (6)		28 ± 6 (6)
86 ± 15 (6)					56 ± 11^a (6)	35 ± 6 (6)

^ap < 0.05, compared to immunoreactive CGRP from respective control half.

Effects of NXN-188, NXN-413, sumatriptan, and the combination of NXN-413 and sumatriptan on capsaicin-induced dilation of rat MMA

Two consecutive capsaicin challenges produced a reproducible dilation of MMA (Figure 5A). The effect of NXN-188, NXN-413, sumatriptan, combination of NXN-413 and sumatriptan, and GR-127935 on MABP and MMA diameter is tabulated in Tables 4 and 5, respectively. NXN-188 significantly blocked capsaicin-induced dilation by 32 ± 5% and 47 ± 8% at 3 mg kg⁻¹ and 10 mg kg⁻¹ doses, respectively (Figure 5B). Sumatriptan, NXN-413, and the combination of the two drugs significantly blocked capsaicin-induced dilation by 22 ± 8%, 16 ± 7% and 37 ± 7% at the 10 mg kg⁻¹ dose, respectively (Figure 5C). No significant difference was observed between different treatments.

Figure 5.

(A) Reproducible increase in dural vessel diameter produced by either capsaicin (10 mg kg⁻¹ intravenous (i.v.) bolus) injection or electrical stimulation (ES) (5 Hz, 1 ms pulse width and of 10 s duration) of the surface of a cranial window in the anaesthetized rat. (B) Effect of NXN-188 on capsaicin-induced dilation of rat dural artery in vivo. Values are given as percentage increase in middle meningeal artery (MMA) diameter from pre-stimulation baseline mean ± SEM. **p < 0.01 (paired t test in between second and third or fourth and fifth capsaicin challenge (n = 6). (C) Effect of NXN-413, sumatriptan and combination of both on capsaicin-induced dilation of rat dural artery in vivo. Values are given as percentage increase in MMA diameter from pre-stimulation baseline mean ± SEM. **p < 0.01 or *p < 0.05 (paired t test in between second and third or fourth and fifth or sixth and seventh capsaicin challenge (n = 6–8).

Table 4.

Effects of various agents on mean arterial blood pressure (MABP). In cases of more than two, a group Kruskal-Wallis test followed by Dunnet’s post hoc test was used, and in cases of only two groups, a Wilcoxon matched paired test was used. Number of experiments is shown in parentheses. Not done (—).

Dose (mg kg⁻¹)	NXN-188	NXN-413	Sumatriptan	Combination of NXN-413 + sumatriptan	GR127935
Saline	0.7 ± 1.0 (15)	0.2 ± 0.3 (8)	0.5 ± 0.4 (14)	1.8 ± 0.4 (7)	−1.2 ± 0.7 (6)
0.1	—	—	—	—	−1.4 ± 1.3 (6)
1	−8.5 ± 1.0 (6)	—	—	—	—
3	−18.8 ± 3.4^a (12)	—	—	—	—
10	−39.5 ± 4.4^a (15)	−37.6 ± 5.0^a (8)	−27.5 ± 3.1^a (14)	−14.4 ± 3.0^a (7)	—

p < 0.05, significantly different from vehicle-induced MABP.

Table 5.

Effects of various agents on rat middle meningeal artery (MMA) diameter. In cases of more than two groups, Kruskal–Wallis test followed by Dunnet’s post hoc test was used, and in cases of only two groups, Wilcoxon matched paired test was used. Number of experiments is shown in parentheses. Not done (—).

Dose (mg kg⁻¹)	NXN-188	NXN-413	Sumatriptan	Combination of NXN-413 + sumatriptan	GR127935
Saline	−1.2 ± 1.4 (15)	0.1 ± 2.4 (8)	0.0 ± 1.3 (14)	−0.7 ± 4.9 (7)	−0.7 ± 0.2 (6)
0.1	—	—	—	—	−0.5 ± 7.0 (6)
1	−11.0 ± 5.8 (6)	—	—	—	—
3	−8.8 ± 4.6 (12)	—	—	—	—
10	−10.0 ± 6.4 (15)	59.6 ± 18.0^a (8)	35.5 ± 7.2^a (14)	25.4 ± 17.8 (7)	—

p < 0.05, significantly different from saline-induced change in MMA diameter.

