**4. New Therapeutic Strategies in Experimental Models**

The first therapeutic approach to migraine is generally symptomatic and aimed to alleviate acute pain, and medications are more effective whether quickly administered. Notwithstanding, there are advances in the development of anti-migraine drugs, and there is a growing need in researching novel therapeutic approaches aimed to treat more effectively migraine compared to actual treatments. In this context, we therefore consider studies on animal models developed with this perspective in this study. The most relevant findings of new compounds and nanoparticles in experimental models are summarized in Table 4.

In a recent study, Moye et al. [92] studied the efficacy of SNC80, a δ opioid receptor (DOR) agonist, in mouse models that replicated different headache disorders. In these models, mice were managed in order to induce CM, post-traumatic headache (PTH), MOH, and opioid-induced hyperalgesia (OIH) [92]. In CM model, mice received NTG by the intraperitoneally intermittent administration. In PTH, mice received isoflurane to be mildly anesthetized and then underwent the closed head weight-drop method in order to induce mild traumatic brain injury, and two weeks after PTH was modelled by low NTG dose intraperitoneally. To model MOH and OIH, animals received intraperitoneally treatment using respectively sumatriptan or morphine. In CM model, animals treated with NTG showed basal peripheral and cephalic hypersensitivity. To evaluate the effect of the activation of DOR, an acute

treatment of SNC80 was performed 24 h after the last injection of NTG. This treatment showed a relevant attenuation of peripheral and cephalic allodynia compared to controls, indicating that pain associated with CM was blocked by DOR activation. In PTH model, basal peripheral and cephalic hypersensitivity were developed in mice treated with NTG compared to controls. Twenty-four hours after the last NTG injection, cephalic allodynia was inhibited by performing an acute SNC80 treatment, indicating that also in this case, the pain associated with PTH was attenuated by DOR activation. In MOH model, basal hind paw and cephalic hypersensitivity were developed in mice treated with chronic administration of sumatriptan. Twenty-four hours after the final injection of medication, mice received an acute treatment with SNC80 that resulted in allodynia attenuation, suggesting that MOH induced by overuse of sumatriptan can be inhibited by DOR activation. In OIH model, mice received chronic treatment with morphine, showing basal hind paw and cephalic hypersensitivity, an effect that was also observed 18–24 h after the last drug injection. After, SNC80 was administered resulting in allodynia effect attenuation induced by morphine treatment. Furthermore, it has been observed that chronic daily administration of SNC80 causes a limited form of MOH, less severe in comparison with mice treated with sumatriptan. These results suggest that DOR agonists might represent a novel therapeutic approach in the treatment in diverse headache disorders showing a different etiology [92].

Pradhan et al., have investigated the therapeutic potential of δ-opioid receptor agonists in mouse migraine models induced with acute and chronic doses of NTG [93]. Animals were treated with three different δ-opioid receptor agonists, SNC80, ARM390 or JNJ20788560, about 1 h 30 min following NTG injection. These receptor agonists were able to significantly reduce NTG-evoked hyperalgesia. In addition, a model of migraine aura was induced by continuous application of KCl in order to evaluate the effects of SNC80 on evoked CSD. SNC80 in the 1 h time interval following administration, was able to reduce the number of CSD events. These data showed the therapeutic ability of the δ-opioid receptor as a promising therapeutic target for migraine [93].

Hoelig et al., studied the effectiveness of NOX-L41, a CGRP-neutralizing mirror-image (L-) aptamer (termed Spiegelmer), in a rat model of electrically evoked meningeal plasma protein extravasation (PPE) [94]. In this study, the authors have tested a Spiegelmer, a molecule synthesized chemically consisting of mirror-image oligonucleotide, which is able to bind to a pharmacologically relevant target molecule. Animals received NOX-L41 as a single dose intravenously or subcutaneously, showing a plasma half-life of 8 h. Furthermore, by means pharmacodynamic studies, after a single administration an extravasation of NOX-L41 from blood vessels in the dura mater and inhibition of neurogenic meningeal PPE for at least 18 h was observed. The Spiegelmer action consisted in binding CGRP, a neuropeptide that promotes meningeal vasodilation, inhibiting CGRP in signaling at its receptor. NOX-L41 showed increased affinity and selectivity for both isoforms, α and β, of human CGRP than a previous studied NOX-C89. The capacity of NOX-L41 to extravasate in surrounding tissues of dural circulation, in order to interact with perivascular vasodilating agents, results in the need to develop compounds able to antagonize CGRP, preventing neurogenic inflammation. This study suggests further the involvement of CGRP in neurogenic PPE, indicating NOX-L41 as a future potential compound for the treatment or prevention of migraine [94].

