**1. Introduction**

Migraine is a complex neurological disease considered the primary headache disorder leading to disabling conditions [1]. The last International Classification of Headache Disorders (3rd edition) describes migraine as a recurrent headache disorder manifesting as a unilateral and throbbing headache with pain intensity from moderate to severe [2]. Common symptoms observed are photophobia and phonophobia, nausea and/or vomiting [2]. Migraine can be distinguished in episodic migraine (EM) and chronic migraine (CM). EM is defined whether the headache days per month are less than 15, and CM, whether headache days are equal or more than 15 for a period of more than three months. Moreover, migraine appears to occur more in women than in men [3]. In addition, it is estimated that about 30% of patients experience an aura that consists in a short period of visual, sensory, or motor disturbances [4]. Mechanisms involved in migraine are not yet entirely clarified. To date, it is thought that the genesis of pain occurs by activation of the trigeminovascular system (TGVS). This system is composed of the cranial vasculature, the trigeminal nerve and the trigeminal nucleus caudalis (TNC). TGVS plays an important role as a major control center in regulating the cerebral blood flow and it is believed as a key conduit for pain transmission [5]. The activation of trigeminal sensory nerve endings induces the release of vasoactive agents, such as calcitonin gene-related peptide (CGRP), substance P and neurokinin A, resulting in vasodilation and dural plasma extravasation, leading to neurogenic inflammation [5]. Current migraine treatments regard the use of drugs aimed to decrease the frequency,

severity, and last of migraine attacks [6]. In case of mild attacks, medications such as acetaminophen and aspirin are used, whereas triptans or dihydroergotamine (DHE) are used for the treatment of a moderate to severe migraine [7,8]. The current pharmacological treatment used in migraine is summarized in Table 1. Our current knowledge about the pathophysiology of this complex disorder is based mostly on animal models developed to study the nociceptive pathways of the TGVS and their ascending projections to the brainstem and the diencephalic nuclei. These models are mostly based on modifications operated in order to try mimicking headache symptoms, requiring manipulations to activate the trigeminal nerve or dural nociceptors [5,9,10]. Although animal models show shortcomings per se, due to difficulties in reflecting all hallmarks of migraine, they have been used as a screening tool for the development of novel anti-migraine drugs, in which serotonin (5-HT) and CGRP receptors have importantly contributed as targets, showing to be involved in pathways that result in headache attacks [11]. Furthermore, drug delivery systems based on the use of formulations composed of nanoparticles could be considered a new attempt to improve the effects of drugs used in migraine treatments. The present manuscript has been generated using PubMed as source of information, with the aim to offer an overview of murine models developed to study migraine pathophysiology. For the promising results obtained in migraine treatment, in this manuscript the pharmacological targets 5-HT and CGRP receptors were evaluated. In addition, the database ClinicalTrials.gov [12] has been used as source of information for the new emerging treatments using agonists of 5-HT receptors and new antagonists of CGRP receptors. Validated animal models of migraine are summarized to provide an overview. At last, new therapeutic strategies and nanoparticles tested in experimental models performed on rats or mice in the last years were considered.



#### **2. Pharmacological Targets in Migraine Treatment: 5-HT and CGRP Receptors**

Research advances are increasingly focusing on the development of anti-migraine drugs. In this context, 5-HT and CGRP receptors have been mainly investigated. 5-HT receptor agonists and CGRP receptor antagonists are providing an important contribution in emerging treatments aimed to counteract migraine attacks [15]. The new emerging treatments, approved or still looking for approval by FDA, are summarized in Table 2.

Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine neurotransmitter, widely distributed both centrally and peripherally, in the human body. It is primarily found in the enteric nervous system located in the gastrointestinal tract. It is also produced in the central nervous system (CNS), specifically in the raphe nuclei where neurons containing 5-HT have been observed [16]. 5-HT biological functions are multiple and complex and its eventual response mainly depends on the nature of the 5-HT receptors implicated [17]. Seven types of 5-HT receptors have been identified, respectively named: 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7. 5-HT1 and 5-HT2 receptors are respectively divided into the following subtypes: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F and 5-HT2A, 5-HT2B, and 5-HT2C [18,19]. Receptors functional characteristics depend on their molecular structure, being G-protein-coupled receptors such as: 5-HT1, 5-HT2, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7, or integral to an ion channel such as 5-HT3 [20]. All 5-HT1 receptor subtypes are coupled to Gi/o, a protein that predominantly inhibits adenylyl cyclase activity, with consequent inhibition of the release of neurotransmitters and reduction in neuronal firing [17,20]. Triptans are agonists of 5-HT1B/1D receptors and showed to be efficacious in the acute treatment of migraine, thus providing an indirect proof suggesting the involvement of serotonin in the pathogenesis of migraine [21,22]. An increased production of 5-HT seems to occur in migraineurs brain compared to control subjects, that might lead to cortical hyperexcitability [23]. A reduction in blood concentrations of 5-HT was observed in subjects with migraine in the absence of aura during the headache phase [24]. Another study indicates that low 5-HT4 receptor binding, that suggests a high 5-HT concentration in the brain, can be considered a trait marker of migraineurs rather than a risk factor that can lead to a conversion from EM to CM. Higher concentrations of 5-HT could lead to an enhancement of the susceptibility to migraine attacks. Therefore, a reduction of 5-HT concentrations could reveal effective to treat migraine, indicating that further studies involving other 5-HT receptor subtypes and modulation of cerebral 5-HT concentrations in migraine subjects are required [25]. The increased understanding of migraine pathophysiology recently resulted in developing novel molecules that are under investigation as emerging options to be used in therapeutics. The novel ditans are serotonin 5-HT1F receptor agonists, a class of drugs that differentiate from the known triptans in showing high selectivity at receptors. The ditan Lasmiditan is currently under investigation in migraine acute treatment [15].

CGRP is a multifunctional neuropeptide that appears to play an important part in migraine mechanism and its origin was predicted observing the alternative splicing of the calcitonin gene [26]. This neuropeptide is known as one of the most potent vasodilators [27]. Two isoforms have been characterized, α-CGRP and β-CGRP. The isoform α is principally expressed in primary sensory neurons, whereas the isoform β is mainly found in intrinsic enteric neurons [27]. The mature form of this neuropeptide is composed of 37 amino acids, and its expression has been particularly noticed in sensory neurons of the dorsal root ganglia (DRG) and trigeminal ganglion (TG) [26,27]. The mature form is stored in vesicles localized in the terminal region of central and peripheral nerve endings. There, their content may be secreted in the dorsal spinal cord or in various peripheral tissues, especially surrounding blood vessels which may modulate vascular tone [28]. In addition, the presence of nociceptors network positive to CGRP in rodent and human meningeal vessels has been observed [29,30], and about 40–50% of TG neurons are positive to CGRP [31,32]. Moreover, in areas of the CNS, such as hypothalamus, thalamus, periaqueductal gray, superior and inferior colliculi, amygdala, trigeminocervical complex (TCC) and the cerebellum, the expression of CGRP has been observed [33,34]. These mentioned brain areas may be associated with migraine pathophysiology, considering the capability of CGRP to change synaptic and neuronal activity at the TCC, and transmission of nociceptive signals to the

thalamus and cortical areas [35,36]. The structure of CGRP receptor is a complex of proteins composed as follows: a G-protein-coupled receptor named the calcitonin receptor-like receptor (CLR) [37]; a single transmembrane accessory protein named receptor activity-modifying protein 1 (RAMP1) [38] (needed to establish the binding of CGRP to CLR), and the receptor component protein (RCP) [39] that characterizes the G-protein associated with the receptor. The expression of the subunits that compose the CGRP receptor complex has been observed in peripheral and central sites [32], e.g., in cell bodies in TG, in the periaqueductal grey, and in the TNC [40,41]. Nevertheless, it is not yet completely established whether the assembly of all these subunits composing the fully functional receptor form is performed in these anatomical structures. Despite this, the expression of the functional form of CGRP-receptor complex has been observed in vascular smooth muscle cells in arteries and arterioles (also in those of the cranial circulation), as suggested, both in vitro and in vivo [27], by the powerful vasodilatory effect of CGRP in these blood vessels. To date, CGRP is considered a new important pharmacological target for migraine treatment [15]. CGRP antagonists act by inhibiting vasodilation and neurogenic inflammation through release blockage of CGRP in the migraine pathway [42].

Emerging CGRP receptor antagonists, such as ubrogepant and rimegepant, are currently under assessment in therapeutics [15]. In addition, the development of new CGRP receptor or ligand antagonists is ongoing, in which monoclonal antibodies (mAbs) such as fremanezumab, galcanezumab and erenumab (all approved by FDA in 2018 for EM or CM), are opening a new approach in therapeutic strategy, representing a valuable support to the solutions already available [15].



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