Effects of NXN-188 and sumatriptan on ES-induced dilation of rat MMA

Two consecutive ES challenges produced a reproducible dilation of MMA (Figure 5A). NXN-188 and sumatriptan blocked ES-induced dilation by 36 ± 10% and 39 ± 6% at the 10 mg kg⁻¹ dose (Figure 6A). Lower doses of NXN-188 did not block ES-induced dilation significantly: 7 ± 8% and 13 ± 6% at 1 mg kg⁻¹ and 3 mg kg⁻¹ dose, respectively (Figure 6A).

Figure 6.

(A) Effect of NXN-188 and sumatriptan on electrical stimulus (ES)-induced dilation of rat dural artery in vivo. Values are given as percentage increase in middle meningeal artery (MMA) diameter from pre-stimulation baseline mean ± SEM. **p < 0.01 (paired t test in between second and third or second and fourth or fifth and sixth or seventh and eighth ES challenge (n = 6–9). (B) Effect of pretreatment of GR127935 on NXN-188 responses on ES-induced dilation of rat dural artery. Values are given as percentage increase in MMA diameter from pre-stimulation baseline mean ± SEM. *p < 0.05 (paired t test in between second and third or fourth and fifth electrical stimulation (n = 6).

Effect of GR127935 on responses of NXN-188 and sumatriptan on ES-induced dilation of rat MMA

GR127935 did not reverse the effect of NXN-188 on ES-induced dilation of rat MMA (Figure 5B). Pre-treatment with GR127935 (100 µg kg⁻¹) significantly reversed the effect of sumatriptan on ES-induced dilation of rat MMA: 14 ± 14% changes in the presence of GR127935 compared to 39 ± 6% sumatriptan alone.

Discussion

Our results demonstrate that NXN-188, a putative anti-migraine drug, inhibits KCl-, capsaicin- and RTX-induced iCGRP release in migraine-relevant cephalic tissues. NXN-188 also blocked capsaicin- and ES-induced increase in MMA diameter. The combination of NXN-413 (a selective nNOS inhibitor) and sumatriptan (a 5-HT_1B/1D agonist) inhibited simulated iCGRP release from skull halves and inhibited capsaicin-induced increase in MMA diameter more than that achieved by each compound alone. The 5-HT_1B/1D receptor antagonist GR127935 reversed the effect of sumatriptan but did not reverse the effect of NXN-188. Thus, the effect of NXN-188 was partially dependent on its nNOS inhibitory property and was independent of its 5-HT_1B/1D receptor agonistic property.

Effect of NXN-188 on KCl-stimulated iCGRP release

NXN-188 decreased KCl-induced iCGRP release from skull halves, TGs and TNCs at concentrations ranging from 10 µM to 100 µM. These concentrations are in line with the concentration of sumatriptan (10 µM to 50 µM) used to inhibit stimulated CGRP release from cultured TG neurons from neonatal rats (20), dura mater of guinea pig isolated skull halves (22) and dura mater of rat isolated skull halves (23). Furthermore, zolmitriptan (10 and 100 µM), a 5-HT_1B/1D receptor agonist, inhibited voltage-activated Ca²⁺ channels of acutely dissociated rat trigeminal sensory neurons (31). Also 5-HT (10 µM) was shown to significantly inhibit potassium-stimulated release of both glutamate and CGRP from primary TG neuron cultures of rats (32). In the human phase І clinical trial, NXN-188 doses ranging from 2 to 800 mg (0.027–11.2 mg kg⁻¹, oral dose) were used. The maximum plasma concentration (C_max) achieved by 800 mg dose was 661.2 ng/ml or 1.6 µM (24), which is similar to the lower concentrations used in the present study.