In recent times, the development of drug delivery systems using nanoparticles has received an increased attention from the scientific community. In this context, Girotra et al. [95–99] conducted studies on nanoparticulates formulations with the aim to increase the brain targeting of pharmacological anti-migraine drugs. Here, we consider some of their investigations.

A dual therapeutic approach was performed on rats and mice in the development of brain-targeted rizatriptan benzoate-loaded solid lipid nanoparticles (RB-SLNs), with the purpose to improve the drug potentiality in counteracting migraine [95]. The formulations have been elaborated by fabricating optimized solutions to obtain particle sizes showing sufficiently high entrapment efficiency and drug release in about 8 h. To evaluate the typical symptoms related to migraine, acetic acid-induced writhing test and light/dark box model were respectively used to induce hyperalgesia and light aversive behavior. Animals that received optimized RB-SLNs by oral administration showed a decrease in migraine-related hallmarks. After 2 h of oral drug treatment, pharmacodynamic evaluations showed that in rats, the brain uptake potential of optimized RB-SLNs was about 18.43-folds greater compared to the free form of the pure drug, whereas in mice showed to cross the BBB resulting in an improvement of its anti-migraine effectiveness. These findings indicate that RB-SLNs showed an improvement in brain target ability, thus providing a potential approach for migraine management [95].

In another study, an innovative approach has been conceived by developing chitosan solid lipid nanoparticles (SLN) that contained sumatriptan succinate (SS) [96]. The SLN formulations optimized in brain targeting. The optimization of the formulations was accomplished by multi-level design factorial in order to obtain a minimize size particles with a high entrapment efficiency and drug concentrations. Rats received the formulations, previously dispersed in deionized water, by oral administration. Behavioral studies indicating a reduction in hyperalgesia in the acetic acid induced writhing test and reduced aversion to light in light/dark box model. The treatment with formulations showed a major availability of SS in the brain in comparison with controls. These results suggest that formulations orally administered consisting in hydrophilic drug SS, loaded in chitosan SLN, were able to cross the BBB, allowing the drug in exerting its pharmacological activity in the brain. Considering their data, nanoparticulate drug delivery systems might represent a future approach to cross the BBB and to improve brain targeting of medications in migraine therapeutics [96].

A further study has been conducted on another formulation for brain targeting of SS, with the aim to evaluate the optimal therapeutic effect of the drug in migraine. For this purpose, nanoparticulate drug delivery system using poly (butyl cyanoacrylate) (PBCA) and bovine serum albumin linked with apolipoprotein E3 (BSA-ApoE) was used [97]. SS was incorporated in the BSA-ApoE NPs and compared with the same drug loaded polysorbate 80 coated optimized PBCA NPs to determine the brain uptake potential of these formulations. The central composite design was used for the formulation of PBCA NPs optimized with minimum particle size, maximum entrapment efficiency along with the sustained drug release. Also, in this study, animals were treated and assessed as described in the previous investigations, and behavioral studies showed similar improvements. The treatments with the nanoformulations prepared in this study showed a high brain/plasma drug ratio 2 h after the oral drug administration. The data obtained by the authors suggest that BSA-ApoE NPs showed a better activity than polysorbate 80 coated PBCA NPs for brain targeting of SS. This technique might offer a perspective, as improved therapeutic approach for the treatment of migraine [97].