The effect of NXN-188 was not reversed by the lower concentrations of GR127935 and SB224289. High concentrations of all these antagonists by themselves significantly increased iCGRP release. These antagonists might be reversing the effect of endogenous 5-HT, thus contributing to the increased iCGRP in SIF at basal level. GR127935 exhibits a very high affinity and selectivity for 5-HT_1B/1D binding sites (pKi = 8.5, 8.9 and 9.9 at rat 5-HT_1B, human 5-HT_1Dα and human 5-HT_1Dβ receptors, respectively), and it antagonizes a number of 5-HT_1B/1D receptor-mediated responses (33,34). SB224289 is a selective 5-HT_1B receptor antagonist (pKi = 8.2 and 6.3 at human 5-HT_1B and 5-HT_1D receptors, respectively), and it antagonizes 5-HT_1B receptor-mediated responses (35,36). It has previously been reported that sumatriptan-induced inhibition of stimulated iCGRP release can be reversed by methiothepin at a 100 µM concentration both in the primary culture of TG (20) and in the guinea pig skull (22). In these studies methiothepin did not induce iCGRP release, but in our experiments methiothepin itself induced significant iCGRP release from all the tissues. Species differences and culture conditions could explain such contrasting observations. Confirming previous findings from our lab (23), sumatriptan inhibited the stimulated iCGRP release in the skull halves and this inhibition was reversed by GR127935. The results from these experiments suggest that the effect of NXN-188 on stimulated iCGRP release is independent of its 5-HT_1B/1D receptor agonistic property.

nNOS immunoreactivity has been found in thin nerve fibre ramifications running along branches of the MMA (4). Very few NOS positive neurons are present in TG and co-localization with iCGRP is rare (37). An increase in NOS-immunoreactive nerve fibres in the rat dura mater has been reported 15–60 minutes after intraperitoneal injection of nitroglycerin (38). After four to six hours of nitroglycerin infusion, an increase of iCGRP and also nNOS-immunoreactive neurons in the rat TG was reported (37). NXN-413, a specific and potent nNOS inhibitor, caused a significant decrease in KCl-induced iCGRP release from the skull halves. The combination of NXN-413 and sumatriptan inhibited iCGRP release more than that achieved by each compound alone. We can speculate that in dura mater the nNOS inhibition and some unknown mechanism of action are responsible for the inhibitory action of NXN-188 on KCl-induced stimulated iCGRP release. NXN-413 did not inhibit KCl-induced iCGRP-release from TGs and TNCs. Thus, NXN-188 seems to inhibit iCGRP release in TGs and TNCs via different modes of action, which needs further investigation. We have used vas deferens to verify the effects of NXN-188. NXN-188 did not decrease stimulated iCGRP in segments of vas deferens, suggesting tissue specificity, excluding the possibility of some ion channel inhibition by NXN-188.

Effect of NXN-188 on vanilloid (TRPV1) receptor agonist-stimulated iCGRP release

Capsaicin activates TRPV1 in primary afferent C and Aδ-fibres and releases sensory neuropeptides, such as SP, neurokinin A and CGRP (39,40). NXN-188 inhibited capsaicin- and RTX-induced iCGRP release from dura. RTX is a highly potent and selective agonist of TRPV-1 receptor (41). Although capsaicin in different experimental systems can activate non-receptor-mediated responses (42), this is unlikely to explain our results, since RTX-induced release was also inhibited by NXN-188. In the rat, both peptidergic and nonpeptidergic nociceptors express TRPV1 (43). Recently, it has been reported that 5HT_1B, 5HT_1D, 5HT_2A, and 5HT_3A receptor mRNA are co-expressed in a subpopulation of TRPV1-positive TG cells (44).

Effects of NXN-188, NXN-413, sumatriptan, and combination of NXN-413 and sumatriptan on capsaicin-induced dilation of rat MMA

Capsaicin has been widely used in animal models to induce cranial vasodilation (45,46). NXN-188 itself did not induce any change in MMA diameter. Capsaicin-induced increase in dural artery diameter was blocked by NXN-188 (47 ± 8%), sumatriptan (22 ± 8%), NXN-413 (16 ± 7%) and the combination of NXN-413 and sumatriptan (37 ± 7%). It is clear that the combination of sumatriptan and NXN-413 has a stronger effect than each compound alone, and these finding are supported by in vitro iCGRP-release data. In dogs, capsaicin-induced external carotid vasodilation was blocked only by intrathecally injected sumatriptan, which is mainly mediated by 5-HT_1B receptors (47).