In another study concerning the use of nanoparticles, Poly (D,L Lactide-co-Glycolide) (PLGA)/ poloxamer nanoparticles (NPs) of the hydrophilic medication zolmitriptan were developed [98]. Randomized factorial design to obtain the critical quality characteristics of minimized particle size and maximized encapsulation efficiency was applied. To determine the brain uptake potential, rats received optimized zolmitriptan encapsulated PLGA NPs as oral administration, and at the different time points, plasma and brain samples were collected. Acetic acid induced writhing test and light/dark box model were respectively used to induce hyperalgesia and aversion to light in mice. After the treatment, the in vivo studies for determining the brain uptake potential showed a 14.13-fold increase in the drug delivered to the brain from the NPs as compared to the free drug. Behavioral tests showed a decrease in the number of writhings, and a significant reduction of light aversion compared to controls. These data suggest that PLGA NPs containing zolmitriptan may represent a future tool in providing systems to cross the BBB for drug delivery that can exert an enhanced anti-migraine effect [98].

Another investigation has been performed using pharmacophore modeling [99]. This technique led to the identification of nystatin as compound active against the receptors iGluR5 kainate receptor (1VSO), CGRP (3N7R), β<sup>2</sup> adrenoceptor (3NYA) and Dopamine D3 (3PBL). Following this result, brain targeted chitosan nanoparticles containing nystatin were prepared, later intraperitoneally administered in rats in order to evaluate brain targeting efficacy. Confocal laser scanning microscopy showed a higher nanoparticles accumulation in the brain than liver and spleen. After treatment with nystatin nanoformulations, behavioral tests performed in mice showed a reduction in hyperalgesia, photophobia and phonophobia compared to controls. This study represents the first approach of the

therapeutic potential of nystatin nanoformulations, suggesting their future application in the treatment of migraine [99].

Wang et al., developed a novel ester derivative of gastrodin (Gas), termed Gas-D, and studied its effectiveness in a model of NTG induced migraine in rats [100]. Gas is a compound obtained by *Gastrodiae Rhizoma* (known in China, also as Tianma), and it has already been used for the treatment of migraine. Rats received Gas-D intragastrically, and subsequently treated by NTG subcutaneously 1 h after the final treatment. The pretreatment with Gas-D showed a reduction in head-scratching behavior, previously induced by NTG treatment. Results obtained from this study require further pharmacokinetic and pharmacodynamic investigations, suggesting that Gas-D, thanks to its anti-migraine effect, might be considered as a future candidate for migraine treatment [100].

Zhao et al., performed a comparative study evaluating two traditional Chinese drugs, gastrodin and ligustrazine, in a rat model of nociceptive durovascular trigeminal activation [101]. In this study, Chinese medications were compared to two Western approaches with propranolol and levetiracetam. Animals underwent surgical procedures for drug administration by femoral vein cannulation. An electrode was applied onto the dura mater above the middle meningeal artery and used to record the electrical stimuli in the TCC. When a reliable baseline to dural electrical stimulation was established, either gastrodin, levetiracetam, ligustrazine, or propranolol was administered. The treatment showed that gastrodin was able to inhibit nociceptive dural-evoked neuronal firing in the TCC, whereas ligustrazine showed no relevant effect on spontaneous activity in the TCC. To perform a comparison with the Chinese drugs, the established migraine preventive propranolol and the ineffective compound levetiracetam were used. As a result, propranolol showed a significant inhibition of dural-evoked responses, whereas the use of levetiracetam showed no effect. Their data suggest that gastrodin showed potential as an anti-migraine treatment, and on the contrary, ligustrazine appeared less promising. Therefore, these findings suggest further investigations about the use of gastrodin in migraine treatment. In addition, the results indicate the usefulness in exploring the traditional Chinese medicine approaches as signposts in developing new drugs for migraine [101].

In the last years, further experimental models have been developed using different approaches. Electrical stimulations have been used in experimental models with the purpose to better understand the mechanisms of migraine. Recently, Zhang et al. developed a repetitive electrical stimulation rat model [102]. In this study, the authors showed a dynamic model that upon stimuli of the dura mater, the TG begins to increase the production of the vasoactive neuropeptide pituitary adenylate cyclase-activating peptide (PACAP). PACAP is released from periphery terminals of the TG to innervating areas such as the dura mater, leading to vasodilation. PACAP is transported through central terminals to the TNC, where PACAP binds to PACAP-preferring type 1 (PAC1) receptor and triggers the excitation of nociceptive neurons with a consequent further increase in PACAP. The new repetitive electrical stimulation model established by stimulating the dura mater in conscious rats can simulate the chronification of frequent onset of acute migraine, from the perspective of cutaneous allodynia and nociceptive behaviors. PACAP appears to have a part in the pathogenesis of migraine potentially via PAC1 receptor. PACAP is co-expressed with CGRP, and therefore shows the potential to be considered a new therapeutic target for migraine [102]. The use of electrical stimulations led to the development of some clinical trials in which their use for the treatment of migraine has been assessed [103,104].