Effects of NXN-188 and sumatriptan on ES-induced dilation of rat MMA

Sumatriptan and NXN-188 decreased the ES-induced increase in MMA diameter at the 10 mg kg⁻¹ i.v. dose. Sumatriptan itself causes a significant decrease in blood pressure and an increase in MMA diameter. Both these observations about sumatriptan were reported previously (29). Pre-treatment with GR127935 significantly reversed the effect of sumatriptan on ES-induced dilation of rat MMA. However, GR127935 did not reverse the effect of NXN-188 on ES-induced dilation of rat MMA. The dose of GR127935 (100 µg kg⁻¹) was previously used to block the 5-HT_1B/1D receptors in the cardiovascular system of the rat (48,49). Recently, it has been reported that the supramaximal dose of GR127935 (310 µg kg⁻¹) abolished the effect of sumatriptan in the pithed rats, claiming 5-HT_1F, but not 5-HT_1A or 5-HT_1D, receptor subtypes inhibit the vasodepressor sensory CGRPergic outflow (50).

In a phase II clinical study, NXN-188 was well tolerated with no serious adverse events and no triptan-like adverse events reported. The primary endpoint in this study was pain relief at two hours; although NXN-188 did not reach significance at this time point (p = 0.0801), a statistically significant response was reported from four through 24 hours (51). In conclusion, NXN-188 inhibited iCGRP release in our preclinical models of migraine but the blockade was not reversed by 5-HT_1B/1D receptor antagonist. This data taken together with the inhibitory effect of NXN-413 on iCGRP release in dura mater suggest that the effect of NXN-188 is mediated via a combination of nNOS-inhibition and 5-HT_1B/1D receptor agonism. The mechanism of action of NXN-188 in TG and TNC is unclear, suggesting involvement of some novel mechanism of action, which may be attractive in relation to migraine prophylaxis.

Footnotes

Clinical implications

NXN-188 inhibited iCGRP release in preclinical models of migraine.

Its effect is most likely mediated via a combination of nNOS inhibition and 5-HT1B/1D receptor agonism in dura mater while the mechanisms of action for inhibition of CGRP release from TG and TNC need to be investigated further.

This novel mechanism may be attractive in relation to migraine prophylaxis.

Conflict of interest

J.S.A. is president and CSO of NeurAxon Inc. J.O. is a member of the scientific advisory board of NeurAxon Inc. D.K.B., S.G. and I.J.-O. have nothing to declare. This study is not funded by NeurAxon Inc.

Acknowledgments

This work was supported by an international mobility grant (Faculty of Health Sciences, University of Copenhagen), Candy’s Foundation, Lundbeck Foundation and the A.P. Møller Foundation for the Advancement of Medical Science.

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NXN-188,a selective nNOS inhibitor and a 5-HT 1B/1D receptor agonist,inhibits CGRP release in preclinical migraine models

Abstract

Background

Methods

Results

Conclusions

Keywords

Introduction

Methods

Animals

Drugs

In vitro immunoreactive CGRP (iCGRP) release studies

Sampling and stimulation

Enzyme immunoassay (EIA) for immunoreactive CGRP

Intravital microscopy on a rat closed-cranial window model

Experimental protocols

ES parameters

Data analysis

Results

Effect of NXN-188 on iCGRP release from skull halves, TGs and TNCs

Effect of different 5-HT1 receptor antagonists on NXN-188-mediated inhibition of KCl-stimulated iCGRP release from skull halves, TGs and TNCs

Effect of NXN-188 on KCl-stimulated iCGRP release from vas deferens segments

Effect of NXN-413 and sumatriptan on KCl-stimulated iCGRP release from skull halves

Effects of NXN-188, NXN-413, sumatriptan, and the combination of NXN-413 and sumatriptan on capsaicin-induced dilation of rat MMA

Effects of NXN-188 and sumatriptan on ES-induced dilation of rat MMA

Effect of GR127935 on responses of NXN-188 and sumatriptan on ES-induced dilation of rat MMA

Discussion

Effect of NXN-188 on KCl-stimulated iCGRP release

Effect of NXN-188 on vanilloid (TRPV1) receptor agonist-stimulated iCGRP release

Effects of NXN-188, NXN-413, sumatriptan, and combination of NXN-413 and sumatriptan on capsaicin-induced dilation of rat MMA

Effects of NXN-188 and sumatriptan on ES-induced dilation of rat MMA

Footnotes

Conflict of interest

Acknowledgments

References

Effect of different 5-HT₁ receptor antagonists on NXN-188-mediated inhibition of KCl-stimulated iCGRP release from skull halves, TGs and TNCs