Furthermore, the effect of electroacupuncture (EA) pretreatment has been investigated. Pei et al. conducted a study regarding the use of EA in rats [105]. In this study, the authors used a conscious rat model of migraine induced by repeated electrical stimulation of the dura mater. Animals treated with EA showed an increase in exploratory, locomotor and eating/drinking behavior and a reduction in freezing-like resting and grooming behavior. In animals that received dural stimulation, an increase of c-Fos neurons in the periaqueductal grey, raphe magnus nucleus, and TNC was observed. This study showed that EA pretreatment may reduce behavioral responses to electrical stimulation of the dura mater in a rat model of recurrent migraine. These findings suggest that EA pretreatment may improve

migraine-like symptoms by altering the descending pain modulatory system. Notwithstanding, further molecular and electrophysiological research is needed to better comprehend the central mechanisms of EA treatment of migraine [105].

Further emerging therapeutic targets are the acid-sensing ion channels (ASICs), which are considered neuronal proton sensors. Amiloride (a non-specific ASIC blocker) is a compound that showed to have benefic effects in animal models of migraine. Verkest et al., investigated the involvement of the ASIC1-subtype in cutaneous allodynia [106]. The authors conducted an investigation on effects of systemic administrations of amiloride and mambalgin-1 (a specific inhibitor of ASIC1a- and ASIC1b-containing channels) on cephalic and extra-cephalic mechanical sensitivity. The treatment was performed on a rat model of acute and CM induced by intraperitoneally administration of isosorbide dinitrate. The systemic administration of these compounds reversed cephalic and extra-cephalic acute cutaneous mechanical allodynia, whereas a single administration caused a delay in the subsequent establishment of chronic allodynia. Established chronic allodynia was also reversed by both mambalgin-1 and amiloride. A single daily administration of mambalgin-1 also showed to have a preventive effect on allodynia chronification. Pharmacological results obtained in this study suggest the involvement of peripheral ASIC1-containing channels in cutaneous allodynia and in its chronification. Furthermore, the results indicate the therapeutic potential of ASIC1 inhibitors in acute and prophylactic migraine treatment [106].

The involvement of TWIK-related spinal cord K<sup>+</sup> (TRESK) channels has also been investigated. These channels are expressed in TG and DRG neurons and are the major background K<sup>+</sup> channels in primary afferent neurons. Mutations in TRESK channels have been associated with familial and sporadic migraine. Nevertheless, whether enhanced TRESK channel activity would reduce the excitability of primary afferent neurons has not been evaluated. Guo et al. [107] observed that the over-expression of TRESK subunits lead to an increase in background K<sup>+</sup> currents, a reduction of input resistance, and a reduction in the excitability of small-diameter TG neurons. The overexpression of TRESK subunits inhibits capsaicin-evoked spikes in TG neurons, suggesting that a TRESK-specific channel opener may exhibit analgesic effect via reducing the excitability of primary afferent neurons [107].

Lengyel et al., investigated in in vitro the effects of chemically modified analogs of cloxyquin, tested on TRESK and other K2P channels. Cloxyquin is known as a specific activator of TRESK (K2P18.1, TWIK-related spinal cord K<sup>+</sup> channel) background potassium channel. In this recent study, among the modified analogues of cloxyquin used, the authors identified A2764, a selective inhibitor of TRESK that can inhibit TRESK in native cells, leading to cell depolarization and increased excitability. This compound may be of use to probe the role of TRESK channel in migraine and nociception [108].


New compounds and nanoparticles in experimental models.

**Table 4.**

#### *Int. J. Mol. Sci.* **2019**, *20*, 2932
